Inflammatory Pathways in Diabetes

Progress in Inflammation Research
Series Editor: Michael J. Parnham
Michael Pugia Editor
Inflammatory
Pathways in
Diabetes
Biomarkers and Clinical Correlates

Series Editors
Michael J. Parnham
Fraunhofer IME & Goethe University
Frankfurt am Main, Germany
Achim Schmidtko
Witten, Germany
The last few years have seen a revolution in our understanding of how blood and
tissue cells interact and of the intracellular mechanisms controlling their activation.
This has not only provided multiple targets for potential anti-inflammatory and
immunomodulatory therapy, but has also revealed the underlying inflammatory
pathology of many diseases. This monograph series provides up-to-date information
on the latest developments in the pathology, mechanisms and therapy of inflammatory
disease. Areas covered include: vascular responses, skin inflammation, pain,
neuroinflammation, arthritis cartilage and bone, airways inflammation and asthma,
allergy, cytokines and inflammatory mediators, cell signalling, and recent advances
in drug therapy. Each volume is edited by acknowledged experts providing succinct
overviews on specific topics intended to inform and explain. The series is of interest
to academic and industrial biomedical researchers, drug development personnel and
rheumatologists, allergists, pathologists, dermatologists and other clinicians
requiring regular scientific updates.
More information about this series at http://www.springer.com/series/4983
Michael Pugia
Editor
Inflammatory Pathways
in Diabetes
Biomarkers and Clinical Correlates
Editor
Michael Pugia
Strategic Innovation
Siemens Healthcare Diagnostics
Elkhart, IN
USA
Additional material to this book can be downloaded from http://extras.springer.com

ISBN 978-3-319-21926-4
ISBN 978-3-319-21927-1
DOI 10.1007/978-3-319-21927-1
(eBook)
Library of Congress Control Number: 2015952543

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Abbreviations
ABP-26
AC
ACC
ACS
Adpn
AdipoR
AG
AGE
AGP
ALP
Akt
AKI
AMI
AMBK
AMPK
ASP
AUC
Bik
BMI
BNP
BSA
CABG
CAD
CBC
CCF
CCL
CD
CHF
CI
CINC
CKD
Adenosine deaminase binding protein 26

Adenylcyclase
Acetyl Coenzyme A Carboxylase
Acute coronary syndrome
Adiponectin
Adiponectin Receptor
Anhydroglucitol
Advance glycation end products
-1- acid glycoprotein
Alkaline phosphatase
Protein kinase B
Acute kidney injury
Acute myocardial infarction
pre-alpha-1-microglobulin/bikunin
5-AMP activated protein kinase
Acylation stimulating protein
Area under the curve
Bikunin
Body mass index
B-type natriuretic peptide
Bovine Serum Albumin
Coronary artery bypass grafting
Coronary artery disease
Complete cell count
Cleveland Clinic Foundation
Chemokine (C-C motif) ligand
Clusters of differentiation
Congestive heart failure
Confidence interval
Cytokine-induced neutrophil chemoattractant
Chronic kidney disease
v
vi
CPB
CTF
CRP
CVD
DGAT
DPP-IV
DM
ECM
EGF
eGFR
ELISA
eNOS
EPO
ESRD
ET
Erk
FABP
Fluo-4
FGF
FFA
FXR
GBM
GDF
GFR
GH
GIP
GLP
GN
GSH
GHSR
GULT
GCD59
HAMA
HbA1c
HDL
HGF
HIF
HMG
HMW
HTN
ICC
IHC
I--I
Ig-CTF
ICC
Abbreviations
Cardiopulmonary bypass
C-Terminal Fragment of Adiponectin Receptor
C-reactive protein
Cardiovascular disease
Acyl CoA diacylglycerol transferase
Dipeptidyl peptidase- IV
Diabetes
Extra cellular matrix
Epidermal growth factor
Estimated GFR
Enzyme linked immunoabsorbent assay
Endothelial NOS
Erythropoietin
End stage renal disease
Endothelin
Extracellular signal-regulated kinase
Fatty acid-binding protein
Calcium binding fluorescence dye
Fibroblast growth factor
Free fatty acids
Farnesoid X receptor a
Glomerular basement membrane
Growth differentiation factor
Glomerular filtration rate
Growth hormone
Glucose-dependent insulinotropic polypeptide
Glucagon-like peptide-1
Glomerular nephritis
Glutathione
Growth hormone secretagogue receptor
Glucose transporters
glycated CD59 (aka glyCD59)
Human anti-mouse antibody interference
Hemogloblin A1c
High-density lipoprotein
Hepatocyte growth factor
Hypoxia-inducible factor
3-Hydroxy-3-methylglutaryl CoA reductase
High molecular weight
Hypertension
Immunocytochemistry
Immunohistochemistry
Inter--inhibitor
Immunoglobulin attached C-Terminal Fragment of AdipoR
Immuno cytochemistry
Abbreviations
IDE
IGF
IGFBP
IGT
IHC
IL
ICAM
JAK STAT
JNK
KIM
KLH
LCN-2
LDL
L-FABP
LMW
LPD
LPS
Lp-PLA2
LXR
LYVE
mAb
MAC
MALDI
MAPK
MCP
MDA
MetSyn
MIC
MIP
MMP
MPO
mTOR
NADH
NAPLD
NGF
NFB
NGAL
NOS
NOX
NPY
ob
OFTT
OGTT
OHdG
OR
vii
Insulin-degrading enzyme
Insulin-like growth factor
IGF binding proteins
Impaired glucose tolerance
Immuno histochemistry
Interleukin cytokines
Intercellular adhesion molecule
Janus kinase/ signal transducer and activator of transcription
c-Jun N-terminal kinases
Kidney injury molecule
Keyhole limpet hemocyanin
Lipocalin
Low-density lipoprotein
Liver type fatty acid binding protein
Low molecular weight
Lipidemia
Lipopolysaccharide
Lipoprotein-associated phospholipase A2
Liver X receptors
Endothelial hyaluronan receptor
Monoclonal antibody
Membrane attack complex
Matrix-assisted laser desorption/ionization
Mitogen-activated protein kinase
Monocyte chemotactic protein
Malondialdehyde
Metabolic syndrome
Macrophage inhibitory cytokine
Macrophage infammatory protein
Matrix metalloproteinase
Myeloperoxidase
Mammalian target of rapamycin
Nicotinamide adenine dinucleotide
Non-alcoholic fatty liver disease
Nerve growth factor
Nuclear factor kappa-light-chain-enhancer of activated B cells
Neutrophil gelatinase lipocalin
Nitric oxide synthase
NADPH oxidase
Neuropeptide Y
Obesity
Oral fat tolerance test
Oral glucose tolerance test
Hydroxydeoxyguanosine
Odds-ratio
viii
oxLDL
pAb
P--I
PAI-1
PAR
PBS
PDF
PDGF
PKA
PKC
PI3K
PLA2
PLC
PNPP
PPAR
PPG
PS
PSS-380
PR
PYY
RAAS
RAGE
RFU
ROC
ROS
S100
sCr
SGLT2
SREBP
SMAD
SOD
sTNFR
TACE
TBS
TFPI
TFA
TG
TGF
TIMP
TNF
TNFR
THP
TLR
TnI
tPA
Abbreviations
Oxidized low density lipoprotein

Polyclonal antibody
pre-interleukin--inhibitor
Plasminogen activator inhibitor
Protease-activated receptors
Phosphate buffered saline
Placental growth factor
Platelet-derived growth factor
Protein kinase A
Protein kinase C
Phosphatidylinositol 3-kinase
Phosphatidylinositol 3-kinase
Phospholipase C
Para nitro-phenyl phosphate
Peroxisome proliferator-activated receptor
Postprandial glucose
Phosphatidylserine
Phosphatidylserine (PS)-binding fluorescence dye
Protienase
Neuropeptide like peptide Y
Renin-angiotensin-aldosterone system
Receptors for AGE
Relative fluorescence units
Receiver-operator characteristics
Reactive oxygen species
Calgranulin
Serum creatinine
Sodium-glucose transporter 2
Sterol regulatory element binding proteins
Sterile alpha motif domain-containing protein
Superoxide dismutase
Soluble TNF receptor
TNF alpha cleavage protease
Tris buffered saline
Tissue factor pathway inhibitor
Trifluoroacetic acid
Triglycerides
Transforming growth factor -
Tissue inhibitor of metalloproteinases
Tumor necrosis factors -
TNF receptor
Tamm-Horsfall protein
Toll-like receptors
Troponin I
Tissue plasminogen activator
Abbreviations
TSP
Tregs
TZD
Uri
uTi
UTI
uPA
uPAR
VCAM
VEGF
WBC
WB
YKL-40
ix
Thrombospondin
Regulatory T cells
Thiazolidinediones
Uristatin
Urinary trypsin inhibitor
Urinary tract infection
Urokinase-type plasminogen activator
uPA receptor
Vascular cell adhesion molecule
Vascular endothelial growth factor
White blood cell
Western Blot
Chitinase-3-like protein 1
Contents
Part I
1
Impact of Inflammation and Innate Immunity Response

in Obesity Mediated Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Michael Pugia
Part II
2
Impact of Complement on Diabetic Disease
Complement and Complement Regulatory Proteins

in Diabetes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jose A. Halperin, Pamela Ghosh, Michael Chorev,
and Anand Vaidya
Part III
Introduction
29
Role of C Terminal Fragment of Adiponectin Receptor

in Inhibition of Insulin Degradation
Development of Adiponectin Receptor C-Terminal

Fragment Bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Michael Pugia and Rui Ma
61
Protease Inhibition and Biological Distribution

of the C Terminal Fragment of Adiponectin Receptor. . . . . . . . . . . .
77
Cell and Biological Models for the C Terminal

Fragment of Adiponectin Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . .
93
C-Terminal Fragment of Adiponectin Receptor

Clinical Correlations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Adrian Vella and Michael Pugia
111
xi
xii
Contents
Part IV
Uristatin Assay for Prediction of Renal

and Other Clinical Events
Uristatin Immunoassay Usage in Glomerular

Nephritis Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Saeed A. Jortani and Michael Pugia
Acute Response of Uristatin in Surgery. . . . . . . . . . . . . . . . . . . . . . . .

Mitchell H. Rosner and Michael Pugia
Cardiovascular and Metabolic Syndrome Impact

on Uristatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
Uristatin Anti-inflammatory Cellular Signaling . . . . . . . . . . . . . . . . .

Manju Basu, Subhash Basu, and Michael Pugia
Part V
11
127
143
157
171
Summary
Progress in New Markers for Diabetes Inflammation . . . . . . . . . . . .

Michael Pugia
193
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
215
On-Line Supplements
ELISA method for IgG-CTF assay

ELISA method for free CTF assay
Western blot methods for studies
Tissue and cell staining methods
Cell culture methods
ELISA method for uristatin assay
xiii
Contributors
Manju Basu Department of Chemistry and Biochemistry,

University of Notre Dame, Notre Dame, IN, USA
Subhash Basu Department of Chemistry and Biochemistry,
University of Notre DameNotre Dame, IN, USA
Michael Chorev, PhD Laboratory for Translational Research,
Division of Hematology, Department of Medicine,
Brigham and Womens Hospital, Harvard Medical School,
Boston, MA, USA
Pamela Ghosh, PhD Laboratory for Translational Research,
Boston, MA, USA
Jose A. Halperin, MD Laboratory for Translational Research,
Boston, MA, USA
Saeed A. Jortani Pathology and Laboratory Medicine, Department of Pathology,
School of Medicine, University of Louisville, Louisville, KY, USA
Rui Ma Siemens Healthcare, Pu Dong New Area, Peoples Republic of China
Michael Pugia, PhD Strategic Innovation, Siemens Healthcare Diagnostics,
Elkhart, IN, USA
Bayer Healthcare Diagnostics, Elkhart, IN, USA
University of Louisville, School of Medicine, Louisville, KY, USA
Department of Chemistry and Biochemistry, University of Notre Dame,
Notre Dame, IN, USA
xv
xvi
Contributors
Mitchell H. Rosner, MD, FACP Division of Nephrology,

Department of Medicine, University of Virginia Health System,
Charlottesville, VA, USA
Anand Vaidya, MD, MMSc Division of Endocrinology,
Diabetes and Hypertension, Brigham and Womens Hospital,
Harvard Medical School, Boston, MA, USA
Adrian Vella, MD Division of Endocrinology and Metabolism, Mayo Clinic,
Rochester, MN, USA
Part I
Introduction
Chapter 1
Impact of Inflammation and Innate Immunity

Response in Obesity Mediated Diabetes
Michael Pugia
1.1
Healthcare Impact of Diabetes
Diabetes has a significant impact on overall healthcare costs. The population of

people in the world who have been diagnosed with diabetes has steadily increased
with the prevalence of obesity over the last 2030 years (See Fig. 1.1). There is a real
need to reduce the number of diabetics and to delay end stage complications. This
need is difficult to address as there is poor diagnostic ability to sort out which prediabetics will become diabetics and which diabetics will progress to complications.
The frequency and duration of hyperglycemia is the commonly used tool.
Hyperglycemia increases as the pre-diabetic patient progresses from impaired glucose tolerance (IGT) to fully glucose intolerant. Diabetic hyperglycemia has been
correlated with complications such as retinopathy, nephropathy, neuropathy, and
increased risk of myocardial infarction, stroke, premature peripheral arterial disease,
atherosclerosis, and cardiomyopathy (Nathan et al. 2014; Forbes and Copper 2013).
Control of hyperglycemia has been extensively correlated to reduced complications
(Nathan et al. 2014). It is now known that diabetic hyperglycemia is clinically correlated with mild inflammation (Jenkins et al. 2008). This chapter is an overview of
the progress made in biochemical relationships of the pathogenesis of diabetes and
inflammation. In Chap. 6 we further explore the tools used in the assessment of
hyperglycemia and diabetic inflammation by reporting clinical testing.
As the pre-diabetic patient progresses to diabetes, significant insulin resistance is
observed. Hyperglycemia worsens with increased insulin resistance, as insulin is
the major controller of glucose metabolism. It is produced in the pancreas where its
secretion is stimulated by a rise in blood glucose. Insulin binds to adipose to inhibit
M. Pugia, PhD
Strategic Innovation, Siemens Healthcare Diagnostics,
3400 Middlebury Street, Elkhart, IN 46515, USA
e-mail: [email protected]
M. Pugia (ed.), Inflammatory Pathways in Diabetes: Biomarkers
and Clinical Correlates, Progress in Inflammation Research,
DOI 10.1007/978-3-319-21927-1_1
M. Pugia
Unmeet need
Mechanism progress
Diabetes in US causes 173 billion in costs

Complication cost of disease growing at >25%
HbA1c insensitive to early diabetes
Need to sort emerging diabetics with high specificity
OGTT is to difficult do and clinicians need a spot test
Fat cell signaling

Inflammation activation
Insulin degradation
Complement activation
Innate immune activation
Increase in Obesity
1990
2010
Leptin ,Adiponectin, etc

TNF activation
IDE, AdipoR1 CTF
gCD59, C1, C3a,b
Adipose involvement, Bik
Pre-diabetics
Type 2
Diabetes
79 mm
23 mm
Type 1
Diabetes
5 mm
Hypertrophic adipose
IGT
Glucose intolerant
Insulin resistance
<10% 15%19% 25%
Beta cell loss
Fig. 1.1 Diabetes impact on health care cost. The increase in obesity in the U.S. over the last 30
years has increased to more than 25 % of the population in many states. The associated rise in
diabetes is now estimated to be 79 million pre-diabetic and 23 million type 2 diabetic. This leaves
an unmet need for diagnostics to sort emerging diabetics with highly specific and sensitive treatments to stall progression and reduce the cost of complications. Mechanistic progress has been
made on several fronts and is reviewed in this chapter
lipolysis and activates the liver and muscle to store glucose as glycogen, and it promotes glucose uptake into muscle and adipose tissues through glucose transporter 4
(GLUT4) (Dugani 2005). The insulin resistant state occurs when insulin receptor
responsiveness is reduced. Significant progress has been made in the identification
of factors that impact insulin sensitivity, such as adipokines, inflammation signaling, complement factors, and immune cell activation. The recently discovered adipokine hormones such as leptin, adipionectin, and ghrelin also impact insulin action.
Additionally, hormones such as growth hormone (GH), insulin-like growth factor
(IGF), insulin-like growth factor binding protein (IGFBP), epinephrine, glucagon,
and cortisone are known to influence glucose metabolism and secretion and impact
insulin action. However, known biomarkers still do not adequately predict insulin
resistance (Goldfine et al. 2011). In Chap. 4 we explore the role of reduced insulin
degradation during insulin resistance.
1.1.1
Glycation Stress
Hyperglycemia causes biological stress that produces advance glycation endproducts (AGEs) that alter proteins, lipids, glycans, and nucleic acids (Goh et al.
2008). AGE are formed either by non-enzymatic glycosylation between reducing
sugars and amine residues or by autoxidation of glucose. Non-enzymatic reactions
Impact of Inflammation and Innate Immunity Response in Obesity Mediated Diabetes
of protein amino groups with glucose form a ketoamine adduct (Amadori product)
that is dependent on incubation time and glucose concentration. Lysine is the preferred non-enzymatic glycation site over hydroxylysine and arginine (Garlick et al.
1988). AGE can form with most molecules in human biological fluids and tissues at
multiple glycation sites (Zhang et al. 2008; Ramrez-Boo et al. 2012). Basal glycation is typically detectable in relatively high concentration with normal patient samples, and diabetic patient samples are about two to fourfold higher (Calvo et al.
1988; Ramrez-Boo et al. 2012).
The discovery that glycated hemoglobin (HbA1c) is an AGE component of
human hemoglobin turned out to be a useful diagnostic tool for diabetic care (Rahbar
2005). Today, HbA1c is well correlated with glycemic control and diabetic complications (Nathan et al. 2014). This correlation is based on the intensive glucose control to reduce carotid intima-media thickness and levels of the HbA1c. HbA1c is
also highly correlated with the extent and duration of hyperglycemia over 23
months. The duration of the prediction is directly due to the natural turnover of red
blood cells. Glycation of human serum albumin (HSA) has also been extensively
studied and shown to increase in diabetic patients (Wa et al. 2007). Non-traditional
AGE markers of hyperglycemia (glycated albumin, fructosamine, 1,5-anhydroglucitol
(1,5-AG)) are responsive to glycemic control over shorter periods (Poon et al. 2014;
Parrinello and Selvin 2014). While nontraditional markers are often linked to vascular complications, they are often found to be inversely related to obesity.
Glycation stress and AGE formation are often proposed as having key mechanistic roles in biological damage. AGE formation on endothelial cells and monocytes
has been tied to nephropathy, retinopathy, and low grade inflammation in humans
(Goh et al. 2008). In the kidneys, AGE formation on glomerular basement membrane (GBM) is correlated with membrane thickening, mesangial matrix expansion,
and increased collagen, fibronectin, and laminin (Throckmorton et al. 1995; Skolnik
et al. 1991). Guanidine-insoluble collagen is the site extensively glycated on GBM
(Garlick et al. 1988). This AGE formation is observed during fibrosis with increased
smooth muscle migration and proliferation. Fibrosis requires activation by connective tissue growth factors and Transforming Growth Factor- (TGF- ). Glycation
of lipids is also detectable. For example, high-density lipoprotein (HDL) levels
increase by about 400 % in diabetic patients (Calvo et al. 1988). AGE of lipid and
proteins are theorized to preserve cellular signal patterns in a pathologically stressed
state until they are cleared from the cell (Ceriello et al. 2009). Inhibition by therapeutic agents has been shown to reduce AGE formation (Goh et al. 2008). However,
how AGE stimulate molecular damage is not completely clear.
Receptors for AGE (RAGE) are often implicated as a pathway for molecular
damage. RAGE are part of the immunoglobulin superfamily of receptors and lectin.
RAGE are found to respond to a wide range of proteins through the glycated lysine
residues (Neeper 1992). However, the binding of these AGE ligands is not sufficient
to induce inflammatory signals. The key ligand primarily responsible for RAGE
stimulation appears to be calgranulin (S100), a protein that is secreted from innate
immune cells (neutrophils and monocytes) independent of glycation (Bierhaus et al.
2005; Valencia et al. 2004). Activation of RAGE appears to be mainly immune cell
M. Pugia
mediated and a pathway for molecular damage. Activation increases vascular endothelial cell expression of intercellular adhesion molecule-1 (ICAM-1) and vascular
cell adhesion molecule-1 (VCAM-1) and causes innate immune cells to secrete
tumor necrosis factors - (TNF-) and TGF- (Valencia et al. 2004; Wautier et al.
1996). These cytokines induce the inflammatory pathways involving the nuclear
factor kappa-light-chain-enhancer of activated B cells (NFB) and the mitogenactivated protein kinase phosphatase (MAPK) pathways (Ramasamy et al. 2011).
Additionally, S100 binding of RAGE on innate immune cells activates phospholipase D2 and phosphokinase C (PKC) which are thought to generate additional reactive oxygen species (ROS) by the conversion of nicotinamide adenine dinucleotide
phosphate-oxidase (NADPH) to NADPH oxidase (NOX) (Pendyala et al. 2009;
Curran and Bertics 2011).
In summary, while glycation stress and glycemic control can be measured by
current AGE markers, there has been only a weak molecular connection between the
AGE markers and the causation of complications. Next generation AGE markers
seek to make stronger connections with the causation of complications. Chapter 2
explores glycated CD59 as one of these next generation markers. The glycation of
CD59 occurs at a specific site that blocks CD59 from inhibiting formation of membrane attack complex (MAC) (Halperin et al. 2000). As a result, glycation increases
MAC cell lysis during complement formation. Chapter 2 also reviews the importance of complement in diabetic complications.
1.1.2
Oxidative Stress
Hyperglycemia is shown to increase oxidative stress and the production of

ROS. Oxidative stress is a normal byproduct of the aerobic metabolism, where oxygen is lost to the formation of superoxide anion free radicals as the predominate
ROS (Pitocco 2013; Seifert et al. 2010). The mitochondria electron transport chain
produces ROS during the production of adenosine triphosphate (ATP) from the glycolytic pathway (citric acid cycle). Hyperglycemia causes the glycolytic pathway to
breakdown into more glucose producing NADH (Yan 2014). Overproduction of
NADH increases mitochondrial oxidative stress byproducts by NADH dehydrogenase. Additionally, the consumption of glycolytic pathway byproducts by the
hexoamine, methlglyloxyal, enediol, and diahydroxyacetone pathways cause additional ROS. Diacylglycerol also activates NOX through PKC and leads to additional
ROS (Pendyala et al. 2009).
The result of ROS is damage to DNA, lipid, and proteins (Piconi 2003; Yan
2014; Seifert et al. 2010). ROS leads to additional free radicals which cause further
damage. For example, production of reactive nitrogen species is formed by free
radical exchange with nitric oxide that is produced by nitric oxide synthase (NOS)
(Pitocco 2013). Oxidative stress is correlated with diabetic inflammation, insulin
sensitivity, and -cell dysfunction (Bashan et al. 2009; Poitout and Robertson 2008).
Oxidative stress is also associated with aging, cancer, atherosclerosis, neuropathy,
nephropathy, retinopathy, obesity, hypertension, cardiovascular diseases, and heart

failure (Lee and Wie 2007; Storz 2005; Lim et al. 2008; Madamanchi et al. 2005;
Kanauchi et al. 2002; Knight 1997; Wilkinson-Berka et al. 2013; Pitocco 2013).
ROS are implicated in causing epigenetic changes, endothelial dysfunction, impacting vascular tone, apoptosis, cell adhesion, and immune responses (Lim et al. 2008;
Lee and Wie 2007; Zhang and Gutterman 2007; Gutterman et al. 2005; Chiarugi
et al. 2003; Grisham 2004). Immune cells cause oxidative stress by increasing ROS
through immune cell NOX (Hou et al. 2008). ROS impact many cellular processes
such as proliferation and apoptosis (Droge 2002). Inflammation, growth factors, and
cytokines also impact ROS formation. Overall, oxidative stress is one plausible
mechanism for biological damage, but it is non-specific to hyperglycemia.
1.2
1.2.1
Impact of Obesity
Adipose Signaling
Obesity clearly leads to diabetes, and it increases the inflammatory response (Brady
et al. 2012; Horakova et al. 2011). Obesity also worsens hyperglycemia and
increases ROS and AGE generation. The current epidemic of obesity has increased
the population of pre-diabetes with impaired glucose tolerance, insulin resistance,
and hypertrophic adipose fat (See Fig. 1.1). The discovery of the hormone, leptin; a
product of the obese (ob) gene, opened the door for discoveries of new regulatory
factors originating from adipose fat (Zhang et al. 1994). Adipose tissue is now
accepted as a source of adipokine signals that impact metabolism and are linked to
obesity mediated diabetes (Preedy and Hunter 2011). Adipose tissue is a complex
matrix and contains various cell types such as adipocytes, macrophages, fibroblasts,
adipocyte precursors, and others (Ahima 2006; Espinoza-Jimnez et al. 2012). Of
the molecules released primarily from adipocytes during obesity, leptin, and adiponectin (Adpn) have the strongest correlated impact on glucose metabolism (Wang
and Nakayama 2010). Leptin and Adpn have the structural characteristics of a cytokine; therefore, they are called adipokines.
Leptin reduces food intake (loss of appetite, satiety) and increases energy expenditure which leads to weight loss (Gordon 2003). It is produced by the ob gene, and its
deficiency causes obesity. Chapter 5 describes leptin resistant animals which become
obese and spontaneously develop diabetes. Leptin signals the level of adipose energy
stores to the brains hypothalamus and causes decreases in food intake and increases
its energy expenditure. This results in more fat oxidization. Leptin activates the Janus
kinase-signal transducer-activator of transcription (JAK STAT) pathway to stimulate
inhibition of neuropeptides and activation of several anorexigenic peptides (Sweeney
2002; Gordon 2003). It has an effect on the central nervous system (CNS), it stimulates fatty acid oxidation in muscle, and it controls triglyceride synthesis in the liver
(Minokoshi et al. 2002; Cohen et al. 2002). Leptin contributes to insulin resistance
and is involved in the release of inflammatory cytokines (Smith 2012).
M. Pugia
Adiponectin has structural similarity to the C1q/TNF family of proteins (Preedy

and Hunter 2011). It activates AMPK and upregulates peroxisome proliferatoractivated receptor (PPAR ) to enhance glucose uptake and fatty acid oxidation in
muscle (Yamauchi et al. 2002) (See Chap. 5 for further details). Adpn suppresses
glucose output in the liver and improves insulin sensitivity through a mechanism
that is not well understood (Wang et al. 2005). Adpn is also present in CNS and
peripheral tissue. Levels of Adpn correlate inversely with obesity; whereas Leptin
levels increase in obesity (Satoh 2004). Decreased Adpn is associated with diabetes
and cardiovascular disease. Adpn also has anti-inflammatory properties, where it
suppresses cytokines, adhesion compounds, and lipid uptake in monocytes
(Yamauchi et al. 2002; Ouchi et al. 2000). Chapters 5 and 10 show how cellular
models can be used to explore cell signal transduction of Adpn and other biochemical substances on the insulin-dependent pathways of phosphatidylinositol 3-kinase
(PI3K), protein kinase B (Akt), and insulin-independent pathways of AMP-activated
protein kinase (AMPK).
Even with well-established cell and animal models in place, the mechanism by
which Adpn delivers improved insulin sensitivity and anti-inflammatory effects is
still disputed. Adpn deficient mice are reported to show small changes in insulin
resistance and glucose intolerance (Kubota et al. 2002), while other reports show that
Adpn deficient mice do not show any improvement (Ma et al. 2002). Additional studies report higher hepatic glucose production in Adpn deficient mice (Meada et al.
2002). The response appears to be impacted by the combination of a high fat diet and
Adpn deficiency. There is also response dependency on the ob gene and PPAR
agonists (Nawrocki et al. 2006). Responsiveness correlates with higher tumor necrosis factors - (TNF-), mRNA expression, and reduced insulin receptor activity via
an unknown mechanism (Meada et al. 2002). Some Adpn oligomers inhibit growth
factors (e.g. platelet-derived growth factor (PDGF), fibroblast growth factor (FGF),
and epidermal growth factor (EGF)), which explains cytokine production and activation of NFB (Wang et al. 2005). Adpn also stimulates caspase mediated cell apoptosis during the pro-inflammatory response (Brkenhielm et al. 2004). The apoptosis
and cytokine stimulation aligns with innate immune mediated events that are further
described in Sect. 1.4.2, rather than a direct stimulation by Adpn. The responsiveness
to insulin secretion might be indirectly related to inflammatory signaling.
Chapters 4 and 5 describe how the adiponectin receptor (AdipoR) has a direct
impact on insulin by inhibition of insulin-degrading enzyme (IDE) and on TNF
release by inhibition of TACE (TNF alpha cleavage protease). This new finding
adds to the previously known functions of the AdipoR to cause glucogenisis and
fatty acid oxidation through the AMPK and PPAR pathways (Kadowaki et al.
2005). Chapter 3 reviews the discovery of free adiponectin receptor C terminal fragment (AdipoR CTF) in human plasma and the development new assays for measurement of this signaling receptor. Chapters 4 and 5 make use of these new assays
in biochemical studies, animal and cell models, and human clinical trials to explore
the impact of AdipoR CTF on the innate immune mediated pathway.
Adipose tissue has additional complex interactions with the brain and other
peripheral organs with additional regulatory factors. Ghrelin is a key component of
this interaction and originates from the gastrointestinal tract and indirectly impacts
the adipose by functioning as the central hunger hormone on the brain (Gumbs
et al. 2005). As a para gut hormone, ghrelin is an endogenous ligand of the growth
hormone secretagogue receptor and stimulates the production of neuropeptide like
peptide Y (PPY) through the JAK STAT pathway. Ghrelin levels increase before
meals, and it is thought to be involved in meal initiation and shows a rapid postprandial decline. Ghrelin administration causes increased caloric intake and initiation of
hunger.
Several additional regulatory factors have weaker connection to glucose metabolism in obesity. Some are primarily produced and secreted by adipose. These include
omentin (interlectin), visfatin (nicotinamide phosphoribosyltransferase), and resistin. Other regulatory factors are secondarily secreted by adipose, like retinol-binding
protein 4 and vaspin. While they all generally correlate with obesity, the relevance
of these regulatory factors is still in question (Preedy and Hunter 2011).
1.2.2
Free Fatty Acid Storage
Adipose is the major site of energy storage in the form of triglyceride (TG).
Adipocyte can release free fatty acids (FFA) and TG when the bodys energy level
demands it. Adipose, FFA, and TG are controlled by several regulatory factors from
adipocytes and other tissue. Acylation stimulating protein (ASP), fatty acid-binding
protein (FABP), adipose factor (aka angiopoietin-like protein 4), and cytosolic fatty
acid synthase (FAS) are examples.
ASP is produced in adipose and other tissue, and it stimulates glucose uptake,
clearance of TG, and it inhibits TG release (Cianflone and Maslowska 1995;
Cianflone et al. 2004). ASP is produced by an interaction with complement C3 (aka
ASP precursor), factor B, and complement factor D (aka adipsin). All can be produced by adipose. ASP increases with increases in weight, diabetes, obesity, metabolic syndrome, and dietary fat (chlyomicrons) (Faraj et al. 2008; Cianflone 2008).
ASP regulates TG synthesis by activating diacylglycerol acyltransferase (DGAT).
However, some researchers report no impact of ASP deficiency in animal models
(Phieler et al. 2013). Other question the role that ASP plays, whether implicated in
complement-mediated injury or in regeneration interaction with complement C3
(Phieler et al. 2013).
In healthy individuals, hypoglycemia decreases insulin levels and increases
lipase breakdown of TG into FFA (Stralfors and Honnor 1989). Albumin and FABP
transport fatty acids to muscle and liver for fatty acid oxidation. FABP is widely
secreted in the body and causes fatty acid uptake and intra-cellular transport
(Chmurzyska 2006). Adipose FABP increases with obesity and correlates with
adipose tissue mass and body mass index (BMI) but is poorly predictive of insulin
resistance (Horakova et al. 2011). Fatty acid oxidation and release of energy in muscle and liver occur by activation and transport into the mitochondria, -oxidation,
and the electron transport chain.
10
M. Pugia
Hyperglycemia increases insulin levels and signals the liver and muscle to stop
fatty acid oxidation and also signals the adipose to store FFA and TG. Under a continued high energy diet in a low exercise environment, increased fat storage and
resulting obesity will occur. Large vacuoles of triglyceride fat accumulate in the
liver and adipose. This causes steatosis in the liver and greater lipid loading of adipocytes and increased adipose mass in the adipose, which leads to hypertrophic and
hypoxic adipose tissue. Adipose factor is produced by adipose and other tissue to
inhibit storage of FFA and TG (Preedy and Hunter 2011). It is produced in the liver
and contributes to the development of diabetic dyslipidemia; it inhibits lipoprotein
lipase in adipose tissue lipolysis; and it raises plasma TG and FFA (Mandard et al.
2006). However adipose factor has poor diagnostic predictive value for diabetic
onset.
Adipose fat synthesis is stimulated by insulin directly by stimulating pyruvate
dehydrogenase and the acetyl-CoA carboxylase enzymes and indirectly by glucose
uptake, which then stimulates nuclear receptors. Synthesis of most fatty acid moieties of the membrane lipids is done by FAS, which synthesizes fatty acids from
malonyl CoA. Synthesis and metabolism of both fatty acids and cholesterol lipids is
through transcriptional regulation by nuclear receptors such as sterol regulatory element binding proteins (SREBP), farnesoid X receptor a (FXR), and the liver X
receptors (LXR). Activation causes transcription of genes for cholesterol synthesis
and the LDL receptor (Ulven et al. 2005; Calkin 2013). Glucose induces FXR gene
expression in a dose- and time-dependent manner. FXR expression decrease triglyceride levels by regulating the expression of several lipid-modulating proteins.
1.3
Inflammation in Obesity and Diabetes
With the development of obesity, there is an increase in adipocyte mass, greater

lipid loading (TG, FFA, chylomicrons), tissue hypertrophy, and reduced vascularization of the adipose tissue. The reduced capacity of the bloodstream to oxygenate
adipose tissue results in hypoxia. Hypoxia then produces a pro-inflammatory
response from adipose through hypoxia-inducible factor (HIF) (Nizet and Johnson
2009; Trayhurn et al. 2008). Alternative complement pathway reviewed in Chap. 2
is activated in hypoxia stress via C1q complement which causes deposits, tissue
injury, and further cellular activation (Collard et al. 1999). Alternative complement
pathway is not triggered directly by glucose or insulin in high fat diets (Peake et al.
2005). Complement activation in adipose tissue, liver, and pancreas occurs under
conditions of oxidative stress with upregulated complement receptors (Phieler et al.
2013). This activation is highly varied and not completely understood. Plasma C3
levels do appear to be higher in patients with diabetes, and this is mainly from adipose activation. However, complement activation is observed in the vicinity of
apoptotic cells in the liver, kidney, and pancreas. Islets cells accumulate extracellular amyloid fibrils which can trigger local complement activation via C1q (Phieler
et al. 2013).
11
Dysfunctional adipose tissue produces cytokines such as TNF, TGF-, and

interleukins (IL-1, IL-6, IL-8 IL-18), as well as chemokines like macrophage inhibitory cytokine (MIC-1), monocyte chemotactic protein (MCP), and cytokine-induced
neutrophil chemoattractant (CINC-1) (Wang and Nakayama 2010; Preedy and
Hunter 2011). Lipocalins such as neutrophil gelatinase-associated lipocalin (NGAL,
aka Lipocalin-2 (LCN-2)) are released by neutrophils in obesity (Preedy and Hunter
2011). Chitinase-3-like protein 1 (YKL-40, aka CHI3L1) is secreted by macrophages in obesity. Regulatory factors that impact the endothelial, such as endothelin
(ET), thrombospondin (TSP-1), plasminogen activator inhibitor 1 (PAI-1), apelin,
and angiotensinogen, are also secreted by adipose in addition to primary excretion
by other tissue. The regulatory endothelial factor adrenomedullin is primarily produced by epicardial fat.
The innate immune and complement responses increase as obesity progresses to
type 2 diabetes (Espinoza-Jimnez et al. 2012; Phieler et al. 2013). The innate cells,
which include neutrophils, eosinophiles, monocytes, natural killer cells, and macrophages, increase in diabetes and obesity. In Chap. 7 we review how diabetics are
prone to urinary tract infection (UTI) and systemic infections like Upper Respiratory
Infection (URI). In Chaps. 7 and 8 we show that non-infective inflammatory activation is common in diabetes and obesity. Chronic mild inflammation in the absence
of infection is now widely accepted and supported by mechanisms for activation of
inflammation, complement, and innate immunity in diabetes (Tilg 200; Phieler et al.
2013; Espinoza-Jimnez et al. 2012).
1.3.1
Endothelial Dysfunction
Clinical and experimental data support a link between systemic inflammation and
endothelial dysfunction in diabetes and obesity (Roberts and Porter 2013).
Endothelial dysfunction occurs in diabetes with impaired endothelial-dependent
vasodilation. Disruption of systems controlling vascular permeation can cause
endothelial dysfunction. The most significant modulator of endothelial vasodilation
is nitric oxide that is produced by endothelial nitric oxide synthase (eNOS) in endothelial and muscle cells. Nitric oxide also inhibits platelet aggregation, cell proliferation, and immune cell adhesion. Cell adhesion molecules (VCAM-1 & ICAM-1)
are reduced. Meanwhile the renin-angiotensin-aldosterone system (RAAS) is the
counter balance system, as it increases vasoconstriction, blood pressure, and fluid
volume when angiotensin-converting enzyme forms angiotensin II.
These systems that control vascular permeation are critical to the innate immune
response and to tissue repair because they allow migration, adhesion, innate immune
cell response, cellular proliferation growth, and remodeling (Pacurari et al. 2014).
During normal inflammation that results from injury or infection, muscle cells
release serine protease and kallikrein, to form kinins, and they cause vasodilation
and hypotension, increased capillary permeability, and infiltration of innate immune
cells to the site of injury (Campbell 2000, 2001). Kinin formation is another mecha-
12
M. Pugia
nism for nitric oxide release and promotion of natriuresis and diuresis. Angiotensinconverting enzyme also reduces kinins. There is a direct correlation between
vasodilation and increased blood flow to the site of inflammation with the loss of
endothelial barrier being a hallmark of inflammation.
Endothelial dysfunction starts in diabetes and obesity when hyperglycemia and
adipose signals exert signaling control on the endothelial barrier that overrides normal physiological control. Increased vascular permeability occurs in all diabetic
vascular complications. Hyperglycemia is shown to diminish normal nitric oxide
signaling and reduce permeability by increasing ROS which reduces nitric oxide
levels (Roberts and Porter 2013). Hyperglycemia also increases vascular permeability by increasing the kallikrein response from smooth muscle (Feener et al. 2013;
Riad 2007). Hypertrophic adipose diminishes nitric oxide through production of
cytokines such as TNF-, which activates NFB and impairs eNOS expression
(Besler 2011). Insulin resistance is another key promotor of vascular dysfunction, as
insulin is less able to play a protective role of controlling eNOS expression via AktPI3K (Aroor et al. 2013).
Fat tissue in an obese individual impacts additional signals that both increase and
decrease endothelial permeability. Adipose angiotensin is secreted during obesity
and activates the RAAS counter balance system in the absence of injury. Adipose
angiotensin increases blood pressure, angiotensin II levels, volume expansion,
sodium retention, and it reduces glucose uptake (Preedy and Hunter 2011; Aroor
et al. 2013). Circulating lipids cause activation of PKC which increases expression
of vasoconstrictor endothelin (ET-1) (Nishizuka 1995). Adrenomedullin is secreted
by adipose and increases with cytokine signaling, and it increases vasodilation and
hypotension by increasing nitric oxide (Preedy and Hunter 2011). Meanwhile, apelin is expressed by adipose tissue in endothelial cells to increase vasodilation and
hypotension by increasing nitric oxide production through the expression of eNOS
during AMPK activation (Preedy and Hunter 2011). Adiponectin activation of
AMPK also causes eNOS expression (Osuka et al. 2012).
Chronic endothelial activation occurs in diabetic and obese individuals even in
the absence of infection caused by hyperglycemic and fatty acid stress (Tsuriya
et al. 2011). This low level chronic activation is thought to be pathological in nature
as it increases vascular dysfunction and contributes to both micro-vascular and
macro-vascular complications (Feener et al. 2013; Kaneko et al. 2011). Vasodilation
and increased blood flow typically allow the innate immune response to preform
tissue repair starting with immune cell migration, adhesion, and response and leading to cellular proliferation signaling, tissue growth, and remodeling (Pacurari et al.
2014). Constant tissue remodeling leads to stiffness in the extra cellular matrix of
the vascular system and loss of the elasticity of endothelium with increased endothelial and muscle cell layers. The elastin to collagen ratio is reduced. This reduced
elasticity allows damaging innate immune cells to continuously infiltrate tissue and
the vascular bed. Cellular proliferation is mediated through the MAPK/extracellular
signal-regulated kinase (MAPK/Erk) pathway. The MAPK/Erk pathway also promotes expression of the potent vasoconstrictor endothelin (ET-1) to reverse the vascular dilation (Weil et al. 2011).
13
Chapter 9 reviews the innate anti-inflammatory response which is activated in

both diabetes and obesity and increases with vascular dysfunction. Chapters 7, 8, 9,
and 10 review the role of Bikunin (Bik) as a key anti-inflammatory component of
this response. Bik is a serpin that inhibits the trypsin family of serine proteases and
inhibits kallikrein, vascular dilation, smooth muscle contraction, and coagulation.
Additionally, Bik inhibits blood coagulation and serine protease degradation of the
endothelial through action of Factors IXa, Xa, XIa and XIIa on plasmin (Kato 2002;
Pugia et al. 2007). In the acute phase of tissue injury, Bik is protective in function,
stalling immune mediated tissue damage (See Chap. 10). In the chronic phase, Bik
stalls tissue repair and cellular proliferation. A prolonged and chronic antiinflammatory response is thought to impair the vascular system. In long standing
inflammatory reactions to chronic diabetic and cardiovascular disease, Bik will be
elevated above the normal range for healthy individuals but at levels below the acute
response range. Tissue factor pathway inhibitor (TFPI) is another Kunitz-type protease inhibitor that inhibits the initial reactions of blood coagulation and PAR (Kato
2002). Adipose tissue has also been implicated as well as several other serpins.
PAI-1 is a serpin that increases in obesity due to cytokine expression and causes
obesity associated fibrinolysis (Preedy and Hunter 2011; Pugia et al. 2007). Vaspin
is a serpin that is released from adipose and found to inhibit kallkrien (Preedy and
Hunter 2011). The impact of prolonged exposure of serpins on innate immunity
activation is a current active topic in chronic disease pathways.
1.3.2
Immune Mediated Apoptosis
Chronic low-grade inflammation in adipose tissue induces an innate immune cell

response in type 2 diabetes (T2D) and obesity (McNelis and Olefsky 2014). Adipose
tissue retains macrophages that are recruited by the pro-inflammatory profile of
cytokines (IL-1, IL-6, IL-18, TNF, TGF-/), chemokines (MIC-1, MIP-2,
MCP-1, CINC-1), YKL-40, and LCN-2. These pro-inflammatory signals are
released by adipose tissue due to fatty acid and oxidative stress (Espinoza-Jimnez
et al. 2012) (See Fig. 1.2). Lipids retained in the sub-endothelial space are a leading
cause of infiltration of macrophages. Activation of complement C3 and C5 also
participates in macrophage recruitment and polarization (Phieler et al. 2013).
Adipocyte macrophages are recently suggested to increase the ability of metabolic
processing of adipocytes. At the same time macrophages are thought to accelerate
the progression of insulin resistance and the development of diabetes (Johnson and
Milner 2012; Patel et al. 2013). Macrophages also release cytokines and chemokines which further recruit other innate immune cells (Tilg and Moschen 2006).
These innate immune cells not only infiltrate the adipose but also increasingly
infiltrate across the vascular endothelium into liver, muscle, and pancreas tissues as
diabetes progresses. Besides the production of the pro-inflammatory profile, stressed
adipose tissue also releases resistin (Preedy and Hunter 2011). Resistin, also known
as adipose tissue-specific secretory factor (ADSF), upregulates adhesion molecule
14
M. Pugia
Growth factor
& cytokine
stress signaling
IL-1
EGFR
Cytokine &
growth factor
release
Extrinsic death
factor & NF-KB
activation
TNF-
IL-1
IL-18
TGF /
TNFR
FasL
Mitochondria
NF-kB
Caspase 8
Serine
protease
release
Innate
immune
cell
Elastase
Granzyme B
Extra cellular
Cell shrinking
and
membrane
blebbing
Cytoplasm
MKK3/6
CytoC
Nucleus Nuclear
receptors
MAPK
p38
Transcription
of cytokine
production
Caspase 9
Caspase 3,6,7
Apoptosis
Cell cycle
apparatus
Transcription
initiation
complex
DNA pol-
helicase
DNA fragmentation and no repair
Fig. 1.2 Innate Immune Mediated Apoptosis. Innate immune cells such as neutrophils, eosinophiles, monocytes, natural killer cells, and macrophages release serine proteases such as elastase,
proteinase 3, cathespin and granzyme B during inflammation. These serine proteases mediate
apoptosis through the extrinsic death receptor and mediate cytokine stress signaling through
release of growth factors and cytokines (e.g. TNF , IL-1, IL-18, and TGF /). The extrinsic
death receptor signals the caspase apoptosis pathways. Growth factor increases MAPK p38 signaling of cytokine transcription. Granzyme B released from immune cells bypass the anti-apoptosis
effect of nuclear transcription factor (NF-B) by directly activating Caspase 3
(VCAM-1 & ICAM-1) and chemokine (C-C motif) ligand 2 (CCL2), which cause
leukocyte recruitment (Olefsky and Glass 2010). Circulating lipids in general cause
activation of PKC, which increases and signaling for immune cell adhesion through
VCAM-1 & ICAM-1 (Nishizuka 1995). Free fatty acids can be recognized by tolllike receptors (TLRs) activation of c-Jun N-terminal kinases (JNK) and NF-B signaling in various leukocytes and macrophages. Levels of both JNK and NF-B are
increased in diabetic patients (Masoodi et al. 2015; de Jong et al. 2014). Activated
JNK can inhibit PI3K and promote insulin resistance in tissue.
Macrophages and other innate immune cells such as neutrophils, eosinophiles,
monocytes, and natural killer cells have a key function to remove apoptotic and
necrotic cells (Espinoza-Jimnez et al. 2012). Immune cells mediate the death of
damaged cells by causing apoptosis. Innate immune cells and macrophages release
serine proteases such as elastase, proteinase 3, cathespin, and granzyme B as a
means to cause immune mediated apoptosis (Yang 1996; Kondo et al. 2013). The
granules of neutrophiles in particular, contain these serine proteases in high concentrations (Mania-Pramanik et al. 2004; Johnson et al. 1988; Nakatani and Takeshita
1999). Degranulation causes release of serine proteases. Cytotoxic T lymphocyte
15
are natural killer cells that also release granzyme (A, B, H, M), tryptase 2, and mast
cell proteases 1 (Trapani 2001; Kam et al. 2000). Human lymphocytes also contain
cell surface elastase. These serine proteases are typically used to remove pathogens
by destruction of cells. Activation of cell death occurs through cleavage of proteins
in inflamed cells.
Immune mediated apoptosis occurs by the extrinsic death receptor (Yang 1996),
and serine proteases release growth factors and cytokines to activate this apoptosis
(See Fig. 1.2). Cytokines activate the extrinsic death receptor and cause signaling of
the caspase pathway. Growth factors increase MAPK p38 signaling of cytokine
transcription. The serine protease, granzyme B, is released from immune cells and
bypasses the anti-apoptosis effect of NF-B by directly activating caspase 3.
Apoptosis is observed in vivo and in vitro in adipose tissue (Sorisky et al. 2000). Bik
is a key pathway for inhibition of immune mediated apoptosis (Pugia et al. 2007)
(See Chap. 10 for further review). The complement system is another major component of cell death (See Chap. 2 for further review), and complement activation in
hypoxia and ischemia-reperfusion oxidative stress results in cell death and impacts
vascular homeostasis (Collard et al. 1999).
Cell death initiates an adaptive immune cell response (Kono et al. 2014).
Macrophages trigger T-cell infiltration into the adipose (Travers et al. 2015).
T-lymphocytes, both CD4+ and CD8+, are found in the adipose of obese and diabetic patients. The role of T-cell activation in the adipose is poorly understood, but
it is clear that T-Cell with class II major histocompatibility complex (MHCII)
increases in fat stressed adipose (Deng et al. 2013). These T-cells have antigen
processing and presentation abilities. Macrophages and T cells further interact with
each other for activation and differentiation and further trigger the adaptive immune
system. Macrophages can express MHC-II molecules (Espinoza-Jimnez et al.
2012). Free fatty acids further activate the proliferation of T-cells. There is emerging
data supporting different innate immune cell subpopulations and activation in the
adipose (Rao et al. 2014; Olefsky and Glass 2010). Regulatory T cells (Treg cells)
that are crucial for mediating immune homeostasis have been observed in the adipose (Pucino et al. 2014). Fatty acids are reported to enhance CD4(+) T-cell proliferation. However, aside from the extremely rare Weber-Christian panniculitis, there
are no reports of auto-immune disease of the adipose (Allen-Mersh 1976).
1.3.3
Fibrosis
The innate immune system also mediates a repair process under normal conditions.
Serine proteases are released to activate protease activate receptors (PAR) (See
Fig. 1.3), and these proteases are released by tissues under inflammatory stress.
Trypsin, thrombin, and plasmin are formed by proteolytic cleavage to activate
protease-activated receptors (PAR) and lead to the cell signal cascade that affects
cell shape, secretion, integrin activation, inflammatory responses, transcriptional
responses, and increased cell motility (Coelho et al. 2003). PAR signaling causes
16
M. Pugia
Trypsin
thrombin
uPA
uPAR Plasmin
activation
PAR
activation
uPAR
Membrane
synthesis
Extra cellular
Cytoplasm
Increased
protein
expression
AC
PLC
PKA
PKC
PLA2
Increased
hormone
production
H
Nucleus Nuclear
receptors
RAS
MAPK/ERK
Cell proliferation
and
differentiation
Inflammation
mediated cell
proliferation
Cell cycle
apparatus
Transcription
initiation
complex
DNA pol-a
helicase
Replication and transcription
Fig. 1.3 Inflammation mediated cell proliferation. Serine proteases trypsin and thrombin are
released in endothelial and epithelial autocrine response during inflammation and activate protease
activate receptors (PAR). Activation causes signaling by phospholipase C (PLC), adenylcyclase
(AC), and phosphatidylinositol 3-kinase (PLA2); protein kinase C (PKC) leads to activation of
MAPK/Erk. MAPK/Erk increases cell proliferation during healing repair through the cell cycle
apparatus controlled by cyclin-dependent protein kinases. PAR activation also increases cytosolic
phospholipase A2 (intracellular hormone) synthesis of prostaglandins, causing nuclear receptor
activation transcription of many proteins. Cell wall bound serine proteases (plasmin) are expressed
and cleave urokinase-type plasminogen activator (uPA) from its receptor (uPAR). Activation causes
the break-down of basement membranes and extracellular matrix during tissue remodeling
phospholipase C (PLC), adenylcyclase (AC), phosphatidylinositol 3-kinase (PLA2),

protein kinase A (PKA), and PKC to activate MAPK/Erk (Cocks and Moffatt 2000;
Cottrell 2002; Miike et al. 2001), and activation of MAPK/Erk increases cell proliferation. PAR activation also increases cytosolic phospholipase A2 synthesis of prostaglandins, an intracellular hormone, causing nuclear receptor activation
transcription of many proteins. Cell wall bound serine proteases (plasmin) are
expressed and cleave urokinase-type plasminogen activator (uPA) and its receptor
(uPAR). Activation of these receptors causes the breakdown of basement membranes and extracellular matrix during tissue remodeling.
Coagulation is another key component of tissue regeneration during inflammation (Pugia et al. 2007). Inflammation triggers coagulation (Kaplan et al. 1982).
Vascular endothelium and muscle cells release coagulation serine proteases such as
plasmin, thrombin, and Factors VII and X in the activation of the coagulation cascade. Coagulation, fibrinolysis, complement activation, and extra-cellular matrix
assembly occur. Factors VII & X cause cleavage of pro-thrombin to thrombin during coagulation. Thrombin forms insoluble fibrin from fibrinogen and forms a mesh
of fibers (a clot) in which blood cells are trapped. Plasmin indirectly causes platelet
17
aggregation and endothelial cell proliferation. Thrombospondin 1 from platelets

mediates cellular attachment and induces angiogenesis and proliferation in adipose
along with activation of TGF- (Kong et al. 2013).
Abnormal tissue repair occurs in diabetic individuals with obesity and leads to
fibrosis. Fibrosis is common in diabetic complications and is found in liver, adipose,
kidney, heart, nerves, eyes, and vascular system (Ban and Twigg 2008). Fibrosis is
characterized by an excessive build-up of the extracellular matrix (ECM). Typically,
a growth factor excess or deficiency occurs in diabetic fibrosis (See Chap. 11 for
further detail). This imbalance can be induced by innate immune cells. For example, macrophage infiltration into the adipose causes fibrosis before adipocyte metabolic dysfunction and cell death (Vila et al. 2014). The presence of functional
Toll-like receptor 4 on adipose tissue hematopoietic cells is necessary for the initiation of adipose tissue fibrosis. This imbalance can also be the result of TGF- promoting fibrosis (See Chap. 11 for further detail).
Several innate immune cell factors that control regeneration and fibrosis are
explored in this book. In Chap. 10, the action of Bik on cell proliferation is explored,
and a new pathway to reducing cell growth is described. Bik is a key pathway for
protecting the extracellular matrix in arterial walls and connective tissues from
abnormal cell growth (Pugia et al. 2007). Bik was previously known to inhibit PAR
and uPA through its action on trypsin, plasmin, and thrombin. Bik also is known to
inhibit coagulation through Factor VIIa (Pugia et al. 2007). In Chap. 10, the action
of Bik on cell proliferation and cell growth reduction is presented. Bik suppresses
cell growth by activating the Akt-PI3K pathway. In Chap. 6, the Adiponectin
Receptor C terminal fragment (AdipoR CTF) is shown to increase intercellular
insulin and reduce insulin sensitivity. These pathways are expected to open new
doorways into the understanding of the impact the innate immune systems has on
tissue regeneration.
1.4
Progression of Diabetes
It has been proposed that all types of diabetes mellitus are a continuous spectrum of
the same disease (Brooks-Worrell and Palmer 2011). In theory, diabetes starts with
insulin resistance and progresses to Type 2 diabetes as a metabolic disease with
glucose intolerance. It then follows with stronger immune system involvement, and
it ends with Type 1 diabetes as an autoimmune disease. To stop progression at the
start of the spectrum is the goal. The initial relationship between obesity and insulin
resistance is well-documented around the world. Lifestyle changes, such as calorierestricted diets, exercise, and behavior modification that result in weight loss of
510 % are the accepted means for significant improvement in insulin resistance
and diabetes (Gumbs et al. 2005).
At the other end of the spectrum, autoantigen-specific inflammatory CD4+ and
CD8+ T cells follow in Type 1 diabetes with destruction of pancreatic insulinproducing cells. Insulin, glutamic acid decarboxylase, and protein tyrosine phos-
18
M. Pugia
phatase are the most common auto-antigens involved in this process (Winter and
Schatz 2011). When the majority of cells are destroyed, the pancreass ability to
secrete insulin in response to blood glucose levels is lost and results in the disruption of glucose homeostasis (Espinoza-Jimnez et al. 2012). As diabetes progresses,
complications such as cardiovascular disease, nephropathy, retinopathy, and neuropathy increase. All diabetic complications share many common mechanistic
behaviors such as oxidative stress, glycation stress, and innate immune involvement, and obesity adds the additional adipose and free fatty acid factors.
1.4.1
Cardiovascular Diseases
Diabetes increases the risk of myocardial infarction, stroke, premature peripheral

arterial disease, atherosclerosis, and cardiomyopathy (Forbes and Copper 2013).
Dyslipidemia, impaired glycemic control, and persistent elevations in blood pressure are common observation in diabetes. Diabetes is the leading contributor to the
88 million patients in US living with one or more types of cardiovascular disease
and to the $444 billion in health care costs. Obesity additionally increases the risk
of mortality and morbidity of cardiovascular disease (CVD) (Wang and Nakayama
2010).
Inflammation, innate immunity activation, endothelial dysfunction, and complement responses are common mechanisms in all diabetes complication. The endothelium is crucial for maintenance of vascular homeostasis and permeability.
Endothelial dysfunction contributes to both micro-vascular and macro-vascular
complications in diabetes. Innate immune cells can bind to the vessel wall and cause
migration, adhesion and infiltration of innate immune cell into tissue. The innate
immune cell response leads to cellular proliferation growth, tissue remodeling,
fibrosis, apoptosis, and T-cell infiltration. The result is impaired muscle growth and
development with reduced muscle mass, reduced myofibril size, decreased skeletal
muscle, capillarization, and angiogenesis (DSouza et al. 2013).
1.4.2
Nephropathy
Kidney disease is a key focus for diabetic disease management as it represents the
largest vascular system in the body. Diabetes is the lead cause of chronic kidney
disease (CKD) and end stage renal disease (ESRD). It leads to 500,000 cases of
ESRD in the US and $33 billion in cost. Infections and nephrotoxic exposure are
additional causes of CKD and ESRD. The diabetic kidney disease mechanism is
similar to that of diabetic cardiovascular diseases, as it starts with low level
inflammation, progresses through innate immune response, and ends with tissue
damage that leads to CKD. Management of kidney disease requires instituting
kidney protective strategies before getting to the CKD stage. The pathology
19
typically starts with inflammation as glomerulonephritis and ends with wide

spread cell loss, fibrosis, vascular dysfunction with GBM thickening and mesangial expansion, complement formation with renal lesions, and auto-immunity
such as IgA nephropathy (Cochrane et al. 1965; Nakatani et al. 2001; Huang et al.
2001; Hotta et al. 1999).
Neutrophilic polymorphonuclear leukocytes and macrophages are accepted as a
primary cause of renal capillary wall injury in glomerulonephritis, and they accomplish this by mediating the release of proteinases (Johnson et al. 1988; Nakatani and
Takeshita 1999). Innate immune cells and ROS lead to TGF formation, increasing
collagen IV synthesis, and glomerular mesangial cell growth that leads to glomerularsclerosis. Serine proteases are key factors that damage the basement membrane
and lead to proteinuria and enlargement of the network structure of the basement
membrane and charge barrier. Platelet coagulation and red blood cells also increase
permeability of the basement membrane to proteins. This results in impaired kidney
cell growth with reduced kidney mass and size, decreased capillarization, and
angiogenesis.
Given the morbidity and mortality associated with kidney disease, there is a need
to improve patient outcomes. Risk stratification requires better resolution of the
causes of kidney injury and better selection of effective therapies. Typically,
determination of a loss of renal function is based on the prediction of the glumerular
filtration rate (GFR). Current biomarkers used to detect and monitor kidney disease
include urinary measurement of RBC, albumin, protein, -1-microglobulin, -2microglobulin, transferrin and creatinine, and blood assays for creatinine, phosphorus, BUN, albumin, protein, cystatin C, and other predictors of GFR. WBC,
bacteriuria and nitrite in urine are used as additional predictors of infection. Chapter
7 discusses how once infection is ruled out, there is a need for assays to manage the
chronic inflammatory phase before GFR changes and progression to CKD.
1.4.3
Retinopathy
Diabetic retinopathy is the leading cause of vision loss in adults (Forbes and Copper
2013). Proliferative retinopathy is the major vision threatening disorder and is similar in mechanism to microvascular injury in CVD and kidney disease. The inner
retina infrastructure supports a high metabolic demand for functions but is limited
in size as light must travel through the inner retina to reach the photoreceptors. The
retina is vulnerable to oxidative stress, hypoxia, and glycemic stress (Antonetti
2006). Degeneration or occlusion of retinal capillaries occurs in diabetes. Diabetes
retinopathy is also characterized by reduced growth and remodeling, fibrosis, retinal
thickening, and cell death due to apoptosis. Macular edema occurs and is characterized by a retinal thickening due to fluid leakage from the breakdown of the blood
barrier. This causes blurred vision with reduced visual acuity (Forbes 2013). Overall
progression of diabetic complications are often studied by retinopathy as the microvascular changes are easily measured in the eye.
20
1.4.4
M. Pugia
Neuropathy
More than half of all individuals with diabetes eventually develop neuropathy
(Forbes and Copper 2013). Diabetics experience sensitivities to vibrations and thermal thresholds. Again, the mechanism of glycation and oxidative stress leading to
endothelial dysfunction and immune mediated cell damage is used to explain this
pathology. Endothelial dysfunction is observed with basement membrane thickening and diminished ability to oxygenate neurons. This dysfunction leads to hypoxia
and nerve fiber deterioration. Neuronal and glial cell death is also associated with
inflammatory signaling and immune mediated cell migration.
1.5
Conclusion
New discoveries in the innate and adaptive immune systems appear key to the relationship of obesity and diabetes. The understanding of the interactions of inflammation in diabetes and obesity is increasing, and several key observations can be made
based on todays understanding. Multiple inflammatory and immune factors impact
insulin sensitivity and glucose tolerance. The initiation of the inflammatory response
by glycemia, adipose, free fatty acid or oxidative stress routes is supported.
Endothelial dysfunction and innate immunity activation mediate tissue damage and
are consistent factors in the diabetes complications. Taken as a whole, inflammation
will remain an important research topic for new avenues in both the management
and treatments of diabetes.
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Part II
Impact of Complement
on Diabetic Disease
Chapter 2
Complement and Complement Regulatory

Proteins in Diabetes
Jose A. Halperin, Pamela Ghosh, Michael Chorev, and Anand Vaidya
Abstract In this chapter we have summarized the body of evidence that supports a
role for the complement system and complement regulatory proteins in the pathogenesis of diabetic complications, with specific emphasis on the novel phenomenon
of glycation-inactivation of CD59. Progress in this area has been remarkable considering that (1) ethical and practical issues preclude conducting in-depth mechanistic studies in humans, especially for chronic conditions such as the complications of
diabetes that take years to develop and (2) non-clinical research is hampered due to
the absence of animal models of diabetes that fully recapitulates diabetic vascular
disease in the intensity and distribution seen in humans.
Similar to other tightly regulated biological systems, phenotypic endpoints
derived from complement activation depend on the delicate balance between complement activators and inhibitors. In the specific case of complement and its regulators, this delicate balance paradigm is well exemplified by the human disease
Paroxysmal nocturnal hemoglobinuria (PNH), an acquired complement-mediated
hemolytic anemia in which blood cells lack glucosylphosphatidylinositol (GPI)anchored proteins including CD59 (Takeda et al., Cell 73:703711, 1993; Parker
et al., Stem Cells 14:396411, 1996). Under normal conditions, the basal tickover activation of complement is sufficient to induce only mild degree of hemolysis. In contrast, when stressors like infections amplify complement activation,
excessive membrane attack complex (MAC) formation on unprotected red blood
cells and platelets lacking CD59 results in paroxysmal crises of hemolysis, platelet activation and potentially lethal thrombotic episodes (Hill et al., Blood 121:4985
4996, 2013; Hillmen et al., N Engl J Med 355:12331243, 2006; Risitano et al.,
J.A. Halperin, MD (*) P. Ghosh, PhD M. Chorev, PhD

Laboratory for Translational Research, Division of Hematology, Department of Medicine,
Brigham and Womens Hospital, Harvard Medical School, Boston, MA, USA
A. Vaidya, MD, MMSc
Division of Endocrinology, Diabetes and Hypertension, Brigham and Womens Hospital,
Harvard Medical School, Boston, MA, USA
DOI 10.1007/978-3-319-21927-1_2
29
30
J.A. Halperin et al.
Immunobiology 217:10801087, 2012). The emerging clinical evidence summarized in this article indicates that comparable mechanisms could be operative in the
pathogenesis of diabetes complications: on the one hand, biological responses to
chronic hyperglycemia like formation of advanced glycated end-products (AGEs)
and fructosamine adducts in proteins as well as auto-antibodies against oxidized
and/or glycated proteins could activate complement while, on the other hand,
glycation-inactivation of CD59 increases the susceptibility of tissues to complement (MAC) mediated damage.
2.1
Introduction
Diabetes is reaching epidemic proportions worldwide and is projected to affect

almost 10 % of the world population by year 2035. However, an epidemic of diabetes is in fact an epidemic of its complications; diabetes is associated with (1) accelerated macrovascular disease resulting in atherosclerotic coronary heart disease,
stroke and peripheral artery disease, and (2) microvascular diseases that damage the
retina leading to blindness, the kidneys leading to end-stage renal failure, and
peripheral nerves leading to severe forms of neuropathy, which combined with
peripheral artery disease are the leading cause of non-traumatic amputations. The
cost of treating complications of diabetes exceeds 10 % of the total healthcare
expenditure worldwide.
Large-scale prospective studies for both type 1 and type 2 diabetes including
the Diabetes Control and Complications Trial (DCCT) study group (DCCT
reports 1995a, b), the UK Prospective Diabetes Study (UKPDS) (UKPDS reports
1998), and the Steno-2 study (Pedersen and Gaede 2003) established that the
complications of diabetes are caused by prolonged hyperglycemia, and that the
extent of tissue damage in individuals with diabetes is influenced by genetic
determinants of susceptibility and by the presence of accelerating factors such as
hypertension and dyslipidemia. A hypothesis summarizing different mechanisms
that may underlie the pathogenesis of diabetes complications proposes that hyperglycemia-induced overproduction of reactive oxygen species (ROS) fuels an
increased flux of sugars through the polyol pathway, an increased intracellular
formation of advanced glycation end-products (AGEs), an increase in reactive
carbonyl compounds, an increased expression of the receptor for AGEs and signaling from its activating ligands; the activation of protein kinase C (PKC) isoforms, and an overactivity of the hexosamine pathway (Brownlee 2001, Giacco
and Brownlee 2010). However, the actual cellular and molecular mechanisms by
which high levels of glucose cause tissue damage in humans are still not fully
understood.
A body of clinical and experimental evidence reported in the past few decades
supports a link between the complement system, complement regulatory proteins
and the pathogenesis of diabetes complications (Acosta et al. 2000; Rosoklija
et al. 2000; Zhang et al. 2002; Flyvbjerg 2010; Mellbin et al. 2012; Hansen et al.
Complement and Complement Regulatory Proteins in Diabetes
31
2004; Hansen 2005; Hovind et al. 2005; Uesugi et al. 2004; Woroniecka et al.
2011; Qin et al. 2004; Engstrom et al. 2005; Wlazlo et al. 2014; Gehrs et al. 2010;
Gerl et al. 2002). Emerging evidence also indicates that the complement system is
involved in several features of cardiometabolic disease including dysregulation of
adipose tissue metabolism, low-grade focal inflammation, increased expression of
adhesion molecules and pro-inflammatory cytokines in endothelial cells contributing to endothelial dysfunction, and insulin resistance (Hertle et al. 2014). Herein
we review the biology of complement with particular emphasis on the membrane
attack complex (MAC) as a potential effector of pathology seen in target organs of
diabetic complications, and of CD59, an extracellular cell membrane-anchored
inhibitor of MAC formation that is inactivated by non-enzymatic glycation forming glycated CD59 (GCD59), an emerging biomarker for the diagnosis and clinical management of diabetes.
2.2
The Complement System
The complement system is an effector of both adaptive and innate immunity. It is

composed of more than 30 plasma and cell-membrane proteins that are synthesized
by hepatocytes or locally in peripheral tissues and normally circulate as inactive
precursors (pro-proteins) (Morgan and Gasque 1997; Laufer et al. 2001; Andoh
et al. 1998; Passwell et al. 1990). Complement proteins interact with one another in
three enzymatic activation cascades known as the classical, the alternative and the
mannose-binding lectin (MBL) pathways (See Fig. 2.1) (Morgan and Harris 1999;
Walport 2001a, b). The three activation pathways eventually converge at the level of
C3 and C5; thereafter, the three complement pathways share a common reaction
sequence involving the late components C6, C7, C8, and C9, leading to the generation of the membrane attack complex or MAC, the main effector of complementmediated tissue damage.
2.2.1
Membrane Attack Complex (MAC)
The formation and function of MAC is described in Fig. 2.1. The MAC is a transmembrane pore that allows influx of salt and water leading to colloidosmotic lysis of
MAC-targeted cells. The formation of the MAC pore, however, seems to be the end
stage of a process that transits through a reversible phase during which MAC pores
can be formed transiently (Halperin et al. 1993, 1988; Acosta et al. 1996) generating
significant changes in the membrane permeability and internal composition of
MAC-targeted cells without compromising their viability (Halperin et al. 1993,
1989; Berger et al. 1993). Figure 2.1 illustrates the large increase of intracellular
Ca++ levels caused by transient, non-lytic MAC pores on isolated guinea-pig cardiomyocytes (Berger et al. 1993). Thus, while the matured MAC pore can kill non-self
32
cells by perforating their plasma membrane, the transient MAC pore can induce
non-lethal biological responses in self cells, and mediate physiological and/or
pathological response (Nicholson-Weller and Halperin 1993).
2.2.2
MAC Cellular Signaling and Complement Activation
The MAC is a mediator of cellular signaling and an effector of organ pathology.

Activation products of complement such as C3b and C4b or the anaphylotoxins C3a
and C5a exert their biological activities through binding to specific receptors
expressed in many cell types. Upon binding to their cognate ligands, these receptors
trigger signaling processes mostly linked to inflammation and immune responses
(Carroll 1998). In contrast, there are no specific receptors for the MAC, which
Classical pathway
Lectin pathway
Antigen-antibody
Pathogen surface
Alternative pathway
C3 hydrolysis
C3
MBL
MASP1
C1qC1rC1s
H20
MASP2
C4
C2
C3bB
C4a
C2b
Ba
C3
C4b2a
C3bBb
C3 convertase
C3 convertase
CR1 CR2
MCP DAF
C3a
C3bBbC3b*P
C4b2a3b
C5 convertase
C5
C6
C5a
C7
C5b
C8
C9n
C5b
C6
C9
C7
Signaling pathways
Pro-inflammatory Pro-thrombotic mitogenic
Anaphylatoxins
C4a
C3a
C5a
Histamine release
C9
C8
Factor H
Factor I
C5 convertase
CD59
Clusterin, S-protein
MAC
Cell lysis
33
Fig. 2.1 The Complement Activation Pathways and its regulators. The Complement System: The
classical pathway is initiated by immune complexes that activate C1. Activated C1 then recruits C4
and C2 to form a C3-convertase (C4b2a) that fragments C3 into C3a, an anaphylotoxin, and C3b,
a molecule at the core of the complement system that binds covalently to hydroxyl groups on carbohydrates or proteins in bacterial surfaces and/or cell membranes. C3b tags invading microorganisms for opsonization and also amplifies the activation cascade acting as a focal site for further
complement activation. The phylogenetically older alternative pathway is activated by low-grade
cleavage of C3 in plasma; the resulting C3b binds factor B, a protein homologous to C2, to form
the C3bB complex. Bound to C3b, factor B is cleaved by the activator factor D, which circulates
in an active form as a serine esterase to form C3bBb: the C3-convertase of the alternative pathway.
Because basal tick over deposition of C3b occurs in all cells exposed to complement, C3b is
always available to prime the alternative pathway. However, continued activation and amplification is normally restricted because cell membranes do not bind factor B efficiently, and the inhibitory factor H prevents the association of factor B with C3b. Amplification of the alternative
pathway occurs when factor H binds to foreign carbohydrates leaving factor B free to complex
with C3b. Specificity for activation of the alternative pathway resides in the ability of factor H to
discriminate between self and foreign carbohydrate determinants (Walport 2001a, b; Morgan
1995). The MBL pathway is initiated when the plasma mannose-binding lectin (MBL) protein,
structurally similar to C1q (Matsushita et al. 1998), in a complex with the mannose-binding lectinassociated proteases 1 and 2 (MASP1 and MASP2) binds to an array of mannose groups on the
surface of microorganisms. This binding activates MASPs, which then cleave C2 and C4 leading
to the formation of the C3-convertase C4b2a in a manner comparable to activated C1 (Walport
2001a). Terminal complement pathway: The three complement activation pathways eventually
converge at the level of C3 with formation of C3b and a C5 convertase that cleaves C5 into C5a,
an anaphylotoxin, and C5b. This is the last enzymatic step of the activation cascades; thereafter, the
complement pathways share a common sequence through the terminal components C6, C7, C8,
and C9, leading to the generation of the membrane attack complex or MAC, the main effector of
complement-mediated tissue damage. The terminal complement components C6 through
C9 -necessary to form the MAC, are constitutively present in plasma. Formation of the MAC is
initiated by C5b followed by the sequential association of C6 into C5b6, and then C7, C8, and C9.
C5b6 is a stable protein that binds to C7 to form the C5b-7 complex that inserts into the lipid
bilayer of the plasma membrane. Membrane-bound C5b67 exposes a binding site for C8 leading
to formation of the C5b-8 complex, which functions as a docking site for C9. A single C9 first
binds to C8 in the C5b-8 complex; then C9 polymerizes forming the C5b-9 complex known as the
membrane attack complex or MAC. The MAC is a circular polymer of variable number of C9
monomers (Podack 1984; Podack and Tschopp 1984; Podack et al. 1982; Podack and Tschopp
1982; Tschopp et al. 1984) with the capacity to insert into cell membranes (Laine and Esser 1989)
and form a transmembrane pore with hydrophobic domains on the outside and hydrophilic domains
in the inside, and an effective internal radius of 57 nm (Mayer 1984; Ramm et al. 1982, 1985).
Restriction of complement activity by complement regulators: In the fluid phase, the C1 inhibitor
regulates the classical pathway by targeting C1, factors H and I regulate the alternative pathway,
and S-protein, clusterin and serum lipids compete with membrane lipids for reacting with nascent
C5b67. In addition to these fluid phase inhibitors, several membrane proteins, decay-accelerating
factor (DAF) (Nicholson-Weller et al. 1985), membrane cofactor (MCP) (Brooimans et al. 1992;
Cervoni et al. 1992) and complement receptors 1and 2 (CR1, CR2) regulate the C3-convertases
(Beynon et al. 1994) a major amplification step in the early complement activation cascade, while
clusterin (McDonald and Nelsestuen 1997), S-protein (Podack and Muller-Eberhard 1978) and
CD59 inhibit formation of the MAC (Fletcher et al. 1994; Bodian 1997; Davies et al. 1989; Huang
et al. 2006; Husler et al. 1994; Meri et al. 1990; Petranka et al. 1992). Massive or unrestricted
activation of complement and MAC formation leads to cell lysis, as seen in immune hemolytic
anemias or PNH. Basal (tick over) activation with or without reduced restriction in nucleated cells
leads to transient MAC pores formation, influx and efflux of ions and macromolecules, and activation of cellular pathways that contribute to proliferation, inflammation and thrombosis as characteristically seen in target organs of diabetic complications
34
inserts directly into the lipid bilayer of the plasma membrane. Insertion of transient
non-lethal MAC pores into cell membranes induces an array of biological responses
that promote cell proliferation, inflammation and thrombosis, as characteristically
seen in all diseases in which focal complement activation is a characteristic pathological abnormality. These responses are mediated by growth factors and cytokines
including bFGF and interleukin-1 (Benzaquen et al. 1994; Acosta et al. 1996) which
are directly released through the MAC pore, and PDGF (Benzaquen et al. 1994),
monocyte chemotactic protein-1 (MCP-1) (Torzewski et al. 1996) and von
Willebrand factor (Hattori et al. 1989) which are released through canonical secretion pathways in response to intracellular perturbations induced by the MAC (See
Fig. 2.2). Acting auto- or para-crinically on extracellular receptors, these leaked/
secreted molecules promote (a) proliferation of fibroblasts, endothelial and smooth
muscle cells (Benzaquen et al. 1994), (b) inflammation through the expression of
pro-inflammatory adhesion molecules such as P-selectin, VCAM-I and E-selectin
(Marks et al. 1989; Libby 2002), and the attraction of monocytes and macrophages
Fig. 2.2 Non-lytic transient MAC triggers a large increase of intracellular Ca++ concentration
([Ca++]i). The figure shows pseudo-color images of isolated adult rat ventricular muscle cell
loaded with fura-2, a fluorescent calcium-indicator dye. Increasing emission intensities were associated with increasing [Ca++]i, coded in a pseudo-color spectrum from blue (low Ca++) to red (high
Ca++). (a) Reference, digitalized phase-contrast image. Before adding complement factors, the cell
shows low emission intensities (yellow-green) (b) that does not significantly change after addition
of C5b6 plus C7 and C8 (c). After addition of C9, the cell showed a marked emission increase
(de), representing an increase in [Ca++]i that lasted 1 min. After an additional 3 min, [Ca++]i
decreased to near baseline levels (fh) (Figure reproduced from Berger et al. 1993)
35
to the site of focal complement activation (Kostner 2004) and (c) thrombosis through
the expression of pro-thrombotic tissue factor, and the transient assembly of the
prothrombinase complex in both the endothelial cell surface and microvesicles shed
from their plasma membrane (Hamilton et al. 1990; Christiansen et al. 1997; Saadi
et al. 1995). Our group has also shown that the MAC promotes in vitro accumulation of cholesteryl esters into mouse macrophages fostering their conversion into
foam cells (Wu et al. 2009a).
Reversible insertion of transient MAC into cell membranes also activates intracellular signaling pathways (Tegla et al. 2011) including increased (a) Ca++ influx
(Acosta et al. 1996; Morgan and Campbell 1985) and Ca++-activated K+ efflux
(Halperin et al. 1989) (b) production of reactive oxygen species (ROS) (Adler et al.
1986; Bellosillo et al. 2001), (c) activation of Ca++-sensitive and Ca++-insensitive
protein kinase C (PKC) (Cybulsky et al. 1990; Carney et al. 1990), (d) activation of
heterotrimeric G proteins of the Gi/Go subfamily (Niculescu et al. 1994), (e) mitotic
signaling through the small G-protein Ras, that induces Raf-1 translocation triggering activation of the ERK pathway (Niculescu et al. 1997), and (f) activation phosphatidylinositol-3 kinase (PI3-K) (Niculescu and Rus 2001). The MAC also
activates the transcription factor NF-B, which induces the production of IL-6 as
well as IL-8 and MCP-1 (Kilgore et al. 1997; Viedt et al. 2000). In glomerular
mesangial cells, insertion of the MAC results in increased production of ROS (Adler
et al. 1986) and the release and/or up-regulation of (a) bFGF and PDGF (Benzaquen
et al. 1994) which induce mesangial cell proliferation and transcriptional up-regulation of TGF- (Torbohm et al. 1990; Lovett et al. 1987); (b) monocyte chemotactic
protein-1 (MCP-1), which attracts monocytes and macrophages to the mesangium
(Stahl et al. 1993); and (c) transcriptional activation of collagen type IV (Wagner
et al. 1994).
Important in the context of this chapter, many mediators and cellular signaling
pathways activated by the MAC are established players in the tissue damage seen in
the target organs of diabetic complications (Kotajima et al. 2000; Adler et al. 2000)
and are common to or intersect with complement-independent mechanisms
up-regulated by hyperglycemia such as overproduction of ROS, up-regulation of
PKC and NF-B (Brownlee 2001, 2005; Giacco and Brownlee 2010).
2.2.3
Complement Regulation Through CD59
Given the potentially devastating effects of uncontrolled complement activation, it

is not surprising that an array of mechanisms have evolved to protect self cells
from complement attack and MAC formation. On the one hand, the inherent instability of the enzymes in the activation pathways and the extremely short half-life of
the activated complement components coupled to the transient nature of the reaction
sequences are intrinsic properties of the complement system that keep its activation
restricted and limit the effects of complement to the vicinity of the activation site.
On the other, a multitude of complement regulatory molecules exist that restrict
critical steps of the pathways (See Fig. 2.1).
36
In particular, CD59 is a specific inhibitor of MAC formation that restricts C9 polymerization. Ubiquitously expressed in mammalian cells, CD59 is an 1820 kDa glycosylphosphatidylinositol (GPI)-linked glycoprotein (Kooyman et al. 1995; Morgan
1995). CD59 inhibits the binding of C9 to C5b-8 by competing for binding to a nascent
epitope on C8 thereby affecting the association of C9 to the C5b-8 complex (Ninomiya
and Sims 1992). CD59 can not only function as an inhibitor of the formation of large
MACs but can also allow cells to eliminate newly formed MACs by blocking the early,
functional channel formation of MAC complexes (Kimberley et al. 2007).
Several lines of evidence indicate that CD59 is the most critical of the complement regulators in protecting human cells from MAC formation and MAC-induced
phenomena: erythrocytes from a family carrying the Inab blood group phenotype
totally lacked decay-accelerating factor (DAF), an inhibitor of early complement
activation, without any associated hemolytic disease (Lin et al. 1988; Telen and
Green 1989; Reid et al. 1991; Sun et al. 1999); in contrast, individuals carrying
CD59 mutations leading to undetectable levels of CD59 on their cell membranes
exhibited an early onset hemolytic phenotype associated with vascular disease or
chronic relapsing polyneuropathy (Yamashina et al. 1990; Nevo et al. 2013).
The GPI anchor in GPI-anchored proteins is introduced as a post-translational modification mediated by a protein called Pig-A that is required for the synthesis of the
GPI-lipid tail (Luzzatto and Bessler 1996; Miyata et al. 1993, 1994; Takeda et al.
1993). Deficiency of GPI-linked proteins including CD59 in erythrocytes and platelets
is phenotypically expressed as a complement-mediated hemolytic anemia, known as
paroxysmal nocturnal hemoglobinuria (PNH) (Halperin and Nicholson-Weller 1989;
Parker 1996; Risitano 2012). This paradigmatic disease, together with the silencing
CD59 mutations described above, illustrates how the delicate balance between complement activation and restriction can be perturbed by the functional deficiency of
CD59, leading to a potentially harmful pathological response even in the absence of
complement-activating stressors.
2.3
Clinical Evidence for a Role of Complement

in the Pathogenesis of Diabetes Complications
Evidence for a potential role of complement in the pathogenesis of diabetes complications was initially provided by the identification of activated complement proteins
in target organs of diabetes complications biopsied or excised from individuals with
diabetes (Rosoklija et al. 2000; Zhang et al. 2002; Falk et al. 1983a, b, 1987).
2.3.1
Diabetic Nephropathy
Falk et al. used antibodies against activated complement proteins including a neoantigen of the C9 portion the MAC to perform immunoelectron (IE) and immunofluorescent (IF) microscopy on kidney tissue from normal humans and individuals
37
with insulin-dependent diabetes (Falk et al. 1983a, b, 1987). Staining of tissues with
specific antibodies directed against the MAC neoantigen is the most direct method
for determining whether complement activation has occurred to completion because
the neoantigen is exposed only when the MAC is fully assembled and inserted into
a cell membrane. IF microscopy of kidney tissue from individuals with insulindependent diabetes and varying degrees of mesangial expansion and glomerulosclerosis demonstrated a positive association between the degree of tissue damage and
the amount of MAC deposited in the mesangium. IE microscopy of specimens from
individuals with diabetic nephropathy revealed that the anti-MAC neoantigen
monoclonal antibody reacted with linear and circular membranous structures within
the mesangium, tubular basement membranes, blood vessel walls, and the glomerular basement membranes (Falk et al. 1983a, b, 1987). Our own studies confirmed
glomerular MAC deposition (co-localized with GCD59, Fig. 2.4b) in kidney biopsies from individuals with diabetes (Qin et al. 2004). We also reported the presence
of extensive MAC deposition in glomeruli and blood vessels in the biopsy of a
kidney transplanted 2 years earlier and diagnosed as recurrent diabetic nephropathy with no signs of rejection (Qin et al. 2004).
Also, several studies have found that serum concentrations of mannose-binding
lectin (MBL), an indicator of the lectin complement pathway, were significantly
elevated in patients with type 1 diabetes (Bouwman et al. 2005; Hansen et al. 2003)
and even more elevated in patients with vascular complications including diabetic
nephropathy (Hansen et al. 2004, 2010; Saraheimo et al. 2005). Further, the
FinnDiane Study showed that the baseline levels of MBL were positively associated
with the progression and prognosis of diabetic nephropathy in type-1 diabetes
(Hovind et al. 2005; Kaunisto et al. 2009). Transcriptome analyses of human kidneys have shown that the canonical complement-signaling pathway is differentially
up-regulated in both glomeruli and tubules of individuals with diabetic nephropathy
and is associated with increased glomerulosclerosis. Woroniecka et al. showed that
increased expression of C3 mRNA and protein in glomeruli was positively correlated
with diabetic kidney disease (Woroniecka et al. 2011). C3 is a key protein central to
all complement activation pathways.
2.3.2
Diabetic Retinopathy
A combination of biochemical, histological and genetic studies suggest that aberrant function of the complement system plays a key role in the etiology of agerelated macular degeneration, a common degenerative ocular disease (Wlazlo et al.
2014). Two independent studies have documented that complement activation
occurred extensively and to completion in the choriocapillaries of eyes from donors
with diabetic retinopathy, while similar deposits were not found in eyes from donors
without retinopathy. In the study by Gerl et al., intense positive staining for MAC
deposits (neoantigens) and C3d was noted without exception in all 50 eyes with
diabetic retinopathy analyzed but only in one of 26 eye donors without diabetic retinopathy (Zhang et al. 2002; Gerl et al. 2002).
38
2.3.3
Diabetic Neuropathy
Nerve damage through complement activation has been reported in several peripheral neuropathies (Kaida and Kusunoki 2010). Interestingly, a recent study showed
that a cell surface deficiency of the complement regulatory protein CD59 due to a
single missense mutation of the CD59 gene was clinically expressed by a form of
chronic peripheral neuropathy in children (Nevo et al. 2013). The presence of activated complement proteins and MAC neoantigen in sural nerve biopsies from individuals with diabetes was first reported by Hays and collaborators (Rosoklija et al.
2000); in the same nerve biopsies analyzed in that study, we observed co-localization
of MAC and GCD59 (Qin et al. 2004).
2.3.4
Diabetic Cardiovascular Diseases
A relatively large body of evidence supports a role of complement in cardiovascular

pathology in general and atherosclerosis in particular (Hertle et al. 2014; Kostner
2004; Wu et al. 2009a, b; Haskard et al. 2008; Niculescu and Rus 1999; Lappegard
et al. 2014; Niculescu et al. 1987; Pang et al. 1979; Schmiedt et al. 1998; Seifert
et al. 1989; Seifert and Kazatchkine 1988). Diabetes and pre-diabetes are major
independent risk factor for cardiovascular disease (Tominaga et al. 1999; Jarrett
et al. 1982). A potential role of C3, its activation products and other components of
the early complement cascade in the pathogenesis of type 2 diabetes has been suggested from associations found in several cross-sectional and longitudinal human
studies, as recently reviewed in (Hertle et al. 2014). Also, cleavage of C3 generates
the anaphylatoxins, C3a and C5a, and further cleavage of C3a by carboxypeptidases
leads to production of acylation stimulating protein (ASP/C3adesArg), a lipogenic
hormone that plays a role in atherosclerosis (Onat et al. 2011; Hertle et al. 2012;
Cianflone et al. 2003). However, the potential role of complement activation in diabetic cardiovascular disease has not been fully explored in human studies. In a subanalysis of the DIGAMI-2 trial, which prospectively studied more than 1,200
individuals with type 2 diabetes who had a myocardial infarction, high blood levels
of soluble MAC at the time of hospitalization strongly and independently predicted
the risk of a cardiovascular event in the subsequent 3.5 years (Mellbin et al. 2012).
In the same sub-analysis, levels of mannan-binding lectin serine peptidase 2
(MASP-2) appeared to be associated with a poor cardiovascular prognosis, although
this did not remain significant after multivariable adjustments.
Together, the findings summarized above suggest an important role of the complement system in the development and progression of diabetic complications.
However, it is important to note that whether the reported associations of elements
of the complement system with diabetes represent causative pathogenic precursors
or are just a collateral consequence of diabetes and its complications is still unclear
and deserves further investigation. Complimentary to the clinical evidence discussed
39
above, in vitro and animal studies seem to suggest that the lectin MBL pathway
could be activated by increased levels of MBL induced by hyperglycemia and by a
cooperative binding of MBL to fructosyllysine, the product of the non-enzymatic
attachment of glucose to - or -amino groups in lysines (Fortpied et al. 2010;
Ostergaard et al. 2013).
2.4
2.4.1
Glycated CD59
Functional Inactivation of CD59 by Glycation
Glycation-inactivation of CD59 is a molecular link between complement regulation

and the complications of diabetes. There are different ways in which the delicate
balance between complement activation and restriction can be unfavorably perturbed in diabetes. On the one hand, autoantibodies to glycated and glycol-oxidized
proteins can activate the classical pathway (Uesugi et al. 2004; Ostergaard et al.
2013; Orchard et al. 1999; Witztum et al. 1983), fructosamines, reportedly activate
the lectin pathway by an increased ligand-receptor binding (Fortpied et al. 2010)
and increase formation of advanced glycation end products (AGEs) on the arterial
walls can serve as neo-epitopes for MBL binding (Flyvbjerg 2010). The presence of
activated products of C3 (C3d and iC3b), C2, and C4 in target organs of diabetic
complications (Gerl et al. 2002; Rooijakkers et al. 2009) is consistent with activation of complement through either or both of these two pathways. On the other
hand, functional inhibition by glycation or reduced expression of complement regulatory proteins such as CD59 (Acosta et al. 2000; Zhang et al. 2002) could lead to
increased complement-mediated damage and MAC-induced biological processes
likely to contribute to the pathogenesis of diabetic complications (Rooijakkers et al.
2009; Zhang et al. 2002; Falk et al. 1983a, b, 1987; Weiss et al. 1990).
Of all the above-mentioned putative mechanisms that potentially contribute to
complement-mediated tissue damage in diabetes, glycation-inactivation of CD59 is
the most extensively studied and documented. Hereafter, we will summarize the
experimental and clinical evidence for a role of GCD59 in the pathophysiology of
diabetes complications and the potential utility of glycated CD59 as a diagnostic
biomarker in diabetes.
Protein glycation on - or -amino groups is a well-established mechanism of
tissue damage in diabetes. Despite the many amino groups exposed on the surface
of either soluble or membrane proteins, very few -amino groups are glycated to a
significant extent. For example, despite the very close sequential proximity of lysyl
residues K61, K65, and K66 in -hemoglobin, only the -amino group of K66 is
preferentially glycated in vivo (Shapiro et al. 1980). This is because significant
-amino glycation requires effective acid-base catalysis, which occurs at glycation
motives such as histidyl/lysyl residues in which the imidazolyl moiety is located at
5A from the -amino group of the lysyl residue or at lysyl-lysyl pairs (Shapiro
40
et al. 1980; Iberg and Fluckiger 1986; Shilton and Walton 1991; Shilton et al. 1993;
Calvo et al. 1993; Flckiger and Strang 1995).
Analyzing the reported NMR structure of human CD59 (Fletcher et al. 1994),
our group predicted that the protein contains within its active site a glycation-motif
formed by amino acid residues K41 and H44 and that glycation could inhibit the
activity of CD59 (See Fig. 2.3a). This idea was further supported by the fact that
K41 is adjacent to W40, a conserved residue that is essential for CD59 function
(Bodian 1997; Yu et al. 1997). Exposing purified or recombinant human CD59 to
glucose, we demonstrated that the anti-MAC activity of CD59 is indeed inhibited by
glycation, as documented with anti-glycated CD59 antibodies (See Fig. 2.3) (Acosta
et al. 2000). Site-directed mutagenesis of human CD59 confirmed experimentally
that (1) human CD59 is inhibited by glycation of its K41 residue and (2) H44 is
required for glycation-inactivation of human CD59 (Acosta et al. 2000) (See
Fig. 2.3b).
a
b
Mutant H44 Q
CD59 activity (%)
100
Mutant K41Q
Glycated
CD59 Ab
50
rWT CD59
Glucose (0.5 M)
25
50
75
Days
100
Fig. 2.3 Glycation of CD59 (a): NMR structure of human CD59 (From Fletcher et al 1994); the
figure highlights the Lys41 (red) and His44 (green) amino acid residues that conform the glycation
motif and the Trp40 (blue) residue, a conserved amino-acid critical for CD59 activity. The His44
of human CD59 is not found in CD59 from any other species sequenced to date. (b) Glycation
inactivates human CD59: the activity of recombinant human CD59 was measured in a hemolytic
assay using guinea-pig erythrocytes (GPE) exposed to human MAC formed with purified terminal
complement components (as reported in Acosta et al. 2000). Incubation of CD59 in the presence
of glucose progressively inhibits CD59 activity (red line). Inhibition of activity is associated with
glycation of CD59, because (a) parallel incubation of the same preparation with the non-glycating
sugar sorbitol did not affect CD59 activity (see Fig. 2 in Acosta et al. 2000). An antibody specific
for the glycated form of CD59 showed the presence of glycated CD59 in the protein inactivated
after incubation with glucose (inset). Site directed mutagenesis of either K41 or H44 generated two
CD59 mutants (mutant K41Q and mutant H44Q) that were active against human MAC but not
inhibited by exposure to glucose (Figure reproduced from Acosta et al. (2000))
2.4.2
41
Animal Studies
The significance of the glycation motif in human CD59 is highlighted by the fact
that the residue H44 is absent in CD59 from any animal species sequenced to date
(See Table 1 in Acosta et al. 2000). This is remarkable because humans are particularly prone to develop diabetic vascular complications, and it is well known that no
single animal model of the disease can recapitulate the extensive vascular disease
that commonly complicates human diabetes (Vischer 1999). This observation has
led to the intriguing hypothesis that the presence of a glycation motif K41-H44 in
human CD59 (not present in CD59 from other species) could contribute to the
unique sensitivity of humans to develop extensive vascular disease in response to
hyperglycemia. To test whether the functional inactivation of CD59 confers higher
risk for diabetes-induced atherosclerosis, our group has generated molecular engineered mice that are completely deficient in CD59 (mouse CD59 knockout
(mCD59KO) mice)), crossed them into the ApoE/ background (one of the models
recommended by the Diabetes Complications Consortium for studying diabetesinduced atherosclerosis (Hsueh et al. 2007)) and rendered them diabetic by injection
of low doses of streptozotocin (an experimental model of type 1 diabetes also recommended by the Diabetes Complications Consortium (Brosius et al. 2009)). The
ablation of CD59 (mCD59 knock out mice in an ApoE/ background) promoted
accelerated atherosclerosis with occlusive coronary disease, vulnerable plaque, and
premature death (Wu et al. 2009a; An et al. 2009). In contrast, either transgenic
overexpression of human CD59 in the endothelium or the administration of a neutralizing anti-C5 monoclonal antibody in these mice significantly attenuated the
development of atherosclerosis (Morgan 1995). These observations in engineered
mice are consistent with a body of experimental evidence supporting a role of complement in the development of atherosclerosis (Haskard et al. 2008; Niculescu and
Rus 1999; Pang et al. 1979; Schmiedt et al. 1998; Buono et al. 2002; An et al. 2009).
Diabetic mice: Initial evidence demonstrated that diabetic mCd59KO/Apoe/ mice
developed a severe and accelerated form of atherosclerosis characterized by significantly larger atherosclerotic lesion areas in the aorta (>2-fold increased lesion area
in en-face aorta preparations) and the aortic roots, as compared to their non-diabetic
littermates. This accelerated atherosclerosis observed in diabetic CD59 deficient
mice was associated with significantly increased MAC deposition (detected with
anti-mouse C9 antibodies) and occurred at levels of hyperglycemia, lipid profiles
and body weights similar to those in diabetic CD59 expressing mice (Liu et al.
2014). Interestingly, diabetic mCD59KO mice had significantly increased MAC
deposition and higher levels of serum iC3b, a byproduct of C3 cleavage during activation of complement, than non-diabetic mCD59KO mice. These two observations
are consistent with the emerging evidence for activation of the complement system
in diabetes (Flyvbjerg 2010; Uesugi et al. 2004; Fortpied et al. 2010; Orchard et al.
1999), as discussed above.
42
2.4.3
Functional Studies of Diabetics
The identification of the cellular and molecular mechanisms by which hyperglycemia causes tissue damage in humans has been hampered by (1) the lack of animal
models that recapitulate the combination and intensity of complications seen in
human diabetes (Vischer 1999), (2) the chronic nature of the complications that can
take decades to become clinically evident, and (3) ethical and practical considerations that preclude conduction of the functional studies required to assess mechanistic hypotheses in humans. In spite of these limitations, two lines of human
experimental evidence seem to support the potential role of glycation-inactivation
of CD59 in the pathogenesis of diabetes complications.
Functional inactivation of CD59 was found in individuals with diabetes. If glycation inactivates CD59 in vivo, one would predict an increased sensitivity to MACmediated phenomena in cells/tissues carrying glycation-inactivated CD59. This
prediction was confirmed in an ex-vivo study using red blood cells (RBC) from
donors with or without diabetes in which we measured both the activity of CD59 in
the RBC membranes and the RBC sensitivity to human MAC. The results showed
that RBC from individuals with diabetes (selected to carry a HbA1c >8 %) exhibited a significantly reduced activity of CD59 and were significantly more sensitive
to MAC-mediated lysis than RBC from individuals without diabetes (HbA1c
<5.5 %) (See Fig. 2.4) (Qin et al. 2004). This reduced activity of CD59 in RBC from
individuals with diabetes is consistent with inactivation of CD59 by glycation and
would explain some reversible hematological abnormalities seen in individuals with
poorly controlled diabetes including increased reticulocyte count and shorter RBC
lifespan (Peterson et al. 1977; Bern et al. 1985; Giugliano 1982), both suggestive of
mild hemolytic anemia. Also, individuals with diabetes experience a high incidence
of thrombotic episodes, their platelets are hyperactive and their plasma contains
high levels of platelet-derived microparticles (PMPs) (Carr 2001). Thrombosis is
also a major cause of morbidity and mortality in individuals with PNH (Halperin
and Nicholson-Weller 1989; Gardner et al. 1958; Hill et al. 2013) whose CD59deficient platelets are exquisitely sensitive to MAC-induced activation and release
of PMP (Wiedmer et al. 1993); platelets from CD59 knockout mice are also activated spontaneously and release PMPs (Qin et al. 2003, 2009a, b). These combined
clinical and experimental observations indicate that inactivation of CD59 by glycation could be one factor contributing to the high incidence of thrombosis in individuals with diabetes.
Diabetic patients exhibited glycated CD59 in organs with complications. An
antibody that specifically recognizes the glycated from of CD59 (GCD59) was able
to identify GCD59 colocalized with MAC in target organs of diabetic complications. The presence of GCD59 was confirmed in renal and sural nerve biopsies from
individuals with diabetes. No detectable amounts of GCD59 were found in biopsies
from individuals without diabetes (See Fig. 2.4b) (Qin et al. 2004). As expected
from the functional inactivation of CD59 by glycation, serial sections of the same

CD59 activity (human RBC)
RBC lysis (human MAC)
(n = 10)
(n = 18)
40
100
50
(n = 18)
Lysis (%)
% CD59 Activity
43
30
20
10
ND
<5.5
>8
HbA1c (%)
GCD59
Kidneys
(n = 10)
ND
<5.5
>8
HbA1c (%)
MAC
Diabetes
No
diabetes
Fig. 2.4 MAC-mediated lysis (a) Reduced activity of CD59 and increased sensitivity to MACmediated lysis in RBC from individuals with diabetes. Red blood cells (RBCs) from individuals
with diabetes (selected by HbA1c >8 %) have an 810 times lower CD59 activity than RBCs from
individuals without diabetes (left panel), and are much more sensitive to cell lysis induced by addition of purified terminal complement components to form the MAC (right panel). (b) Co-localization
of MAC and GCD59 in renal biopsies from individuals with diabetes. GCD59 co-localized with
MAC in diabetic human kidneys. Serial sections of the same glomerulus in a kidney from an individual with and another without diabetes stained with anti-MAC or antiGCD59 antibodies.
Arrows indicate positive staining areas for glycated CD59 and MAC (For details on results depicted
in this figure see Qin et al. (2004))
renal and nerve biopsies showed co-localization of GCD59 with MAC deposits
(Qin et al. 2004). Increased levels GCD59 and MAC have also been observed in
other tissues extracted from individuals with diabetes, namely skin biopsies and
saphenous veins removed for grafting of occluded arteries (unpublished observation
(Halperin)).
44
2.5
Complement-Targeted Therapeutics
Progressively recognized as a major mediator of a variety of clinical disorders that

include both acute and chronic diseases and affect a wide range of organs, modulation of the complement system with either small molecules or biologics is now
considered a promising strategy in drug discovery (Ricklin and Lambris 2007,
2013). Fueled by (a) a wealth of high resolution protein structures (Forneris et al.
2010; Janssen et al. 2005, 2006; Torreira et al. 2009; Wiesmann et al. 2006; Wu
et al. 2009b), functional assays (Rooijakkers et al. 2009; Garlatti et al. 2010;
Laursen et al. 2010; Shaw et al. 2010; Gingras et al. 2011; Kajander et al. 2011;
Morgan et al. 2011, van den van den Elsen and Isenman 2011; Hadders and Gros
2012) and genome-wide association studies (Degn et al. 2011), which combined
offer new insight into the molecular details and ligand interaction sites of complement components; (b) the approval and therapeutic success of the anti-C5 antibody,
eculizumab, (Alexion, Cheshire, CT, USA) for the treatment of PNH (Hillmen et al.
2006, 2007; Rother et al. 2007) and more recently of atypical hemolyticuremic
syndrome (Legendre et al. 2013); and (3) the evidence that even small changes in
the activity of individual complement proteins may add up to significantly affect
disease progression, the field of complement-targeted drug discovery has seen a
surge in approaches to therapeutically intervene at various levels of the complement
cascades. Potential therapeutic approaches for interventions include inhibition of
C3 activation, inhibition of C3 convertase assembly, acceleration of C3 convertase
decay, promotion of C3B proteolysis, inhibition of C3 and/or C5 convertase activities, inhibition of membrane attack complex assembly by CD59, and reestablishment of normal alternative complement pathway control with protective Factor H
protein. Compounds directed against some of these targets that are currently in
early clinical trials for other indications include: compstatin/POT-4, a peptide
inhibitor that inhibits the central step of the complement cascade by preventing
cleavage of C3and ARC1905, a pegylated, aptamer-based C5 inhibitor that inhibits
the cleavage of C5 into C5a and C5b. Other complement pathway-modulating compounds currently being considered for and/or are under preclinical development
include a humanized anti-factor D antibody (TNX-234), a Fab fragment of an anticomplement factor B (CFB) antibody (TA106), a complement receptor 2 (CR2)factor H hybrid proteins, a small molecule C5aR peptidomimetic (JPE-1375), and
anti-properdin antibody, an inhibitor of C1 esterase inhibitor (C1-INH) and consequently the classical pathway, and a soluble form of complement receptor 1 (sCR1).
Based on the extensive clinical and experimental evidence supporting a role of
complement in the pathogenesis of diabetes complications, it is conceivable that in
the future, complement inhibition could offer an early point of intervention that
could potentially retard, prevent, or even reverse progression of those complications. However, given the pleomorphic biological activities of the complement system, risk-benefit analyses of therapeutic application targeting complement to
prevent or treat complications of diabetes will have to evaluate cautiously the potential systemic effects of chronic complement pathway modulation.
2.6
2.6.1
45
Glycated CD59 as a Biomarker for Human Diabetes

Available Biomarkers to Assess Glucose Physiology
The diagnosis of diabetes, appropriate medical management of diabetes, and risk

stratification of diabetic complications all require reliable biomarkers that reflect
integrated glucose handling. The most intuitive potential biomarker is circulating
glucose itself; however, circulating glucose concentrations vary significantly
based on the temporal relation to meals, physical activity, and stress. Therefore,
standard practices include the measurement of a morning blood glucose while
fasting, as well as more provocative physiologic maneuvers including measurement of glucose following a glucose challenge (such as the 75 g oral glucose tolerance test). A fasting morning blood glucose measurement provides
cross-sectional circulating glucose concentrations independent of a prandial challenge, while the oral glucose tolerance test provides insight into the cross-sectional ability to handle a prandial glucose load. While both of these biomarkers of
glucose handling are valuable, neither assesses long-term or integrated glucose
handling over time.
The hemoglobin A1c (HbA1c) assay overcomes this limitation as it reflects the
approximate average circulating glucose levels over a three month period (Nathan
et al. 2008). In this regard, HbA1c has become one of the most utilized tests to survey for chronic hyperglycemia, in particular to monitor patients with diabetes on
medical therapy, and is increasingly used as a crude screen to diagnose diabetes
(American Diabetes Association 2015). Similarly, other less utilized biomarkers of
chronic glucose handling include fructosamine, glycated albumin, and
1,5-anhydroglucitol. While these assays reflect long-term circulating glucose concentrations, none of them are involved in the pathogenesis of diabetes complications
related to hyperglycemia; rather these biomarkers represent pathologically unrelated bystanders that are collaterally glycated.
In this context, glycated CD59 (GCD59) represents a novel biomarker for human
glucose handling. The measure of GCD59 reflects glucose levels that are present in
extracellular fluids and result in glycation and thereby inactivation of CD59. In
addition, since glycation-inactivation of CD59 results in a dysinhibition of MAC
formation and complement mediated tissue damage, GCD59 levels may also reflect
a measure of hyperglycemia-induced complement-mediated tissue injury (or the
risk for developing diabetic complications).
2.6.2
GCD59 Levels in Diabetes
The assessment of GCD59 as a practical marker of human blood glucose and potential predictor of risk for diabetic complications was not possible until the recent
development of a highly sensitive and specific ELISA to measure serum and plasma
46
human GCD59 (Ghosh et al. 2013). Though CD59 is a cell membrane-bound protein, a soluble form of CD59 that is shed from cell membranes by phospholipases is
present in human blood, urine, saliva and other bodily fluids (Hakulinen and Meri
1995; Lehto et al. 1995; Meri et al. 1996). Ghosh and colleagues reported the optimization of a novel sensitive ELISA for measuring circulating blood GCD59 in
individuals with and without diabetes: GCD59 was significantly higher in nearly all
participants with diabetes when compared to those without diabetes and demonstrated a high sensitivity for the diagnosis of diabetes (Receiver operating characteristics area under the curve = 0.88). The development and availability of this assay
enabled a number of subsequent human studies that highlighted the value of GCD59
measurements to diagnose and monitor human diabetes.
2.6.3
GCD59 as a Predictor of Human Glucose Handling
In a series of human studies, Ghosh et al. evaluated how well GCD59 correlated
with HbA1c and measures of glucose handling following provocative interventions
(Ghosh et al. 2014). In their first study, they showed that in a cohort of 400 individuals with (196) or without (204) type 2 diabetes, the mean circulating GCD59 was
nearly fourfold higher among those with than among those without diabetes (See
Fig. 2.5). Further, a strong and positive association between GCD59 and HbA1c
was seen not only among individuals with diabetes, where mean HbA1c levels were
8.4 % (r = 0.58, P < 0.0001), but also among individuals without diabetes where
mean HbA1c levels were only 5.6 % (r = 0.45, P < 0.0001). Overall, the authors
observed that across the entire spectrum of the population, a positive linear relationship between GCD59 and HbA1c existed (r = 0.67, P < 0.0001) and was independent
of age, gender, and diabetes diagnosis.
1.2
n = 196
Fig. 2.5 Blood levels of GCD59 classify

individuals with and without diabetes.
Cross sectional evaluation of GCD59
levels in individuals without (HbA1c
<6.5 %) vs individuals with (HbA1c
>6.5 %) diabetes. Bars represent
means SEM. Students t test
p value = 3.7 1023
GCD59 (SPU)
1
0.8
0.6
0.4
p < 0.0000001
n = 204
0.2
0
HbA1c 6.5 %
HbA1c 6.5 %
47
In a separate study, Ghosh et al. assessed how single fasting plasma GCD59
measurements predicted the glucose response to a 2 h 75 g oral glucose tolerance
test (OGTT) (Ghosh et al. 2014). They focused on a medication-naive population of
participants who carried no prior diagnosis of diabetes (n = 109) but were all deemed
to have a high-risk for developing diabetes based on family history of diabetes,
personal history of obesity or gestational diabetes or at-risk ethnicity (Hispanic,
African-American, or Native-American). They observed a clearly positive and linear relationship between GCD59 and the 2 h glucose response to a 75 g OGTT;
more importantly, this relationship was independent of other predictors of glucose
handling such as age, gender, race, body-mass index, fasting plasma glucose, blood
pressure, HbA1c, and lipoprotein concentrations ( = 19.8, adjusted P = 0.03).
In the context of the aforementioned clinical studies suggesting that higher
circulating human GCD59 concentrations strongly predict higher HbA1c and 2 h
glucose responses to an OGTT, Ghosh and colleagues hypothesized that interventions to lower circulating glucose would result in parallel reductions in GCD59.
In an intervention study whereby poorly controlled patients with diabetes were
enrolled in a highly monitored program to initiate and titrate subcutaneous insulin
therapy to normalize hyperglycemia, the investigators measured serial GCD59
levels. In total, 21 patients with diabetes (8 with type 2 and 13 with type 1) with a
mean HbA1c approximating 10 % were initiated on insulin therapy and followed
for 8 weeks. Patients were instructed to measure their blood glucose by finger
stick four times a day for 7 days a week (28 readings per week), and were visited
by a trained nurse every week to titrate insulin doses based on the weekly glucose
log aiming for pre-prandial blood glucose measurements of 80120 mg/dL. Blood
samples were collected while fasting every week and GCD59 and HbA1c were
measured from these weekly blood samples. Within the first two weeks of aggressive insulin therapy, the average weekly glucose (average of 28 weekly home
measurements) declined as expected from 176.4 mg/dL to 149.4 mg/dL and
remained at approximately 150 mg/dL for the remaining 6 weeks of the study.
Strikingly, along with the rapid reduction in average weekly glucose, a parallel
reduction in GCD59 was observed from 0.94 SPU to 0.73 SPU that also remained
stable similarly to glucose for the remaining 6 weeks of the study. Not unexpectedly, the HbA1c measurements did not significantly change during these first two
weeks of insulin therapy, however, did decline to approximately 8 % by the end of
the 8 week study period (Ghosh et al. 2014).
Together, these initial human studies provide tantalizing preliminary evidence
that supports a number of features of plasma GCD59 levels: (1) single plasma
GCD59 levels accurately and consistently reflect mean circulating plasma glucose
levels, (2) single plasma GCD59 levels may be a sensitive biomarker for the diagnosis of diabetes, and (3) plasma GCD59 levels may represent a potential biomarker for the chronic management of diabetes patients on oral or injectable
therapies.
48
Other complement proteins that have been reportedly used to study the pathways
of complement activation and could be potentially explored as diabetes biomarkers
include: C1q, C3, C4d (classical and lectin pathways), factors D and Bb (alternative
pathway), 3a and C5a (general complement activation), soluble MAC complexes
(terminal complement activation), and soluble forms of complement regulatory proteins DAF, MCP and CR1 (Hillmen and Richards 2000).
2.7
Conclusions
Based on the evidence summarized above, it is tempting to propose a pathogenic

model of human diabetic complications in which the delicate balance between complement activation and restriction is broken by a combination of glycationinactivation of CD59 and complement activation by autoantibodies, increased
activity of the MBL pathway and possibly other still unidentified mechanisms. This
results in an increased MAC deposition as observed in target organs of diabetes
complications. Increased MAC deposition activates pathways of intracellular signaling, including increased production of ROS, activation of PKC and up-regulation
of NF-B, and induces the release of pro-inflammatory, prothrombotic cytokines,
and growth factors. These complement-dependent mechanisms together, perhaps
synergistically, with the multiple complement-independent mechanisms summarized by M. Brownlee, promote inflammation, proliferation and thrombosis as characteristically seen in the target organs of diabetes complications (Brownlee 2001;
Giacco and Brownlee 2010; Brownlee 2005). Figure 2.6 summarizes these
complement-dependent and complement-independent mechanisms highlighting the
points of convergence common to both mechanisms such as increased ROS, and
activation of PKC and transcription factor NF-B.
Future research in the area should comprise studies to elucidate further (1) the relative roles of these complement related mechanisms in the pathogenesis of the different
diabetes complications, (2) whether differences in complement activity or in expression of complement regulatory proteins such as CD59 could explain the increased risk
of diabetes complications among ethnic minorities, (Lanting et al. 2005) and (3) how
complement-induced biological phenomena and other hyperglycemia-induced cellular responses interplay in the pathogenesis of diabetes complications. Since the complement-related mechanisms described in this review are driven by hyperglycemia,
its conceivable that they will operate similarly in type-1 and type-2 diabetes, a notion
to be tested in future human studies. Such research will aid in the development of
novel strategies for early stratification of individuals with diabetes who are at higher
risk of complications, improve prevention of complications and develop new complement-targeted therapeutic approaches for the complications of diabetes that represent
a major cause of morbidity and mortality in adults.
49
Fig. 2.6 A model of the cellular and molecular mechanisms. A model of the cellular and molecular mechanisms leading to cell proliferation, inflammation and thrombosis characteristically seen
in the target organs of diabetic complications is shown. High levels of glucose in diabetes glycates
and thereby inactivates CD59 and also increases complement activation. Combined, these effects
of hyperglycemia on the complement system trigger more MAC deposition on cell membranes.
The figure illustrates the different cellular signaling pathways induced by high glucose: the complement/MAC-dependent mechanisms summarized in this review are shown on the right side of
the figure, and the complement-independent mechanisms reviewed in (Brownlee 2001, 2005) are
shown on the left side of the figure. The center of the figure highlights the common signaling pathways reportedly triggered by either high glucose or the MAC. The inset illustrates how the delicate
balance between complement activators and inhibitors can be broken by either increased activation
or decreased restriction of the complement pathways. The evidence summarized in this review
indicates that both mechanisms are operative in the pathogenesis of diabetes complications
50
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Part III
Role of C Terminal Fragment

of Adiponectin Receptor in Inhibition
of Insulin Degradation
Chapter 3
Development of Adiponectin Receptor

C-Terminal Fragment Bioassays
Abstract The presence of adiponectin receptor C terminal fragment (AdipoR

CTF) in human blood was discovered using immune-affinity mass spectroscopy
techniques (Pugia et al., Clin Proteomics 5:156162, 2009). Deficiency in diabetic
patients was observed and followed up by enzyme linked immunoabsorbent assays
(ELISA) with generation of a set of antibodies for AdipoR1 and R2 CTF. Additional
western blot analysis identified the majority of AdipoR CTF in human, rat and rabbit blood was covalently bound to immunoglobulins (Ig-CTF) as the primary native
form. Bound CTF in patient plasma was found at 954900 ng/mL and to be <0.2 %
of all plasma IgG. Free CTF was found at 0.055.0 ng/mL. CTF was shown to be
covalently bound to immunoglobulin and only released upon basic conditions and
not simply by breaking the disulfide bonds. Mass spectroscopy peptide enrichment
assays utilizing trypsin digests were unable to digest bound CTF and basic treatment was required to liberate free form. Antibodies for AdipoR1 and R2 CTF were
paired in sandwich ELISA suitable for measurement Ig-CTF bound forms for clinical patient testing and free CTF forms for animal model testing. Analytical validation demonstrated <10 % between sample and <10 % within patient reproducibility.
Calibration, precision, and stability were acceptable. No cross reactivity or interference was observed. Simple sample preparation was demonstrated as a benefit over
mass spectroscopic peptide enrichment assays analysis.
M. Pugia, PhD (*)

3400 Middlebury Street, Elkhart IN 46515, USA
R. Ma
Siemens Healthcare, 278 Zhouzhu Road, Pu Dong New Area 201318,
Peoples Republic of China
DOI 10.1007/978-3-319-21927-1_3
61
62
3.1
3.1.1
M. Pugia and R. Ma
Introduction
Adiponectin Response
Adiponectin (Adpn) is secreted by adipose and regulates energy metabolism and

inflammation (Scherer et al. 1995; Maeda et al. 1996; Spiegelman et al. 1996;
Nakano et al. 1996; Gil-Campos et al. 2004) with its insulin-sensitizing (Kadowaki
et al. 2006; Ouchi and Walsh 2007), anti-inflammatory (Szmitko et al. 2007) and
cardioprotective (Kubota et al. 2002) properties. Reduction of Adpn in obese mice
and humans correlates with diabetes, cardiovascular disease and insulin resistance
(Kadowaki et al. 2003). Adpn is very abundant in the bloodstream with a sequence
homology to complement C1q without the Fc binding site. It is found in three major
oligomeric isoforms, with the high molecular weight (HMW) form most often cited
as clinically relevant (Shapiro and Scherer 1998; Menzaghi et al. 2007). Surprisingly,
X-ray crystallography analysis of adiponectins globular head showed similarity to
tumor necrosis factor- (TNF-) structure without significant amino acid sequence
homology (Maeda et al. 2002). These similarities suggest the possible regulation
mechanism of Adpn in the process of inflammation and complement.
In a healthy patient, plasma Adpn increases with weight as adipose tissue
increases and in response to insulin and fatty acids (See Fig. 3.1). Adpn is released
into the plasma and signals metabolism in the muscle and liver. Adpn increases
glucose uptake in muscle, decreases gluconeogenesis in liver, and increases fattyacid oxidation in both (See Fig. 3.1). This action occurs through the activation of
acetyl coenzyme A carboxylase (ACC) and 5-AMP activated protein kinase
(AMPK) (Ma et al. 2002; Kadowaki et al. 2002; Mao et al. 2006; Sattar et al. 2006;
Daimon et al. 2003). Adpn is also an insulin sensitizing agent. Fatty tissues become
hypertrophic in obese patients due to lipid loading. Hypertrophic adipose tissue
secretes less Adpn. Lower plasma Adpn correlates with increased risks of hyperglycemia, diabetes, insulin resistance and cardiovascular disease (Shapiro and
Scherer 1998; Hara et al. 2006; Phillips et al. 2003; Mao et al. 2006). Plasma Adpn
can be increased with thiazolidinediones (TZD) treatment (Tsao et al. 2002). This
anti-diabetic activates the peroxisome proliferator-activated receptor (PPAR )
increasing Apdn secretion and sensitizing insulin. How Adpn sensitizes insulin is
unclear.
The clinical sensitivity of Adpn to diagnose and monitor diabetes is poor with
many false negatives and positives. A greater understanding of the Adpn pathway is
needed to improve clinical accuracy. It is unknown how Adpn impacts insulin sensitivity. Adpn alone has no effect on protein kinase B (Akt) phosphorylation (Tsao
et al. 2002). There is also no explanation for Apdn inflammatory behaviors. Adpn
inhibits tumor necrosis factors - (TNF-) induced adhesion molecule expression,
inhibits nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) activation in endothelial cells and reduces macrophage production OD TNF- (Ouchi
and Walsh 2007; Yamauchi et al. 2003). Adpn also activates mitogen-activated protein kinase (MAPK p38) and induces endothelial apoptosis (Brkenhielm et al. 2004).
Development of Adiponectin Receptor C-Terminal Fragment Bioassays
Adipocyte
Fat
Adiponectin
(multimeric)
Full-length
Adiponectin
AdipoR2
PPAR
Insulin
receptor
Therapies
(TZD)
63
Glucose
production
Fatty acids
AdipoR1
Globular
adiponectin
Hepatocyte
Liver
Insulin
sensitivity
AMPK
ACC
Myocyte
Muscle
Glycolysis
and fatty acid
oxidation
Fig. 3.1 Adiponectin Receptor System. In normal patients, the adiponectin receptor system allows
adiponectin to reduce glucose production in the liver (AdipoR2) and causes glycolysis and fatty
acid oxidation in muscle (AdipoR1) through signaling cell metabolism via 5-AMP activated protein kinase (AMPK). Adiponectin also activates cell growth (MAPK p38) and anti-atherogenic
responses (PPAR ). AdipoR2 reacts to full length adiponectin in low, medium and high molecular
weight forms but not globular adiponectin (gAdpn). AdipoR1 is more responsive to gAdpn. In
metabolic syndrome patients, adipocytes become hypertrophic due to lipid accumulation. The
result is less adiponectin is produced and a reduced the AMPK response in muscle and liver. In the
liver heptocytes, glucose production is not shut down and in the muscle myocytes, less glycolysis
and fatty acid oxidation occurs
3.1.2
Role of Adiponectin Receptor
The adiponectin receptor is a membrane bound G protein-coupled receptor with

seven transmembrane domains; the N-terminal in the cytosol and the C-terminal in
an unexpected extra-cellular position (See Fig. 3.2). Two adiponectin receptors have
been identified. AdipoR1 is ubiquitously expressed and abundant in skeletal muscle.
AdipoR2 is expressed mostly in the liver (Kadowaki et al. 2003, Yamauchi Kadowaki
et al. 2003; Brkenhielm et al. 2004). T-cadherin is a novel receptor found for highmolecular-weight (HMW) adiponectin multimers (Hug et al. 2004). The internal
N-terminus of AdipoR1 was found to interact with the adaptor protein containing a
pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper
motif to initiate the downstream cascade AMPK (Ouchi and Walsh 2007). The
external C-termini is of unknown function. It is unclear if Adpn interacts with the
C-terminus or the loops between the transmembrane domains.
Our goal was to investigate the function of the C -terminal fragment (CTF) as a
potential new avenue in this mechanism. We previously reported that the C-terminal
64
M. Pugia and R. Ma
376
Area of
interest
344
CO2H
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g
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C-terminal fragment
CTF
307
298
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l
v
h a l l g f
t
w i n g t e
t
s i f r i a h
k
f c a r f s p
m
l h g h r p p
r v i p y d v l p d w l k d n d y l
w
r r g e w v k y v f e e m k e m a h h a q l p l t
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i a n p p k a e e e q t c p v p q e e e e e v r v
v
r k g k e e l l p g l e a l e v t d a e r n s a p
a
m s s h k g s v v a p g n g
Extracellular
matrix
Transmembrane
179
Cytoplasm
Fig. 3.2 AdipoR1 inverted G protein-coupled receptor. The adiponectin receptor is an inverted
membrane bound G protein-coupled receptor with seven transmembrane domains and the
N-terminal in the cytosol and the C-terminal in an unexpected extra-cellular position. The
C-Terminal for G protein-coupled receptors is typically on cytoplasm side
fragments of adiponectin receptor (named as AdipoR CTF) existed in human

plasma, and the free CTF was low or missing in most diabetic patients tested using
immuno-affinity mass spectroscopy (Pugia et al. 2009). CTF was significantly
higher (P > 0.001) in non-diabetics (n = 10, ave 3.1 ng/ml, sd 0.6) than diabetics
(n = 10, ave 0.4 ng/ml, sd 0.1). This chapter demonstrates methods used to translate
a proteomic discovery into a clinical assay. We report methods for measurement of
Adiponectin receptor (AdipoR) in blood which offer tools to for researches to further explore this pathway (Pugia 2012; Pugia and Ma 2013).
3.2
3.2.1
Methods
AdipoR1 and R2 CTF Peptide Preparation
Peptides were made to match the clinical form, to cover the cleavage site, to serve
as soluble standards, to produce antibodies and to study the inhibition of proteases.
(See Table 3.1 and supplemental table on CTF peptide sequences). AdipoR1 and R2
peptides were either synthesized by Siemens Healthcare Diagnostics (Walpole MA)
or under contract to Celtek Biosciences LLC (Nashville TN). The 17 mers were
obtained from Phoenix Pharamaceuticals Inc (Belmont, CA). All peptides were
characterized by an HPLC C-18 column and matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy and typically >90 % pure.
65
Table 3.1 Adiponectin receptor C- terminal fragment peptides

Peptide name
AdipoR1 CTF343-375
AdipoR1 CTF344-375
AdipoR2 CTF344-375
AdipoR1 CTF343-375
AdipoR1 CTF341-370
AdipoR1 CTF341-355
AdipoR1 CTF351-375
AdipoR2 CTF351-375
AdipoR2 CTF351-375
AdipoR1 CTF358-375
AdipoR2 CTF358-375
AdipoR1 CTF351-362
AdipoR1 CTF344-353
AdipoR1 CTF367-375
AdipoR2 CTF367-375
Number of amino acids

(mer)
33
32
32
33
30
15
25
25
13
17
17
12
10
9
9
Description of peptide
R1 Longer form
R1 Clinical form
R2 Clinical form
Used for polyclonal antibodies
Covers the cleavage site
Covers the cleavage site
R1 Soluble portion
R2 Soluble portion
R2 used for standard
Non inhibitory domain
Inhibitory domain (active site)
Tail (end of C terminal)
Tail (end of C terminal)
The peptides matching the masses in human clinical blood were previous identified by immune-affinity mass spectroscopy techniques as the 32 mers of AdipoR1
CTF343-375 (Pugia et al. 2009). These 32 mer peptides had poor solubility due to ValVal-Ala-Ala (Gly)-Ala-Phe-Val sequence and therefore were of lower purity and
difficult to work with. The 25 mer peptides AdipoR1 CTF351-375 had good solubility
and were suitable materials for standards. This material was dissolved at 2 mg/mL
in 60 % DMSO-water. All peptides containing the cysteine at position 269 readily
dimerized unless dissolved in 60 % acetonitrile-water with 0.1 % TFA. Mixing peptides in the absence of trifluoroacetic acid (TFA) allowed the R1-R2, R1-R1 and
R2-R2 25 mer dimers to be synthesized in high purity.
3.2.2
CTF Antibodies
Mouse monoclonal antibody (mAb) clones 444, 515, 510, and 461 were raised
against R1 25-mer (soluble section), R1 9-mer (tail), R2 9-mer (tail) and R1 15-mer
(cleavage site) peptides using standard methods (See Table 3.2). Biacore testing
against the R1 and R2 peptides for 9-mer, 12-mer, 25-mer and 32-mers showed all
mAb to be medium affinity except 515 which was low affinity. A high affinity rabbit
monoclonal was made to R1 12-mer (active site) conjugated to keyhole limpet
hemocyanin (KLH) carriers for immunization (Epitomics Inc. Burlingame, CA).
Binding kinetics were measured using surface plasmon resonance (Biacore et al.
3000 instrument).
66
M. Pugia and R. Ma
Table 3.2 Adiponectin receptor CTF antibodies & cross reactivity and pairing
Antibody
Mouse mAb 444
R1CTF
Mouse mAb 515
R1CTF
Mouse mAb 510
R2CTF
Mouse mAb 461
R1CTF
Rabbit mAb R1CTF
Rabbit pAb R1CTF
Rabbit pAb R1CTF
Binding site
Active site
Raised against
AdipoR1 CTF351-375
Tail
AdipoR1 CTF367-375
Tail
AdipoR2 CTF367-375
Cleavage site
AdipoR1 CTF341-354
Active site
Soluble portion
Active site
AdipoR1 CTF351-362
AdipoR1 CTF351-375
AdipoR1 CTF351-362
Clone identification
ATCC 444-1D12.1H7
PTA 12564
ATCC 515-4D6.1B10
PTA 12563
ATCC 510-6B6-1G1
PTA 12565
ATCC 461-4H11.2A4
PTA 12562
SAT-56-1 IgG
NA
NA
Antibody
ATCC
444-1D12.1H7
Reacted with
R1 12-, 25-, 32-mer
peptides
No cross reaction with

R2 peptides or the R1 9-mer
peptide
ATCC
515-4D6.1B10
ATCC
510-6B6-1G1
ATCC
461-4H11.2A4
Rabbit monoclonal
R1 9-, 25-, 32- mer

peptides
R2 9-, 25-, 32- mer
peptides
R1 32- mer peptide
R2 peptides or the R1 12-mer

peptide
R1 peptides or the R2
12- mer peptide
R2 peptides or the R1 9-,
12- or 25- mer peptides
R2 peptides or the R1 9- mer
peptides
R1 12-, 25-, 32mer peptides
Pairing
pAb against CTF
25- mer
mAb 461 or 515
pAb against CTF

25- mer
mAb 461 or 515
ATCC deposited antibody cells lines are available from ATCC upon request at [email protected]
Legend: Antibodies and their antigenic sites are shown along with cross reactivates and pairing.
Note pairing with mouse mAb 515 was dependent on conjugation, N-hydroxysuccinimide conjugation method did not allow pairing but carbodiimide conjugation did
Rabbit polyclonal antibodies (pAb) were raised against CTF R1 33-mer and
25-mer using standard methods (See Table 3.2). Rabbits did not produce antibodies
to 33 mers conjugated with bovine serum albumin (BSA) but did with conjugation to
KLH. Both BSA and KLH conjugates to 25 mer produced antibodies. Cross reactivity and paring studies were conducted on all antibodies by ELISA (See Table 3.2).
The sequence for rat was only one amino acid different and all clones cross-reacted.
3.2.3
Conjugated Antibodies and Standards
Conjugates were made for ELISA, Western blot (WB), immunohistochemistry

(IHC) and immunocytochemistry (ICC) analysis. ALP and FITC conjugated
antibodies were tested for uses and cross reactivity (See Table 3.3).
Immunoglobulin CTF standard was made by conjugating AdipoR1 CTF375-351 and
R2 CTF 375-363 to human IgG. AdipoR1 CTF375-351 was used as standard for free
67
Table 3.3 Antibodies conjugates and uses

Conjugates and standards
Anti- R1CTF pAb25 mer-ALP
Binding site
Multiple
Anti- R2CTF mAb510-FITC
Tail
Cleavage
site
Active site

Anti human IgG mAb to
ALP
Anti- R1CTF mAb444-ALP
Fab
Active site
Std
R1 25
mer
Pair
with
444
Use
ELISA,
WB
IHC, ICC
IHC, ICC
IHC, ICC
Ig-R1
CTF
Ig-R2
CTF
444
ELISA
510
Assay for
R1CTF
R2 CTF
Ig- R2CTF
R1CTF
R1 CTF
Ig- R1CTF
Ig- R1CTF
Ig- R2CTF
WB
R1CTF
Ig- R1CTF
CTF ELISA. All materials were stored in 0.1 M Tris, 0.25 M NaCl, pH 7.3, 0.5 %
BSA and 0.1 % NaN3.
3.2.4
Western Blot Methods
Western methods for measuring CTF R1 or R2 and the related protein were done per
standard denatured SDS PAGE gels or native gel techniques (See on-line supplement for full procedure at www.extras.springer.com/2015/978-3-319-21926-4).
Low molecular weights peptides were analyzed with 20 % Tris -Tricine SDS PAGE
gels. Transfer membranes were incubated with antibodies for CTF R1 or R2 conjugated to alkaline phosphate and developed with substrate.
3.2.5
ELISA Methods
ELISA methods for measuring the immunoglobulin bound form of CTF R1 or R2

were developed by are coating ELISA plates with anti CTF R1 or R2 antibody,
washing the plate with tris buffered saline (TBS) before blocking the plate (See online supplement for full procedure at www.extras.springer.com/2015/978-3319-21926-4). Samples are collected in sterile BD Vacutainer tubes containing
10.8 mg K2EDTA. Samples were diluted 1:48 in phosphate buffered saline containing 1 % BSA, 0.1 M citric acid, 0.27 g/dL EDTA and pH adjusted to 6.4. A second
monoclonal antibody for Anti-Human IgG produced in mouse (binds human Fab)
and conjugated to alkaline phosphatase was used for detection with para-nitro
68
M. Pugia and R. Ma
phenyl phosphate (PNPP) substrate. Calibrators and standards were made by conjugating IgG (Fitzgerald 30-AI17) to AdipoR1 CTF375-351 (25 mer).
ELISA methods for measuring the free forms of CTF R1 utilized a polyclonal
rabbit antibody raised to CTF 25 mer and conjugated to ALP (See on-line supplement for full procedure at www.extras.springer.com/2015/978-3-319-21926-4). The
R1 CTF 25 mer is used for calibration and control. Samples were diluted 112 in
TBS containing protease inhibitor. For human plasmas, Protein A is used to remove
Ig-CTF from human plasma prior to assay for free form.
3.2.6
Mass Spectroscopy Peptide Enrichment Assays
Immuno-affinity enrichment after trypsin digestion using magnetic particle was

conducted to measure AdipoR CTF. The SISCAPA method (SISCAPA Assay
Technologies Inc.) and the immuno-affinity MALDI (iMALDI) methods (MRM
Proteomics Inc.) were followed according to literature (Whiteaker et al. 2007;
Camenzind et al. 2013). In brief, both methods used magnetic particles with antibodies to enrichment for peptides of interested. The protocols starts first with serum
or plasma samples being denatured, reduced, alkylated and digested with trypsin to
yield sample digests; stable-isotope labeled synthetic versions of target peptides are
added in known amounts as internal standards; target-specific anti- AdipoR CTF
peptide antibodies are captured on protein G magnetic beads; the magnetic bead
bound antibodies are added directly to the sample digests where they capture target
peptides; and the beads are then washed to remove unbound peptides.
Both methods used arginine isotope labels (13C6; U-15N4) in a heavy form of the
target VHFYGVSNLQEFR peptide as an internal standard with a 10 D mass difference from native endogenous peptide. The heavy peptide was >90 % purity and
stable in 30 % acetonitrile and 0.1 % formic acid at 80 C. Limits of detection were
generated by titrating unlabeled light peptides from 10 pmol to 56 amol, while the
heavy version of the peptide remained constant at 500 fmol.
In the SISCAPA approach, bound heavy and light target peptides are eluted from
the antibody by low pH and the target peptides and their respective internal standards
are quantitated by mass using a LC-MS/MS. ZORBAX 300 SB-C8 column held at
40 C on an Agilent 6490 triple quadrupole mass spectrometer. The target peptides
were separated using a 3-min gradient with 0.1 % formic acid in water as solvent A and
90 % acetonitrile in 0.1 % formic acid in water as solvent B. Source conditions included
drying gas at 200 C, sheath gas at 250 C and 11 L/min flow for both drying and
sheath gases. Eight transitions were monitored using optimized collision energies: four
equivalent transitions for each of the light (endogenous) and heavy (IS) peptides.
In the iMALDI approach, beads with bound heavy and light target peptides are
placed directly on the MALDI target plate, without prior elution. The beads were
washed three times with 15 % ACN, 25 mM ammonium bicarbonate prior to addition. After washing, a final 3 L of 25 mM ammonium bicarbonate were added to
the beads, the solution centrifuged briefly and spotted onto a MALDI target plate
69
using an AB/Sciex 4800 MALDI-TOF/TOF system (Applied Biosystems, Foster

City, CA, USA). Next, 1 L of CHCA matrix was added on top of the dried spot.
The samples analyzed in reflector positive mode with delayed extraction over a
mass range of 8004000 m/z, collecting 1250 laser shots per spot. The AB
4000-Series Explorer software was used for instrument control and the AB 4000
Series Data Explorer (v4.9) was used for data analysis.
3.3
3.3.1
Results
Mass Spectroscopy Discovery Technique
The AdipoR1 C-terminal fragment (AdipoR CTF) was discovered in plasma

using immunoaffinity mass spectroscopy and anti-CTF antibodies without any
protease treatment of sample (Pugia et al. 2009). The original SELDI-MALDI
method (Ciphergen) detected the native free peptide mass in circulation without
protease digestion (See Fig. 3.3). Two CTF masses observed were at 3378
3381 Da and 34503498 Da corresponding to 32 amino acid peptide (AdipoR
3498
Normal, n = 5
3381
3100
3200
3300
3400
3500
3600
Diabetic, n = 10
5 X mag
3100
3200
3300
3400
3500
3600
Fig. 3.3 Mass spectra of peptide after antibody separation from plasma. SELDI-MALDI mass
spectrograph is shown after using antibody for surface capture for AdipoR CTF344-374 as native free
peptide without protease digestion mass in plasma of five diabetes is compared to five nondiabetics. Two CTF masses observed were at 33783381 Da and 34503498 Da corresponding to
32 amino acid peptide (AdipoR CTF344-374 of 3475 Da) and 31 amino acid peptide (AdipoR
CTF343-374 of 3376 Da)
70
M. Pugia and R. Ma
CTF344-374 of 3475 Da) and 31 amino acid peptide (AdipoR CTF343-374 of 3376 Da).
The masses were confirmed with the new two monoclonal antibodies spanning
the length of the 32 amino acid peptide (ATCC 444-1D12-1H7 & ATCC
461-4H11.2A4). Mass spectroscopy showed CTF dimers in plasma. The R1-R1,
R2-R2 and R1-R2 dimers were confirmed in plasma by western blot and by
ELISA. Finally, this method showed CTF 32-mer did not bind to immobilized
adiponectin supporting the fragments function was not part of receptor binding
of adiponectin.
3.3.2
Discovery of Immunoglobulin CTF
The mass spectra discovery was followed up with western blots using the monoclonal antibodies to CTF. Native gel and denatured gels of plasmas from patients with
normal, impaired glucose tolerance (IGT), and diabetes by oral glucose tolerance
testing (OGTT) showed cross reactivity to Anti R1 CTF 25 mer pAb ALP (See online supplement data for western blot method at www.extras.springer.com/2015/9783-319-21926-4). Antibodies bound to unknown high molecular weight plasma
components at 27, 42, 50 and higher kDa.
These forms were identified as CTF bound to immunoglobulin (Ig CTF). Native
and denatured gels showed the CTF bands to cross react to antibodies for immunoglobulin chains. These high molecular weight forms of CTF were also found in rat,
mouse and rabbit blood. While free CTF was observed in plasma it was primarily as
the dimer. The full receptor was only found in tissue samples and not plasma. While
free CTF is often undetected in diabetics, the Ig CTF forms was found in the plasma
of normal, pre-diabetic, and diabetic patients. Similar results were also observed for
the Adiponectin Receptor 2 fragment.
Commercial grade immunoglobulins (IgG) showed cross reactivity to AntiR1CTF mAb 444 ALP. The western blot of these materials exhibited binding to
the same high molecular weight fragments (27, 50 and higher kDa) seen in human
plasmas. Filtration to remove proteins >10 kDa, also removed any detectable cross
reactivity. Denaturing gels under SDS-PAGE reducing gel 18 % with 2-mercaptoethanol did not break bonds to liberate CTF from immunoglobulins.
3.3.3
Immunoglublin Binding Mechanism
CTF was thought to be covalently bound to immunoglobulin (Ig CTF) as it was not
released by simply breaking the disulfide bonds. Alkaline conditions (250 mM Tris
buffer, pH 11.5, and heated at 37 C at 2 h) did liberate CTF from immunoglobulin
(See on-line appendix for western blots at www.extras.springer.com/2015/9783-319-21926-4). Re-blotting same gel showed IgG remained intact on gel and still
reacted to anti-IgG but not to anti-CTF. Alkaline conditions are also required to
71
break the ester between C3b and immunoglobulin chains (Sim and Twose 1981;
Lutz and Stammler 1993; Suhn and Pangburn 1994). Therefore it was reasoned that
CTF could behave in a similar mechanism. Native complement component C3
binds C3b to IgG by means of A reactive thioester as a means for immune complex
solubilization, clearance of soluble immune complexes, phagocytosis of opsonized
target cells and in stimulation of the alternative complement pathway.
The thioester of C3b is between Cysteine and Glutamine in the peptide sequence
Gly-Cys-Gly-Glu-Glx-Asn-Met. These reactive thioesters are capable of forming
esters or amides with peptides when immunoglobulin is brought into proximity. A
mechanism for CTF attachment to IgG that is the same as C3b would require an
internal thioester to be formed between cysteine and glutamic acids in the peptide
R1 CTF sequence Glu-Gly-Gly-Cys-Thr-Asp-Asp-Thr-Leu-Leu and R2 CTF
sequence Gly-Gly-Gly-Cys-Ser-Glu-Glu-Asp-Ala-Leu.
3.3.4
Immunoglublin Chain Identification
Analysis of purified human IgG showed CTF attaches to the 50 kDa gamma heavy
chain and 26 kDa kappa/lambda light chains (see on-line appendix for western blot
at www.extras.springer.com/2015/978-3-319-21926-4). CTF also attached to the
light chains of IgA and IgM. Smaller light chain fragments of 15, 13 and 12 kDa
also contained CTF. The Fc effectors function domain of the gamma heavy chain
did not contain CTF. This indicates that CTF is attached to gamma and kappa/
lambda chains in the Fab region of IgG.
ELISA confirmed CTF is integrated into IgG, IgA, IgM, IgD and IgE isotypes
using an anti- kappa and anti CTF monoclonal sandwich assay to measure immunoglobulin bound CTF (Ig-CTF). Anti-gamma chain and anti CTF monoclonal sandwich assay measured bound CTF only in the Ig isotype. Similarly, other specific
monoclonal antibodies allowed sandwich assays that measured CTF bound to IgG,
IgA, IgM, IgD or IgE (See on-line appendix for ELISA method for immunoglobulin
bound CTF at www.extras.springer.com/2015/978-3-319-21926-4). It appears
Ig-CTF is polyclonal as both IgG and IgM bands in native and denatured western
blots were diffuse bands.
3.3.5
Mass Spectroscopy Peptide Enrichment Assays
Immuno-affinity enrichment after trypsin digest using magnetic particles has

become a common approach to develop clinical assays by mass spectrometers. Two
such methods, the SISCAPA and iMALDI techniques were tested as potential CTF
assays (Anderson et al. 2004; Whiteaker et al. 2007, 2010; Eyford et al. 2009;
Camenzind et al. 2013; Anderson et al. 2012). These methods are optimized to
detect fM levels of peptides in the 4003000 Da range. We found both methods
72
M. Pugia and R. Ma
required high-affinity anti-peptide antibodies and typical affinity mouse mAb were
not effective. Either a rabbit mAb or rabbit pAb proved effective. The rabbit mAb
clones were selected based upon nanomolar affinity and slow off-rates. The best
binding kinetics were: ka of 1.38E6 M-1s-1, kd of 1.24E-4s-1, KD of 0.09 nM and
half off-time of 93.0 min.
Both SISCAPA and iMALDI methods, used the antibodies against the 12-mer
fragment (AdipoR1 CTF351-362) as this peptide was ionized very efficiently and
detection at lower concentrations . The 32-mer, 25-mer, or 19-mer fragments did not
ionize well by electrospray or by MALDI using a variety of matrices. As a result the
clinical 32-mer was not detectable in plasma at clinical concentrations and required
over 1835 ng in the sample.
Instead both methods had to form the 12-mer fragment by trypsin treatment of
the 32-mer. The SISCAPA method showed excellent quantification for 12-mer
added to human plasma with a LOD of 11.1 fmol and LOQ of 24.6 fmol. The
iMALDI method had an LOD of 1.28 fmol for 12-mer in buffer but only an LOD of
500 fmol in plasma. Non-specifically bound plasma peptides may be causing ionization suppression of the 12-mer signal or the complex nature of the plasma digest
inhibited the polyclonal antibodies ability to bind and retain the 12-mer through the
iMALDI procedure.
However, endogenous CTF (bound or unbound) in the native sample could not
be found. It was confirmed that the immunoglobulin linkage of CTF interfered with
tryptic cleavage. The 12-mer CTF peptide was formed after dissociating the endogenous CTF from the immunoglobulin by bringing the sample to pH ~11.5 using
Tris- Base and incubating at 37 C for 2 h prior to digesting the sample. However,
this increased the complexity of the assay and proved no more sensitive than ELISA.
3.3.6
Analytical Validation of ELISA
3.3.6.1
Cross Reactivity
Low molecular weight peptide analysis gels (1020 % Tris-Tricine) showed the free
CTF peptide standards readily dimerized and were detected at ~67 kDa and
contained no masses at 27, 42, 50 kDa or higher. The R1 antibodies showed no R2
cross reactivity nor did the R2 antibodies show any R1 cross reactivity.
3.3.6.2
Calibration Reproducibility and Stability
Calibrator reproducibility and stability were achieved by use of citrate-phosphateBSA buffer at pH 6.4. The calibrator stock used for daily dilution was stable stored
58 C for 6 months and at 20 C for 4 months. Calibrators had to be fresh daily
and used within 24 h at 58 C. Freezing dilute calibrators depressed values.
3.3.6.3
73
Analytical Validation
Precision and Accuracy testing was conducted on the final Ig-CTF ELISA
method over 3 days for ten sample levels on four separate plates for each day.
Each plate used with separate calibration curves. The overall repeatability of
the assay was a 2.6 % and the overall accuracy was 11.2 % in the reportable
range of 954900 ng/mL. Accuracy was reduced at lower concentrations. The
sensitivity limit was 13.5 ng/mL. No prozone effect was observed up to
21,200 ng/mL.
3.3.6.4
Within Patient Reproducibility and Stability
Specimens were collected in EDTA, heparin and sodium fluoride tubes on day 1
as non-fasting samples, days 2 and 45 after overnight fasts. Patient weight, height
and fasting blood glucose meter readings were taken (10 % were diabetic and
40 % were obese). Samples were kept at 5 C for <8 h until whole blood and
plasma were separated and sample dilutions made. Dilutions were stable for
1 day at 525 C and for 6 months at 20 C. Samples were stable for 20 C for
12 months. Three freeze/thaw cycles were tested. Variance was tested by three
separate dilution and duplicate determinations were made for each specimen
condition.
3.3.6.5
Recovery
Recovery was tested on specimens collected from seven patients across the reportable range. The samples were diluted 1:12: 1:24, 1:48, 1:96 and 1:187. The target
dilution was 1:48. Results at twice the target dilution and half the target dilution
recovered as expected. Whole blood and plasma from the same patient showed the
same recovery. The recovery fell off at dilution were 1:12 and 1:187. The recovery
at 1:12 was 46 % of expected values, while recovery at 1:187 was 71 % of expected
values.
3.3.6.6
Interference Testing
Interference testing was conducted by contriving six plasma samples with the
potential interference. Hemolysis, icterus, lipemia, anti-coagulates, proteinemic and
human anti-mouse antibody interference (HAMA) impact was tested by addition of
500 mg/mL hemoglobin, 60 mg/dL bilirubin, 500 mg/dL cholesterol, 10,000 mg/dL
triglycerides, 2 IU/mL heparin, 12.0 g/dL of albumin, 60 mg/mL goat and mouse
IgG. Human immunoglobulin (IgG and IgM) may contain natural CTF-Ig (show by
western blot) and is not suitable for cross reactivity testing.
74
M. Pugia and R. Ma
3.3.6.7
Blood Levels of CTF and Ig-CTF by ELISA
Analysis of patient plasma by ELISA was possible for immunoglobulin bound CTF
(Ig-CTF) and for free CTF (See on-line appendix for ELISA method for free CTF
at www.extras.springer.com/2015/978-3-319-21926-4). Bound CTF in patient
plasma was found to be <0.2 % of all plasma IgG. This percentage was similar
whether measuring Ig CTF R1 or Ig CTF R2. The percentage of IgM with CTF was
typically lower, <0.03 % of all plasma IgM. The number of CTF attached to IgG
was typically 12, in most patients with a range of 15.
Assays for bound Ig-CTF and free CTF by ELISA were possible. To measure free
CTF, the bound form was removed by Protein A column as the antibodies do no distinguish between the free and the bound forms. The amount of Ig-CTF in plasma,
954900 ng/mL was significantly greater than amount of free CTF, 0.055.0 ng/mL
with less than 0.1 % in free from. While the free CTF ELISA assay works for rat blood,
the interference in human blood was too large to develop a clinical assay for free CTF.
3.4
Conclusion
Discoveries based on peptides detected by immune affinity mass spectroscopy

method such as SELDI can be useful discovery tools when trypsin is not used.
However these methods can be misleading as to true complexity of protein forms in
native samples. Mass spectroscopy based on trypsin digestion of native samples
such as SISCAPA and iMALDI were misleading in this case as native forms were
resistant to treatment trypin hydrolysis. The mass spectroscopy peptide enrichment
was unable to detect the free peptide until the bound CTF was released by base
treatment to liberate free form which could be trypsinized.
Traditional bioassay such as Western blot and ELISA are recommended. These
methods were suitable for testing of patient and biological samples (See on-line
supplement for full procedures at www.extras.springer.com/2015/9783-319-21926-4) and can be used for studies on Ig CTF formation, absorption into
tissue (liver, pancreas, brain), and CTF mechanism action. These assays could be
useful for screening of compounds that block or lead to Ig CTF formation, and for
investigation of mechanism action.
Acknowledgements We would like to acknowledge the work done by Siemens Healthcare
Diagnostic people including Mary Foltz, Jill Perry, David Brock, Qingping Jiang, Yu Hui Lin, Jim
Freeman and others who helped prepare antibody, conjugates, peptides, and assay formats. We
would like to acknowledge the mass spectroscopy work done by the SELDI group of Dr. Saeed
A. Jortani, Department of Pathology and Laboratory Medicine, University of Louisville, School of
Medicine, by the SISCAPA group of Dr. Terry W. Pearson and Dr. Matthew Pope of SISCAPA
Technologies at the University of Victoria BC Canada Victoria., and by the i-MALDI group of Dr.
Christoph Borchers & Dr Andrew Munk of MRM Proteomics at the University of Victoria BC
Canada Victoria.
3.5
75
Supplements
CTF Peptide Sequences

ELISA Method for IgG-CTF assay
ELISA Method for free CTF assay
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Chapter 4
Protease Inhibition and Biological Distribution

of the C Terminal Fragment of Adiponectin
Receptor
Abstract Protease inhibition studies showed Adiponectin Receptor C- Terminal

Fragment (AipoR CTF) inhibited insulin-degrading enzyme (IDE) and TNF alpha
cleavage protease (TACE). Cleavage of Adiponectin Receptor by TACE to form
CTF was demonstrated by western blot assays analysis of recombinate AdipoR1
treated with TACE and by inhibition studies. Tissue and blood analysis by standard
immuno histochemistry (IHC) and immuno ctyochemistry (ICC) methods demonstrated CTF is primarily absorbed into the liver, pancreas and brain tissue where it
is co-located with IDE. CTF formation by TACE appears to occur in fat and muscle
tissues and to be carried in circulation by an immunoglobulin bound form (Ig-CTF)
that freely circulates and is immune cell bound in both blood and lymph nodes.
Tissue from animal models showed levels of CTF increase with disease progression
from non-diabetic to diabetic states. All of these data unveiled a novel regulation
mechanism of AdiopR CTF in its free and Ig-associated forms which could impact
insulin sensitivity and prevent insulin degradation by IDE in tissue and cells.
4.1
Introduction
This chapter demonstrates methods to determine the function and biological locations of peptides such as 32 amino acid peptide (AdipoR CTF344-374) discovered in
the plasma. Identification of peptidases likely to interact with the peptide is a useful
starting point. The peptide sequence can be compared to the peptide cleavage
M. Pugia, PhD (*)
R. Ma
Siemens Healthcare, 278 Zhouzhu Road,
Pu Dong New Area 201318, Peoples Republic of China
DOI 10.1007/978-3-319-21927-1_4
77
78
M. Pugia and R. Ma
sequences for known peptidase. Once identified, peptidases of interest can be used
to explore the mechanism of peptide formation and function. Integrated data bases
source such as MEROPS that have a hierarchical classification system that allows
identification of homologous sets of peptidase and protein inhibitor sequences
(Rawlings et al. 2006). A MEROPS search identified insulin-degrading enzyme
(IDE) and TNF alpha cleavage protease (TACE) as potential peptidases for CTF
(See Sect. 4.3.1). As Adiponectin has insulin sensitizing properties and ability to
inhibit TNF signaling that cannot be fully explained, both IDE and TACE are
candidates of significant interest to offer new understandings of the pathway (GilCamposa et al. 2004; Ouchi et al. 2000).
4.1.1
Insulin-Degrading Enzyme (IDE)
Insulin-degrading enzyme (IDE) is also known as -secretase (BACE) or insulysin

and fragments the , chains of insulin at nine cleave sites (Duckworth et al. 1998).
Insulin degradation is a regulated process controlling the removal and inactivation
of the hormone. Degradation occurs through an endosomal degradation pathway
which controls the levels of intracellular insulin. Intracellular insulin impacts the
recycling of the insulin receptor and receptor sensitivity (Kahn et al. 1973). While
circulating insulin is primarily cleared in the liver and kidneys, IDE is present in all
insulin sensitive tissues and is alternative feedback mechanism for insulin clearance
and receptor sensitivity (Kuo et al. 1993).
Pancreatic secretion increases blood concentrations of insulin and c-peptide in
obesity (Tura et al. 2001). The patient presents with hyperinsulinemia without glycaemia and is categorized as insulin resistant. The degree of insulin resistance
worsen through stages of worsening glucose intolerance and is measured using frequent sampled insulin and glucose values during an oral glucose tolerance test
(Sathananthan 2012). As obesity progresses to diabetic glucose intolerance, glycaemia starts to be routinely observed and insulin secretion starts to decrease below
normal levels (Bonora et al. 1983).
Insulin clearance rates decrease in obesity and diabetes and correlate with
reduced IDE effectiveness to degrade insulin (Duckworth et al. 1998; Abdul-Hay
et al. 2011). Deletion of IDE in animal models does cause insulinemia without
immediate glycaemia. Insulin resistance with reduced insulin receptor and reduced
glucose tolerance does occur as the animals ages in response to chronic hyperinsulinemia (Abdul-Hay et al. 2011). Inhibition of IDE may constitute a valid approach
to the treatment of diabetes and Alzheimers disease with synthesized inhibitors
(Leissring et al. 2010; Maianti et al. 2014)
IDE also cleaves on insulin-like growth factor I and II (IGF), transforming growth
factor (TGF), A protein precursor (APP), - amyloid peptide (A), and glucagon
(Duckworth et al. 1998; Kuo et al. 1993). Alzheimers disease (AD) is caused by
aggregates of the amyloid peptide (A), which is generated by cleavage of the A
Protease Inhibition and Biological Distribution of the C Terminal Fragment
79
protein precursor (APP) by IDE followed by -secretase (Farris et al. 2003).

Therefore IDE contributes to A 42 oligomers noted in amyloid plaques and tau
pathology in cognitive defects (Tamboli et al. 2010). Breakdown of extracellular A
by insulin-degrading enzyme in microglia is thought to be central to Alzheimer
diseases (Tamboli et al. 2010). However while advances in synthetic IDE inhibitor
design are reported natural inhibitors of IDE remain unknown (Maianti et al. 2014;
Olson et al. 2007)
4.1.2
TNF-Converting Enzyme (TACE)
TNF-converting enzyme (TACE) processes pro- tumor necrosis factor - (TNF-)

to active TNF- in the bloodstream and is central to the inflammatory response
(Black 2002). Insulin sensitivity, glucose, and lipid metabolism are associated with
TNF-. Deletion of TACE or TNF- in animal models protect from insulin resistance
and obesity (Serino et al. 2007; Black 2002). TNF- mediates cytokine signaling and
cytokine activation of c-Jun N-terminal kinase (c-JNK) which can phosphorylate the
insulin receptor substrate and inhibit insulin signaling (Lorenzo et al. 2008). TNF-
neutralization decreases circulating free fatty acids in obese animals and lowers
plasma glucose concentrations and weight increases (Weickert and Pfeiffer 2006).
Adipose tissue of obese humans with insulin resistance also expresses elevated levels
of TNF- mRNA relative to lean individuals (Hotamisligil 1994) .
4.1.3
Goal
Our goal was to investigate the function of the C -terminal fragment (CTF) as a
potential new avenue for the mechanism of action of adiponectin. We explored
TACE enzyme as the likely upstream protease to release CTF and IDE as the likely
downstream protease to be inhibited by CTF. We also wanted to determine the
potential tissues for formation and accumulation of CTF.
4.2
4.2.1
Methods
Protease Identification
The 32 amino acid peptide (AdipoR CTF344-374) found in plasma was submitted to
the MEROPS peptidase database (http://merops.sanger.ac.uk/) for potential protease search. TNF-converting enzyme (TACE, also known as A disintegrin and
metalloproteinases 17, ADAM17) was the only recorded protease to cleave peptide
80
M. Pugia and R. Ma
sequence X-X-X-X + Val-Val-Ala-Ala matching AdipoR CTF344-374 cleavage site.

Another protease able to cleave AdipoR CTF344-374 and sequences of X-X-XLeu + Val- X-X-X is IDE (insulin-degrading enzyme or Insulysin). Other protease
able to cleave this simple sequence include, pepsin (A&B), renin, cathepsin (D, E,
L, G & H), leucolysin, enprilysin, endothelin converting enzyme, aminopeptidase
Ap1, deutrolysin, camelysin and peptidase C1 & K.
4.2.2
Inhibition Studies
Inhibition studies were performed using InnoZyme Insulysin-IDE

Immunocapture Activity Assay Kit and InnoZyme TACE Activity Kit purchased
from EMD Chemicals, Inc. (Gibbstown, NJ). Recombinant human TACE standard
was purchased from R&D Systems Inc. (Minneapolis, MN). IDE standard was
from the kit. CTF peptides at concentrations of 0, 12.5, 25, and 50 mg/L were
incubated with IDE or TACE standard coated on the assay plate along with the
fluorescent substrates and controls. The assay was read over 0300 min to monitor
the change of relative fluorescence units (RFU) recorded at Ex325/Em405 using
an FLx800 Fluorescence Microplate Reader (BioTek Instruments, Inc., Winooski,
VT).
4.2.3
Tissue Sample Preparation
At termination, animals were perfused with saline. Brain, pancreas, liver, lymph
nodes, fat and quadriceps muscle were harvested from rats graded as normal (n = 3),
short term diabetic (n = 3), on long term diabetic (n = 3). Tissues were also harvested
from rabbits which did (n = 3) and did not (n = 3) produce polyclonal antibodies to
CTF. After PBS perfusion tissues was embedded for frozen tissue slices with OCT
compound, placed in disposable base in ice bath and then frozen at 70 C (RamosVara 2005). Cyro slices at 8 m were made on to permafrost Fisher slides (AML
laboratories). Separate slices (n = 20) were obtained for each tissue sample with
additional slices for left, right cerebral hemisphere and cerebellum. Slides with tissue slices were stored at 70 C.
4.2.4
Blood Sample Preparation
Blood samples from rats and human were collected fresh in EDTA tubes and stored
at 4 for less than 8 h.
4.2.5
81
Tissue and Blood Analysis
Tissues and blood sample were stained using standard immuno histochemistry
(IHC) and immuno ctyochemistry (ICC) methods (See on-line supplement for full
procedures at www.extras.springer.com/2015/978-3-319-21926-4). The slide area
containing tissue or a 10 L whole blood smear was circled with a hydrophobic pen
to create an area for holding liquid. Then 100 L of 5 % formaldehyde in phosphate
buffered saline (PBS) was added to the holding area followed by incubating for
15 min at RT. The slide was permeabilized in PBS with 0.2 % Triton-X 100 for
515 min. After air drying, the Fc receptors were blocked with 50 L IgG in PBS
(5 mg/ml G4368 Sigma Aldrich) for 15 min RT, washed in PBS with 0.1 % Tween
20 at 5 min RT. Slides were stained with 50 L of 4,6 diamidino-2-phenylindole
dihydrochloride (DAPI) (0.01 mg/mL) & 100 L of one or more antibody with
fluorophore (FITC, TR or CY5). Each antibody was at 0.1 mg/mL in PBS. The slide
was incubated for 15 min at 37 C and washed in cell wash buffer (PBS/Tween 20
at 0.1 %) in a wash bath for 5 min. Cover slip was applied with 5 L DAPCO.
Phase contrast and fluorescence microscopy was conducted with Leica DM5000
and using the filter sets for reading four fluorophores (DAPI, FITC, TR and CY5)
signals. The slides are scanned at 40 X using 50300 ms exposures. A grading system was established for consistency across all slides in an experiment.
4.2.6
Cleavage Study
Analysis of recombinant AdipoR1 (62 kDa from Novus) before and after treatment
with TACE (R&D Systems) was conducted by Western Blot techniques (See on-line
supplement for full procedure at www.extras.springer.com/2015/978-3-319-21926-4)
using with 100 ng AdipoR1 and 1 ug TACE incubated 37 C and 24 h followed by
western blot denatured gel with Anti R1CTF25 pAb-ALP (5 g/ml).
4.3
4.3.1
Results
Protease Inhibition Studies
Both ADAM17/TACE and IDE activity showed inhibitory effects due to AdipoR1
CTF344375 (See Figs. 4.1 and 4.2). A structure reactivity study was conducted for a
series of AdipoR1 and R2 CTF peptides as inhibitors for ADAM17/TACE and IDE
(See Table 4.1). As a negative control, the lack of elastase or trypsin inhibition by
AdipoR1 CTF344-375 was confirmed.
82
M. Pugia and R. Ma
120.0%
% TACE Control Activity .
100.0%
80.0%
60.0%
40.0%
CTF 32 mer 0g/ml

CTF 32 mer 12.5
g/ml
CTF 32 mer 25
g/ml
20.0%
0.0%
30
60
90
120
150
180
210
240
270
300
Time (Minutes)
-20.0%
Fig. 4.1 Effects of AdipoR1 CTF344-375 on the activities of ADAM17/TACE. Synthetic peptide
Adiponectin Receptor C terminal fragment 32 mer peptide (AdipoR1 CTF344-375) at concentrations
of 050 g/mL was incubated with ADAMI7/TACE from 10 to 120 min. The percent activities
relative to that of the control sample (0 mg/L CTF1) were plotted
120%
100% Control Activity
100%
80%
60%
40%
CTF 32 mer
CTF 32 mer
CTF 32 mer
CTF 32 mer
20%
0g/ml
12.5g/ml
25g/ml
50g/ml
0%
0
50
100
150
200
250
300
Time (Minute)s
Fig. 4.2 Effects of AdipoR1 CTF344-375 on the activities of IDE. Synthetic peptide Adiponectin
Receptor C terminal fragment 32 mer peptide (AdipoR1 CTF344-375) At concentrations of 050 g/
mL was incubated with IDE from 60 to 300 min. The percent activities (RFU) relative to that of
the control sample (0 mg/L CTF1) were plotted
83
A sharp dose- and time-dependent inhibition of ADAM17/TACE activity was

observed with only the full length 32 mer sequences (See AdipoR1 and R2 CTF344-375
in Table 4.1 and Fig. 4.1). Activity resumed and remained at about 50 % after 60 min
of incubation. The acute decrease of activity and recovery supported TACE is
the possible cleavage protease. The sequence of AdipoR1 and R2 CTF344-375 of ValVal-Ala-Ala at the cleavage points matches the known cleavage sequence for
ADAM17/TACE.
Additional experiments supported that ADAM-17/TACE was the cleavage protease. Western blots of recombinant AdipoR1 (62 kDa from Novus) before and after
treatment with TACE (R&D Systems) showed AdipoR1 cleaved into a 27 kDa fragment and a 7 kDa fragment matching a CTF dimer (See on-line supplemental western blot data at www.extras.springer.com/2015/978-3-319-21926-4).
Table 4.1 Adiponectin Receptor CTF peptide inhibition data

Peptide
name
AdipoR1
CTF344-375
AdipoR1
CTF351-375
AdipoR1
CTF358-375
AdipoR1
CTF367-375
AdipoR1
CTF344-353
AdipoR1
CTF351-362
AdipoR2
CTF344-375
AdipoR2
CTF351-375
AdipoR2
CTF367-375
AdipoR1
CTF351-375
AdipoR1
CTF351-375
AdipoR2
CTF351-375
AdipoR2
CTF351-375
AdipoR1
CTF351-375
AdipoR2
CTF351-375
Number of
amino acids
(mer)
32
Form
Monomer
25
Description
R1 clinical form
ADAM-17
inhibition
(observed)
Positive
IDE inhibition
(concentration at
90 % inhibition)
150.0 g/ml
Monomer
R1 soluble portion
Negative
25.4 g/ml
17
Monomer
Negative
Negative
Monomer
Negative
Negative
10
Monomer
Negative
Negative
12
Monomer
Negative
20.2 g/ml
32
Monomer
Non inhibitory
domain
Tail (end of C
terminal)
Non inhibitory
domain
Inhibitory domain
(active site)
R1 clinical form
Positive
140.0 g/ml
25
Monomer
R1 soluble portion
Negative
9.0 g/ml
Monomer
Negative
Negative
25
Homodimer
Tail (end of C
terminal)
R1 CTF25 R1
CTF25
Negative
12.5 g/ml
25
Homodimer
R2 CTF25 R2
CTF25
Negative
9.0 g/ml
25
Heterodimer
R1 CTF25 R2
CTF25
Negative
5.2 g/ml
84
M. Pugia and R. Ma
A time-independent inhibition of IDE activity was observed with all sequences

of CTF containing AdipoR1 CTF351-362, considered the active inhibitor site (See
Table 4.1 and Fig. 4.2). The impact was not reversed with time. The amount of CTF
needed for inhibition of 90 % (IC90) of the IDE activity was determined. The soluble 25- and 12-mer peptides were more active inhibitors, with the 12-mer identified
as the key inhibitory site.
The homology between the R1 CTF and R2 CTF is very high with key differences only in the last nine peptides of the C terminal. No large inhibitory differences
were observed between R1 and R2. R2 was slightly more inhibitory. The dimers for
CTF were not stronger inhibitors than monomers. The hetero-dimer of R1-R2 is
slightly active than the homo-dimers.
Structural comparison of insulin beta chain and CTF 32 mer was made as both
bind IDE. There was no sequence homology between them. Molecular modeling
showed structural similarity in the size and orientation of the helixes (see Fig. 4.3).
The inhibitory domain AdipoR1 CTF351-362 is located the in bend between sheet
located at AdipoR1 CTF344-354 between the helix located at AdipoR1 CTF356-364 .
The interaction point between the sheet and helix is Phe 353 and Arg 361. IDE has
multiple insulin cleavage sites at multiple sites for insulin (Duckworth et al. 1998).
The inhibitory domain of the CTF inhibitor could association with IDE and block
insulin binding.
Adiponectin Receptor
C terminal fragment (CTF)
Insulin
Fig. 4.3 Molecular structure comparison of AdipoR CTF to insulin. I-TASSER structure for CTF
32-mer heterodimer of CTF R1 and CTF R2 is shown. The two cysteines were placed in docked
orientation (ZDOCK). The helixes of AdipoR1 and R2 located at CTF356-364. are shown along
with the between sheets AdipoR1 and R located at CTF344-354. Insulin chain dimer complex of
4-hydroxybenzamine was taken from the protein database (PDB-ID: 2bn3, Resolution 1.40 ).
The helix of insulin chain is from Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-AlaLeu-Tyr-Leu-Val-Cys-Gly (Images are shown as captured in RasMol viewer)
4.3.2
85
Tissue Forms and Distribution
Multiple forms of bound CTF found were found in tissue and cell cultures by western blots with monoclonal antibodies to CTF (See Table 4.2 and on-line supplement
data for western blots at www.extras.springer.com/2015/978-3-319-21926-4). CTF
bound to Ig, Fab2, IgG or IgM immunoglobulin (Ig CTF) was only observed in
liver, muscle, and myocyte cell culture. The full receptor was found in fat, muscle
and liver tissue samples and not plasma. Lower molecular weight forms of CTF
bound to gamma chain or kappa light chain were observed in pancreas and mesenteric fat tissues. TACE and IDE were found in liver, pancreas, muscle and mesenteric fat of normal and diabetic rats. Normal and diabetic rat treated in one exposure
with AdipoR1 30 and 32 mer exhibited no changes in tissue forms.
4.3.3
Plasma Forms
All Ig CTF and free CTF forms were observed in plasma (See Table 4.2). Follow up
with immuno ctyochemistry (ICC) of human whole blood showed Ig-CTF was
freely circulating in plasma As well As on washed white blood cells (WBC) but not
red blood cells (RBC) (See on-line supplement for cell and tissue staining procedure at www.extras.springer.com/2015/978-3-319-21926-4). WBC binding to
Ig-CTF occurred on some neutrophils with polymorphonuclear pattern, basophils,
T cell (CD3 positive by counter staining) and Beta cell lymphocytes (CD 19 positive by counter staining). As the Fc receptor interaction was blocked, the WBC
binding was assumed to be through CTF binding or absorption into cells. It was not
determined whether CTF was in free form or IgCTF bound form when associated
with WBC. Levels of CTF detected were above any expression level of AdipoR1 on
WBC surface (Golz et al. 2007). Additional free circulating level TACE and IDE
were also found in human blood by westerns blot with some differences noted in
molecular weight between normal and diabetic samoles.
Table 4.2 CTF Immunoglobulin in human tissue and blood
Ig-CTF band (KD)

IgM-CTF
IgG-CTF
Fab2-CTF
Ig-CTF
Gamma heavy chain-CTF
AdipoR1
Light chain CTF
Light chain CTF
CTF dimer
Observed kDa in Western blots

Human Fat
Liver
Muscle
plasma
tissue
tissue tissue
250
200
160
100
100
110
70
80
55
55
3745 43
3745
25
25
25
25
22
22
22
22
7
Pancreas
tissue
50
25
22
Myocyte
cell culture
200
100110
60
47
M. Pugia and R. Ma
86
4.3.4
Primary Tissues of Action
Immuno histochemistry (IHC) of rabbit and rat tissue showed that Ig-CTF is most
strongly absorbed into the liver followed by the brain and pancreas but not the adipose
(See Table 4.3 and on-line supplement for cell and tissue staining procedure at www.
extras.springer.com/2015/978-3-319-21926-4). The liver is therefore a primary organ for
CTF absorption. Counter staining for IgG showed also immunoglobulin built up. The
liver, brain and pancreas are also where IDE primarily resides in agreement with IDE as
the site of CTF action. The muscle and fat were the primary location for TACE in agreement with these tissue being primary sites for releasing CTF.
4.3.5
Fat Morphologic Observations
CTF was not detected intracellular and was located on outer membrane of white adipose. Cells were IgG negative on counter staining with anti-IgG antibody and CTF
observed appears to be due to AdipoR. CTF was also clearly seen in outer membrane
of brown fat and connective tissue. TACE counter staining was positive on the cell
surface and co-located with CTF. Fat appears to be a source of CTF from but not a
target of free CTF or CTF-Ig.
4.3.6
Muscle Morphologic Observations
CTF was not detected intracellular and was located on outer membrane muscle.
Cells were IgG negative. Staining for CTF with mAb 461 was negative as this antibody only detects membrane released CTF. Staining for CTF with mAb 444 was
positive as this antibody detects CTF still bound to full AdipoR receptor. TACE
Counteres staining was positive on the cell surface and co-located with CTF. Muscle
appears to be a source of CTF Ig but not a target of free CTF or CTF-Ig.
Table 4.3 Intracellular CTF observed in IHC fluorescent staining
CTF
TACE
IDE
IgG counter
staining
Observed immuno histological stain results

Cerebral right
Liver
Cerebellum & left
Pos. 5+
Pos 1+
Pos 1+
Neg.
Neg.
Neg.
Pos
Pos
Pos
Pos where Pos where
Pos where
CTF found CTF found CTF found
Pancreas
Pos1+
Neg.
Pos
Neg
Fat (adipose)
Neg.
Pos
Neg.
Neg
Muscle
Pos 1+
Pos
Neg.
Neg
CTF Staining grade on scale of 15 across all tissue on same day with replicates of three animal
per determination. Constant microscopic exposure times were used to grade to same scale. For fat
tissues like white adipose too fragile for making slice, a tissue homogenization method was used
to make the slide
4.3.7
87
Liver Morphologic Observations
CTF was detected in intra cellular regions of liver tissue with both CTF antibodies
and was detected even after Fc blocking. TACE staining was negative. IDE staining
was positive. IgG was detected inside and outside the cells. Liver could not be as
source of CTF IgG but could a target of CTF or CTF-Ig to act on IDE.
4.3.8
Pancreas Morphologic Observations
CTF was not generally detected in intra cellular areas with either CTF antibodies
even after Fc blocking. CTF was detected in the interstitial space, especially in areas
which had some inflammation with observed lesions and white blood cells. These
WBC cells were IgG and CTF positive. There was IgG detected migrating into the
tissue with CTF positivity near lesions. This behavior was observed with both CTF
antibodies. CTF detected as receptor free epitope using mAb 444 was more often
extra cellular as IgG-CTF and CTF detected as the membrane epitope using mAb
461 was more often inter cellular. This tissue was TACE negative and therefore not
a source of CTF-Ig but a target of CTF Ig.
4.3.9
Brain Morphologic Observations
CTF was detected in both intra and extra cellular areas with either antibodies and all
types of Fc blocking. No differences in right or left cerebral or cerebellum were
observed. The brain tissues were TACE negative and IDE positive therefore not a
source of CTF-Ig but a target tissue. CTF R1 free fragments (mAb 461) were negative for free CTF. CTF-Ig (mAb 444) was detected in interstitial areas surrounding
the tissue especially around endothelial. WBC migration into the interstitial CSF
space was detect, especially in areas which had some inflammation lesions. These
interstitial WBC cells and areas were positive for IgG and CTF co-location. The
brain tissues were negative for IgG inside the cells.
4.3.10
Tissue Absorption in Disease
Staining of tissue from a diabetic rat model showed an increase in CTF absorption
as rats became diabetic (See Chap. 5 for description of animal moedels). The
increase occurred in the liver, brain and lymph nodes (See Table 4.4; Fig. 4.4).
Absorption into lymph nodes occurred earlier while liver absorption continued to
increase with length of diabetes. Cell binding of CTF was clearly visible in the
lymph nodes. Diseased liver tissues contained immune cells with CTF such as macrophages and monocytes.
M. Pugia and R. Ma
88
Table 4.4 CTF in tissue by immunuo fluorescent staining
CTF
Liver
Lymph
Normal rats
N=3
Neg
Pos 2+, Neg. Neg.
Neg
Pos 5+, Neg, Neg.
Short term diabetics rats

N=3
Pos
Pos 5+, Pos 4+, Pos 3+
Pos
Pos 10+, Pos 11+, Pos 8+
Long term diabetic rats

N=3
Pos
Pos
Pos 5+, Pos 11+, Pos 5+
Staining of tissue from a diabetic rat model showed CTF was absorped into tissues as rats became
more diabetic over 16 weeks. Staining was graded on scale to 140
The impact of an immune response to CTF was shown using rabbits which did
and did not produce antibodies to CTF. The immune response greatly increased
CTF absorption into the liver (See Table 4.5). Circulating IgG immune complexes
are primarily eliminated by the liver (Datta-Mannan et al. 2007). The serum halflife of IgG is ~330 h and absorption into endothelial cells is regulated by the Fc
receptor (Johansson et al. 1996). Antibodies binding CTF would bring CTF into the
liver. The presence of CTF in liver of diabetic animals is therefore more likely due
to natural degradation of immunoglobulin. No antibodies to CTF were observed in
rat models or human patients.
4.4
Conclusion
This data that supports that CTF could contribute to inhibition of insulin degradation through IDE. The 32-mer CTF peptide was less soluble due to the N- terminal
hydrophilic peptides. The iso-electric point of 4.1, net charge at pH 7.0 of 3 and
average hydrophilicity of 0.4 is favored by the acidic endosome environment.
Therefore it was proposed AdipoR1 CTF344-375 is formed inside the endosome by
TACE cleavage allowing CTF regulation of insulin concentration by inhibiting
insulin degradation by IDE (See Fig. 4.5).
This could prevent IDE from lowering intracellular insulin. Higher insulin would
be expected to decrease insulin receptor sensitivity to bind plasma insulin (See Fig.
4.5). At the same time TACE activity is needed for CTF formation. Competitive
binding of CTF to TACE could reduce free TNF- formation and decrease its
inflammatory effects.
In abnormal patients such as long term diabetics and chronic inflammatory diseases, AdipoR1 CTF formed in tissue and blood is expected to inhibit insulin degradation. The result could be CTF build up in liver, pancreas and other tissue causing
a reduced insulin response. The CTF absorption would be expected to increase as
diabetes progresses. Additionally AdipoR1 CTF is carried on antibodies and carried
into cells to tissue. It is also possible this mechanism is used to alter the immune cell
response in the lymphnodes. Any resultant increase in cellular insulin would cause
reduced insulin receptor response (Kahn et al. 1973).
89
Table 4.5 CTF in tissue by immunuo fluorescent staining

Non producers
N=3
Producers
N=3
CTF in liver
Pos
Strongly pos
Pos 35+, Pos 40+, Pos
30+
CTF in brain
Neg
Pos 1+, Neg, Neg
Pos
Pos 10+, Pos 11+, Pos
8+
CTF in pancreas
Pos
Pos
Pos 8+, Pos 12+, Pos 8+
Staining of tissue from a rabbits with and without producing CTF polyclonal antibodies showed
CTF was absorption into tissues. All rabbits were exposed to CTF antigen for 3 months and some
liver absorption occurred with our immune response. Staining was graded on scale to 40. Example
images shown above, Blue is DAPI stain showing nuclei and green is CTF
Liver
Lymph nodes
Normal
Early
Diabetic
Late
Diabetics
Fig. 4.4 Tissue staining for Adiponectin receptor C terminal fragment (AdipoR CTF) . Absorption
of AdipoR CTF into tissue as with diabetic disease progression is shown. Images are of IHC staining of tissue from diabetic rats which were normal or diabetic for short or long periods of time.
Blue color is DAPI staining showing nuclei and green color is AdipoR CTF from antibody to
AdipoR CTF labeled with green fluorescent dye. Images shown are at 400 magnification. Frames
show CTF was absorption into tissues as rats became more diabetic over 16 weeks
90
M. Pugia and R. Ma
Adiponectin
TNF-
Insulin
Receptor
response
TACE
Pro-TNF-
AdipoR1
IDE
Insulin
Degrading
Enzyme (IDE)
CTF
IDE
Endosome
Insulin
Fig. 4.5 Proposed role of Adiponectin receptor C terminal fragment (AdipoR CTF) in insulin
receptor response. The proposed mechanism of AdipoR CTF formation and function in regulating
insulin resistance is shown. The cleavage of CTF by ADAM17/TACE in the endosome is proposed.
Intercellular CTF would inhibit insulin degradation through insulin degradation enzyme (IDE) and
increase cellular insulin levels. Increased intercellular CTF and insulin are observed and proposed
to reduce insulin receptor response. The inhibition of TACE by interaction with AdipoR CTF could
also inhibit TNF- release. Adiponectin (Adpn) is normally present due to fat cell signaling. Adpn
binds to AdipoR potentially modulating CTF release by TACE with other unknown factors.
Adiponectin secretion is suppressed in diabetes and obesity, as is CTF whereas tissue levels of
CTF increase. Tissues with CTF inhibiting (IDE) would be less responsive to insulin
The CTF compound can be useful for increasing insulin (proliferation). The
assays can be useful for diabetic drug screening, for screening of compounds to
block CTF absorption into tissue (liver, pancreas, brain) and formation (adipose,
muscle and lymph nodes). The IHC and ICC methods are useful for study of CTF
on immune cells (from blood and lymph nodes) and in tissues for study of immune
activation and tissue absorption in disease. A recent study showed a contradictive
result of effect of IDE inhibition by synthetic inhibitors on the oral glucose tolerance and glucose injection (Maianti et al. 2014), which may suggest the inhibition
of IDE activity can result in an complicated insulin desensitizing effect worth further story.
Acknowledgments We would like to acknowledge the work done by Siemens Healthcare
Diagnostic development people including Stoney Jackson, Karen Marfurt and others who helped
with animal tissues harvesting. We are grateful to Dr Carl Marfurt of Indiana University School of
Medicine-South Bend for his help on methods for tissue preparation. We thank Boston Universitys
Zlab for the generation of the TASSER structures for AdipoR1 CTF344-375 and AdipoR2 CTF344-375
91
Supplements
IHC and ICC Cell Staining Method
References
Abdul-Hay SO, Kang D, McBride M, Li L, Zhao J, Leissring MA (2011) Deletion of insulindegrading enzyme elicits antipodal, age-dependent effects on glucose and insulin tolerance.
PLoS One 6(6):e20818
Black RA (2002) Tumor necrosis factor-alpha converting enzyme. Review. Int J Biochem Cell Biol
34(1):15
Bonora E, Zavaroni I, Coscelli C, Butturini U (1983) Decreased hepatic insulin extraction in subjects with mild glucose intolerance. Metabolism 32:438446
Datta-Mannan A et al (2007) Monoclonal antibody clearance impact of modulating the interaction
of IgG with the neonatal Fc receptor. J Biol Chem 282(3):17091717
Duckworth WC, Bennett RG, Hamel FG (1998) Insulin degradation: progress and potential.
Endocr Rev 19(5):608624
Farris W et al (2003) Insulin-degrading enzyme regulates the levels of insulin, amyloid beta -protein, and the beta-amyloid precursor protein intracellular domain in vivo. Proc Natl Acad Sci U
S A 100(7):41624167
Gil-Camposa M, Canetea R, Gilb A (2004) Adiponectin, the missing link in insulin resistance and
obesity. Clin Nutr 23:963974
Golz S, Bruggemeier U, Geerts A (2007) Diagnostic and therapeutic for diseases associated with
GPCR AdipoR2 and R1. US2007/0053913, US2007/0037226
Hotamisligil GS, Budavari A, Murray D, Spiegelman BM (1994) Reduced tyrosine kinase activity
of the insulin receptor in obesity-diabetes. Central role of tumor necrosis factor-alpha. J Clin
Invest 94(4):15431549
Johansson AG et al (1996) Liver cell uptake and degradation of soluble immunoglobulin G immune
complexes in vivo and in vitro in rats. Hepatology 24:169176
Kahn R, Neville DM, Roth J (1973) Insulin-receptor interaction in the obese-hyperglycemic
mouse. A model of insulin resistance. J Biol Chem 248:244250
Kuo WL, Montag AG, Rosner MA (1993) Insulin degrading enzyme is differentially expressed
and developmentally regulated in various rat tissues. Endocrinology 132(2):604611
Leissring MA et al (2010) Designed inhibitors of insulin-degrading enzyme regulate the catabolism and activity of insulin. PLoS One 5(5):e10504
Lorenzo M et al (2008) Insulin resistance induced by tumor necrosis factor-alpha in myocytes and
brown adipocytes. J Anim Sci 86(14):E94E104
Maianti JP et al (2014) Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by
multiple hormones. Nature 511:9498
Olson RE, Marcin LR (2007) Chapter 3. Secretase inhibitors and modulators for the treatment of
Alzheimers disease. In: Macor JE (ed) Annual reports in medicinal chemistry, vol 42. Elsevier
Inc, Wallingford CT, USA, pp 2748. ISSN 978-0-12-373912-4
Ouchi N et al (2000) Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial
NF-kappaB signaling through a cAMP-dependent pathway. Circulation 102(11):12961301
Ramos-Vara JA (2005) Technical aspects of immunohistochemistry. Vet Pathol 42(4):405426
Rawlings ND, Morton FR, Barrett AJ (2006) MEROPS: the peptidase database. Nucleic Acids Res
34:D270D272
Sathananthan A (2012) A concerted decline in insulin secretion and action occurs across the spectrum of fasting and post challenge glucose concentrations. Clin Endocrinol (Oxf)
76(2):212219
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Serino M et al (2007) Mice heterozygous for tumor necrosis factor-alpha converting enzyme are
protected from obesity-induced insulin resistance and diabetes. Diabetes 56(10):25412546
Tamboli IY et al (2010) Statins promote the degradation of extracellular amyloid beta peptide by
microglia via stimulation of exosome-associated insulin-degrading enzyme (IDE) secretion. J
Biol Chem 285(48):3740537414
Tura A, Ludvik B, Nolan JJ, Pacini G, Thomaseth K (2001) Insulin and C-peptide secretion and
kinetics in humans: direct and model based measurements during OGTT. Am J Physiol
Endocrinol Metab 281:E966E974
Weickert MO, Pfeiffer AF (2006) Signaling mechanisms linking hepatic glucose and lipid metabolism. Diabetologia 49:17321741
Chapter 5
Cell and Biological Models for the C Terminal

Fragment of Adiponectin Receptor
Abstract The impact of the peptides such as 32 amino acid peptide AdipoR1
C -terminal fragment (CTF) (AdipoR CTF344-374) can be studied in cell and animal
models provided the conditions are defined for the biological system tested. In this
work, calcium depleted cardiomyocytes (C2C12) were found useful as cell model
for study of Ig-CTF formation and release. This cell model could be a useful tool for
screening of compouns to block formation of CTF. A breast cancer cell model
(SKBR-3) with labeled insulin was found useful for measurement of cellular loading and inhibition of insulin degradation. This cell model could be a useful tool for
screening of compounds to block CTF inhibition of IDE. Leptin resistant rat models
were shown to become hyper-insulinemic when treated with CTF allowing a model
for inducing insulin resistance. The progression of leptin resistant rats to diabetes
was measure-able by their oral glucose tolerance and plasma CTF levels. This animal model supported that AdipoR CTF raises intracellular plasma insulin but not
glucose and is an important factor in diabetic progression. Together these models
allow exploring formation and signaling of new inhibitory plasma peptides and
demonstrate how to explore bioactivity of proteomic discoveries with cell and animal models.
5.1
Introduction
Metabolic syndrome is a collection of conditions including glucose intolerance,

obesity, hypertension, hypertriglyceridemia, and hypercholesterolemia that precede
diabetes mellitus and cardiovascular disease (Shin et al. 2013). The ability of insulin
to signal glucose transport by the glucose transporter (GULT1/4) is reduced.
M. Pugia, PhD (*)
R. Ma
Siemens Healthcare, 278 Zhouzhu Road,
Pu Dong New Area 201318, Peoples Republic of China
DOI 10.1007/978-3-319-21927-1_5
93
94
M. Pugia and R. Ma
Reduced insulin response impairs the mitochondrial metabolism of glucose and cell
survival though the phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT)
and extracellular-signal-regulated kinase (ERK) pathways (Chang et al. 2004;
Dugani and Klip 2006; Copps and White 2012; Siddle 2011).
Exercise increases mitochondrial metabolism of glucose generating fuel-depleted
cells with increased intracellular calcium levels (Ojuka 2004; Hardie 2004). Glucose
transport by GULT1/4 is activated during exercise via the AMP-activated protein
kinase (AMPK) (Jessen and Goodyear 2005). Activation of AMPK also increases
fatty acid oxidation by inactivating acetyl-CoA carboxylase (ACC) and lowering
malonyl-CoA content (Hayashi et al. 2000). Adipokines such as leptin and adiponectin are key insulin-independent activators of AMPK (Carling 2005; Yamauchi
et al. 2002). Lipid loading during obesity causes hyperplastic-hypertrophic fat producing less adipokines and lessening AMPK activation.
Cardiomyocytes have become important cellular models for the study of AMPK
activation (An 2006). Cellular growth rates and kinase activation are carefully monitored while the cultures are exposed to various stimulates. Adiponectin activates
cardiomyocyte AMPK, but inhibits cell proliferation by suppressing extracellularsignal-regulated kinase (ERK) activation and inducing apoptosis (Wang et al. 2005;
Shibata et al. 2004; Brkenhielm et al. 2004).
Metabolic syndrome can also be studied by animal models that develop disease
prematurally in life (de Artiano and Miguel Castro 2009). This allows rapid assessments of new markers, pathways and treatments. The degree of metabolic syndrome
worsens through the life of animal with increased hyperglycemia, hyperinsulinemia,
weight gain and glucose intolerance (Schmidt 2003). Rats resistant to leptin are one
of the most widely used metabolic syndrome model. Breeds such as ZDF, Zucker,
BB/ZDR, JCR, SHHF, SHROB and ZSF were created by classical breeding methods and all shown to carry a defective leptin receptor with a gene mutation in fa
allele (Clark et al. 1983; Kava et al. 1990; Tirabassi et al. 2004).
Leptin resistance animals become obese and spontaneous develop diabetes, metabolic syndrome, nephropathy, neuropathy, hepatic steatosis and congestive heart
failure at an age of 3 months (An 2006; Schmidt 2003; Rodrigues 2006). Leptin is
synthesized in adipose cells inducing satiety and weight loss by acting on leptin
receptors in the hypothalamus and central nervous system (Gorden and Gavrilovay
2003). Leptin replacement improves hyperglycemia, hyper-insulinemia, and triglyceride levels reducing hyperplastic-hypertrophic fatty in adipocytes.
Leptin resistance rat models have been used to show the benefits of many new
treatments pathways. The value of incretines, a major pharmacological agent for
diabetic care, was first shown in the ZDF rat model (Doyle and Egan 2007).
Incretines improved glucose tolerance through GLP-1 receptor signaling mediated
by increased cell proliferation and decreased apoptosis allowing islet regeneration
and include gastric inhibitory peptide (GIP), glucagon-like peptide-I (GLP I) and
pituitary adenyl-cyclase-activating peptide. Dipeptidyl peptidase IV (DPPIV)
inhibitors preventing incretine degradation have similarly been studied. Nesfatin for
appetite control (Shimizu et al. 2009), antagonist of the cannabinoid CB1 receptor
(Pavn et al. 2008), and the impact of free acid amide hydrolase FAAH activity
(Cable et al. 2014) have been demonstration in the ZDF rat model.
Cell and Biological Models for the C Terminal Fragment of Adiponectin Receptor
95
The goal of any new metabolic syndrome treatment is the improvement of energy
metabolism resulting in increased glucose uptake, glycolysis, pyruvate oxidation
and fatty acid consumption. Our goal was to investigate the impact of the AdipoR1
C -terminal fragment (CTF) to determine potential benefits. Therefore we explored
the impact of CTF in cardiomyocytes and cancer cell lines by measuring formation
of Ig-CTF, intracellular calcium and insulin, and cellular proliferation. We also measured the impact of CTF on plasma insulin and glucose tolerance in a leptin resistant
rat model and measured the plasma CTF with progression to a diabetic state.
5.2
5.2.1
Methods
Cell Culture Methods and Bioassays
The materials and bioassay used for AdipoR CTF were as described in previous
chapter. Globular Adpn (16.8 kDa) was obtained from Alpco Diagnostics (Salem
NH) and confirmed to be 1418 kDa by western blot. Full length Adpn (25.4 kDa)
was purchases a human recombinant protein from BioVendor, LLC (Candler, NC)
and R&D Systems (Minneapolis MN) and confirmed to be 2636 kDa by western
blot. As a result of glycosylation, the recombinant protein can migrate as high as
approximately 36 kDa band in SDS-PAGE under reducing conditions. The low,
middle and high molecular weight oligomeric forms of Adpn activate different
signaling pathways through the full length and globular peptide domain. Both
Adpn forms were tested. Bikunin was obtained from Scipac Ltd (Sittingbourne,
Kent UK).
5.2.2
Cell Lines
Cell culture mouse myocytes (C2C12 CRL1772) and human cancer cells (SKBR-3)
(American Type Cell Culture, ATCC, Manassa VA) were maintained in culture.
IMDM + 10 % FBS growth media was used for SKBR-3, while C2C12 were maintained with Dulbeccor Modified Eagles medium (MEM), glutamine and 10 % fetal
bovine serum. Serum contains native insulin and adiponectin. Cells were grown at
37 C in an atmosphere of 95 % air and 5 % CO2 and propagated by splitting with
one to four dilutions using PBS/EDTA at cell growth.
5.2.3
Cell Culture Treatment
Myocytes are then grown in Modified Eagle Medium (MEM) supplemented with
2 mM glutamine in addition to penicillin (50 units/ml), streptomycin (50 g/ml) and
10 % fetal bovine serum in an atmosphere of 95 % air and 5 % CO2. The cells were
96
M. Pugia and R. Ma
propagated by sub-culturing at their confluence (45 days) in one to four dilutions

using either trypsin or PBS/EDTA. Cell confluence was around five million cells per
T-75 cm2 flask. Semi-confluent cell cultures were synchronized twice using 0.5 mM
hydroxyurea (sterile) in MEM, containing 2 mM glutamine without serum, for two
24 h period. After synchronization the hydroxyurea was removed, cells were harvested asceptically using trypin-EDTA, counted, and replenished with serum free
MEM at 0.3 106 cells/ml into either multiple T-25 cm2 flasks or by adding 100 L
to separate micro-titre-plate wells.
The cells are grown for another 24 h serum free MEM followed by induction with
various concentrations of Bik (0, 20, 40 g/mL), Adpn (0, 20, 40 g/L), CTF 25 mer
(0, 20, 40 g/L) and combination in serum free MEM under sterile condition. The
extracellular Ca2+ concentration is raised to 0.5 mM with calcium chloride and the cells
were allowed to grow additional 24 h before harvesting using PBS/EDTA. The cells
were harvested with trypsin, and centrifuged for 5 min @ 2000 rpm followed by
decanting supernanant and adding small amount (~200 L) media. The cells are suspended in 1:3 ratio with PBS after saving an aliquot for cell counts and later analysis.
5.2.4
ELISA and Western Blot Analysis
ELISA for Adpn was performed using monoclonal antibody (mAb) directed against
the globular domain in kits from B-Bridge International Inc (Mountain View, CA).
CTF and CTF-Ig were measured as previously described. Analysis for p38 MAPK
was performed using rabbit monoclonal antibodies from Cell Signaling Technology
Inc (Danvers MA) by western blot method. Measurements of the relative increase in
ERK phosphoralyation were preformed according to the ELISA manufacturers
instruction (Assay Designs, Ann Arbor MI).
5.2.5
Intra-cellular Calcium Analysis
Measurements of cytosolic calcium were done by infusing cells with the membrane
permeable calcium sensitive Fluo-4 AM dye (Molecular Probes, Eugene, OR).
Packed viable cells (106 cell/L) in 100 L PBS were treated with 5 L of fluo-4,
AM dye solution. The dye solution was made by dissolving 50 g of Fluo-4 AM
into 100 L of a 20 % solution Pluronic F-127 in DMSO. Cells were incubated for
30 min at 37 C to allow permeabilization of the dye. Cells were centrifuged for
10 min at 7000 rpm (12,000 g) and pellets resuspended in 100 L PBS to wash out
the extra-cellular fluorescent dye. Intracellular proteases hydrolyzed Fluo-4 AM
which caused fluorescence in the presence of calcium. A 5 L sample was analyzed
on a glass slide with an attached covers using fluorescent microscopics.
Fluorescent images were obtained at room temperature using a Leica DM5000
confocal microscope with a 63 1.2 NA objective in a darken room. Phase contrast
97
images were used to locate regions of intracellular space and measure fluorescent
signals. The Fluo-4, excitation and emission wavelengths were 488 nm and 510
590 nm, respectively. Fluorescent images were collected with a frequency of 0.6
1.0 frame/s for 5 min. Calibration was done by measuring the fluorescent after
mixing 5 L fluo-4 dye solution, 5 L of 1 N NaOH with 100 L calcium calibration
solution at 0 or 100 nM calcium chloride.
5.2.6
Intracellular Insulin Measurements
Human cancer cells (SKBR-3) were grown to confluency. A CTF 25 mer 1 mg/mL
stock solution in DMSO was used to make 0.5 mg/mL in 1 % DMSO/media while
0.1 mg/mL insulin-FITC (Sigma Aldrich) in media was made by diluting 1 mg/mL
stock with ten-fold with cell growth media. Media was filtered through 0.22
syringe filter unit after addition of CTF or insulin-FITC. Cells were treated with the
fresh media. SKBR-3 cells were treated with different concentrations of insulinFITC (0, 1, 10 g/mL), CTF 25 mer (0, 50 g/L) and with the combination added
under sterile condition. The cells were incubated for ~2 days @37C, 5 % CO2 mix
cells in microtiter plate shaker on low speed. Cultures were split into triplicates by
placing 0.4 mL into separate wells of a Lab-Tek II 4-well chamber slide (Sigma
Aldrich). All slides were measured at 1, 2, 3 days for uptake of insulin-FITC after
washing with 2 1 mL PBS. Fluorescent images were obtained at room temperature
using a Leica DM5000 and the L5 filter set for FITC (excitation 494 nm/emission
518 nm). The slides are scanned at 40 using 50300 ms exposures.
5.2.7
Cell Proliferation Counts
A 10 l cell suspension was added to 80 ul PBS and 10 ul Trypan blue (1 %) and 4

squares counted on the slide in hemacytometer (area: 0.1 cm 0.1 cm 0.01 cm).
Count all nine chambers and repeat the count with second dilution in trypan blue
calculation: total # cells in 9 chambers 4 10,000/9 = # cells per mL, Typically
cells were grown to 5.5 105 cells/mL.
5.2.8
Animal Models
Animal model studies were performed at PreClinOmics, Inc. (Indianapolis, IN).

Normal and diabetic rats were injected with CTF as part of a glucose tolerance challenge. The concentrations of glucose, insulin, Adpn, and free CTF were measured predose and at 30, 60, 90, and 120 min after injection. Normal Hsd:SD rats were divided
into two groups (n = 7 as controls and n = 8 for CTF vehicle). Diabetic ZDF rats (Charles
98
M. Pugia and R. Ma
River Laboratories) (n = 12) were also divided into two groups (n = 6 for each control or
CTF vehicle). The disease rats were at 438593 g and on diabetic diet initiated at
2526 weeks age. The animals were fasted for 16 h overnight before injection with
CTF and glucose. Injection vehicle was prepared by weighing 210 mg of AdipoR1
CTF341-370 (30 mer) or AdipoR1 CTF344-375 (32 mer) as compound into 5 ml sterile saline with 0.4 g/ml (2 g/kg) glucose with 1 % albumin (Sigma, Lot #: 067K0136).
5.2.9
Disease Progression Study
Groups of non-diabetic (n = 6), short term diabetic (n = 6), and long term diabetic (n = 6)
were determined by glucose tolerance. Rats (ZDSD) used were at 438593 g and on
diabetic diet imitated at 2526 weeks age. At time of study animals were dosed with 2 g/
kg glucose (Sigma, Lot #: 067K0136), p.o. following a 16 h overnight fast. Whole blood
was collected from the tail vein at pre-dose, 60 and 120 min following glucose challenge. The glucose tolerance curve in the OGTT varied depending on the age, strain,
degree of diabetes and other test conditions. The glucose typically peaks at 30 min at
175200 mg/dl in a normal rat. Insulin will typically peak somewhat later at several
times the baseline level. Observation of the test subject occurred once daily during the
accumulation and study period. Body weight was measured on Day 0 for sorting and on
Day 1 to determine dose volumes. Food consumption was not measured.
5.2.10
Biomarker Measurements
Whole blood was collected from the tail vein at 0, 30, 60, 90, and 120 min (150 L
for each time point into Li-Hep) following glucose challenge. Whole blood glucose
was measured by Beckman CX4. HbA1c was analyzed on an AU480 clinical chemistry analyzer. Insulin was analyzed on serum samples by MSD (K112BZC) or
ELISA (Alpco, Salem, NH). A sample of 50 L of whole blood was collected and
immediately plated into a 15 ml conical tube and diluted with 3.6 mL TBS and frozen at 20 C until measurement of free CTF by ELISA (See previous section). An
additional 100 L of whole blood was collected for the measurements of adiponectin (Adpn) levels were measured with a rat ELISA kit from Alpco (Salem, NH).
5.3
5.3.1
Results
Cardiomyocyte Models
Cardiomyocytes (C2C12) produced Ig-CTF when grown in culture with fetal bovine
serum containing immunoglobulins. Western blot analysis detected IgM-CTF,
Fab2-CTF, Ig-CTF and AdipoR1 in cell membranes lysates (See Table 4.3 of Chap. 4
99
for western blots data). Ig-CTF remained membrane bound. Cardiomyocytes did
not release any detectable free CTF or Ig-CTF into culture media measured by
ELISA.
The causes or inhibition of Ig CTF formation and release was examined by
inducing cardiomyocytes culture media with additional biochemical factors. Adpn
was present in fetal bovine serum culture media but was not observed in washed cell
membranes. Treating cell cultures with additional Adpn did drive receptor binding
and Adpn was observed in washed cells membranes. This additional Adpn did not
increase or inhibit Ig-CTF formation or cause CTF release from the membrane.
Binding studies showed neither full-length nor globular Adpn bound directly to the
CTF of the Adiponectin receptor.
Intracellular calcium (Ca2+) was also measured on cardiomyocytes using a Ca2+
sensitive dye (Fluo-4 AM) after exposure of viable cells to media containing Ca2+
and washing to remove extra-cellular and unabsorbed Ca2+ (See Table 5.1). Green
fluorescence from Fluo-4 AM was observed inside the cell indicating the presence
of calcium and membrane permeation. Microscopic analysis of the phase contrast
images and fluorescence images was used to determine the amount and intensity of
fluorescence inside the intracellular area of the cell in the range of 0.5
100 mM. Calibration solutions were used to quantitate the intracellular Ca2+ concentrations. Cells were treated with CTF and Apdn to determine impact.
Cardiomyocytes (C2C12) cultures typically maintain intra-cellular Ca2+ in an
8090 nM constant range. Intra-cellular Ca2+ decreased to >5 nM with when cells
were exposed to 20 g/mL bikunin (Bik). The addition of bikunin (Bik) served as a
means to strip cells of intra-cellular calcium and allowing means to diminished Ca2+
to zero base line for measuring increases in viable cells (See Chap. 12 for further
information). Cells lysates showed Bik was maintained on cell surfaces during this
treatment. Cells were further treated with Adpn or CTF 25 mer in presence and
absence of Bik.
The addition of Adpn to Ca2+ depleted cell to restored intra-cellular Ca2+ to 91
nM (Table 5.1). Adpn binding of AdipoR signals activation of AMPK which regenerates the ATP major pathway and promotes the cytosolic localization of liver
kinase B and a minor pathway (the phospholipase Ca2+/calmodulin-dependent protein kinase kinase-dependent pathway) that stimulates Ca2+ release from intracellular stores restoring cytosolic calcium (Jessen and Goodyear 2005; Zhou et al.
2009). Thus Ca2+ restoration is a good measure of Adpn activation of AMPK. The
addition of AdipoR1357-375 CTF had no impact on intra-cellular Ca2+ whether cells
were depleted by Bik or loaded with calcium (See Table 5.1). The Adpn restoration
is useful tool to determine that receptor function is not impaired during CTF formation and release.
Over all, the mechanism of Ig-CTF formation and release remains unclear.
However calcium depleted cardiomyocytes cell model do appear useful for study of
enzymes and proteins required for Ig-CTF formation and release. As a pathway tool,
cardiomyocytes could be grown with enzyme inhibitors and blockers in serum free
media to examine factors required for Ig-CTF formation and release. Cardiomyocytes
also allow monitoring Adpn activation of AMBK via normal adiponectin receptor
function.
100
M. Pugia and R. Ma
Table 5.1 Intra-cellular signals after exposure to CTF and Adpn

Protein and Exposure level (g/
mL)a
Bik
0
0
0
20
0
0
0
0
20
20
20
Adpn
0
20
40
0
0
0
20
40
20
0
20
AdipoR1357-375
0
0
0
0
20
40
20
40
20
20
0
Intra-cellular signals
Calcium
MAPK p38c (%
(nM)b
change)
93
8%
82
14 %
Not measured 10
4
17 %
84
0%
Not measured 13 %
Not measured 19 %
Not measured 14 %
72
nm
9
13 %
91
10 %
MAPK ERKc (%
change)
0%
7%
33 %
5 %
5%
6%
7%
38 %
nm
6%
14 %
a
Synchronized cultures of myocyte (C2C12) were incubated 5 h with Bikunin (Bik), Adiponectin
(ADPN) and/or AdipoR1357-375 at 0, 20, or 40 g/mL. Cells were harvested for western blots and
whole cell used for intra-cellular calcium measurement
b
Intra-cellular calcium was measured by incubating with cell the membrane permeable calcium
sensitive Fluo-4 AM for 30 min at 36 C to allow intracellular hydrolysis to cause fluorescence in
the presence of calcium. Cells were washed and centrifuged to remove extra-cellular dye and fluorescent images captured
c
Measurements of the relative change in phosphorylation of MAPK ERK by ELISA and MAPK
p38 by Western Blot were measured at 0, 6, 12 and 24 h after cells were revived in media containing serum
5.3.2
Cellular Insulin Uptake
Breast cancer cells (SKBR-3) did not contain any detectable CTF or AdipoR1 but
did contain IDE and TACE (See on-line supplement for cell and tissue staining
procedure at www.extras.springer.com/2015/978-3-319-21926-4). As CTF is absent
in the cell but the target downstream protease IDE are present, SKBR-3 makes a
good model for measuring the impact of CTF on intra-cellular insulin. Treatment of
SKBR-3 with CTF increases the amount of intercellular insulin (See Fig. 5.1). The
number of insulin loaded cells double when exposed to 10 ug/mL of CTF. The insulin loaded cells clearly showed intercellular fluorescence from the labeled insulin.
The insulin loading cell model allows screening of compound to block CTF inhibition of IDE.
Cells treated with CTF maintain viability (proliferation) with no observable cell
death (apoptosis) (See Fig. 5.1). Cardiomyocytes exposed to CTF also did not modulate phosphorylation of ERK and MAPK-p38 or slow proliferation (See Table 5.1). No
observable cell death was noted. As insulin signaling of the PI3K/AKT/mTOR pathway increases proliferation and decreases apoptosis, insulin loading of cells would be
expected to have reduce insulin receptor response and proliferation. However no
Effect of CTF on Cell Viability
Effect of CTF on Insulin Uptake

120
100
100
80
cell viablility (%)
insulin uptake (# cells)
101
80
60
40
60
40
20
20
0
0
1 ug/mL
10 ug/mL
insulin,
day 1
insulin,
day 1
10 ug/mL
insulin,
day 2
1ug/mL insulin
(no CTF)
10ug/mL insulin
(CTF)
Control (no CTF)

+0.5mg/ml CTF
Fig. 5.1 Effect of CTF-25 mer on insulin uptake and cell viability in SKBR3 cell model. Breast
cancer cells (SKBR) did not contain any detectable CTF or AdipoR1 but did contain IDE and
TACE (by ICC method). This makes SKBR a good model for measuring the impact of CTF on
cellular insulin degradation. Treatment of SKBR with CTF doubled the cellular labeled insulin.
Cells treated with CTF maintain viability (proliferation) with no observable cell death (apoptosis).
Insulin loading was demonstrated by fluorescence inside cells due to label on insulin
impact to proliferation observed. It appears that CTF does not inhibit PI3K/AKT/
mTOR intercellular signaling. This could be due to the excess of insulin used in cell
culture media. An insulin straved model was not done. This proliferation effect contrasts the apoptotic impact of Adpn, which did suppress phosphorylation of ERK in
cardiomyocytes (See Table 5.1) and restored intra-cellular Ca2+. Restored intra-cellular
Ca2 would inhibit calcium dependent PI3K pathway and desensitize insulin response.
5.3.3
Impact on In-Vivo Insulin
Normal and diabetic rats were injected with CTF as part of a glucose tolerance challenge (See Tables 5.25.4 and Fig. 5.2). Normal and diabetic rats showed different
behaviors. In normal rat showed substantial plasma insulin increases compared to
control (See top two graphs of Fig. 5.2). Glucose was not reduced and insulin levels
in plasma doubled in normal rats. This behavior matched an insulin resistance
behavior. Diabetic rats which already had with impair insulin production (<10 %
plasma insulin levels of normals) did not exhibit an increase in plasma insulin upon
Animal #
Minutes after dose
ZDSD rat 1
ZDSD rat 2
ZDSD rat 3
ZDSD rat 4
ZDSD rat 5
ZDSD rat 6
week
0
0
0
0
0
0
Animal ID
815004001
815004002
815004003
815004004
815004005
815004006
Mean
St. Dev.
SEM
Body weight (g)

544
428
544
531
483
552
513.67
48.76
19.91
HbA1c %
5.25
4.43
4.74
5.33
5.17
5.50
5.07
0.40
0.16
Glucose (mg/dl)
pre
60
105.0 263.0
101.0 246.0
110.0 236.0
105.0 257.0
99.0
304.0
108.0 261.0
104.7 261.2
4.1
23.3
1.7
9.5
120
164.0
172.0
180.0
234.0
250.0
176.0
196.0
36.4
14.9
Table 5.2 Glucose, insulin, HbA1c and CTF response in normal and diabetic rats: non diabetic rat group
Insulin (pg/mL)
pre
60
595.6 1086
575.4 874.5
868.4 1307
490.6 770.1
595.1 808.4
559.9 791.9
614.2 939.5
130.5 213.6
53.3
87.2
120
984.4
963.4
1167
1093
876.4
1222.
1051.
132.1
53.9
CTF (pg/mL)
pre
60
1.3
113.8
41.8
261.7
67.0
319.7
78.0
350.5
111.8 184.0
28.5
182.0
54.7
235.3
39.1
90.9
16.0
37.1
120
285.7
110.0
181.3
139.8
74.8
135.5
154.5
73.3
29.9
102
M. Pugia and R. Ma
AdipoR1 CTF344-375 32 mer
(2mg/kg)
103
AdipoR1 CTF341-370 30 mer

(2mg/kg)
Glucose
Insulin
0%
0
100%
Glucose
Insulin
0%
20
40
60
80
Minutes
100
120
100%
20
40
60
Minutes
80
100
120
100%
100% of Control
Diabetic rats
100% of Control
100%
200%
Glucose
Insulin
100% of Control
Normal rats
100% of Control
200%
Glucose
Insulin
0%
0%
0
20
40
60
80
Minutes
100
120
20
40
60
80
Minutes
100
120
Fig. 5.2 Glucose and insulin response in disease. A study was conducted by dosing animal ZDF
diabetic rats and HD normal rats with Adiponectin Receptor C- Terminal fragment (CTF 32 mer
and CTF 30 mer). Normal and diabetic rats were injected +/ CTF just prior to a glucose tolerance
challenge. The concentrations of glucose, insulin, Adpn, and free CTF were measured pre-dose
and at 30, 60, 90, and 120 min after injection. Adpn did not significant change with time or diabetes. CTF was significantly lower in diabetic rats (63.7 3.3 ng/mL) than in normal rats (98.8 8.1 ng/
mL) and rose 2035 ng/mL over 120 min with CTF injection. Substantial insulin increases
occurred for normal rats (top two graphs) but glucose was not reduced and insulin resistance
behavior occurred. Diabetic rats only produced <10 % of insulin of normal rat and did not show
substantial insulin increases when dosed with CTF
CTF exposure (See bottom two graphs of Fig. 5.2). It was further shown that diabetic rats post treatment had CTF loading in liver, pancreas, brain and lymph nodes
where as normal rats did not (See Chap. 4).
The levels of CTF in the animals were monitored during this study. Plasma free
CTF was significantly (P < 0.003) lower in diabetic rats (63.7 3.3 ng/mL) than in
normal rats (98.8 8.1 ng/mL) prior to the glucose tolerance challenge. In contrast, no
difference in plasma adiponectin was detected in normal (24.1 3.7 g/mL) or diabetic rats (20.9 3.9 g/mL) prior to the glucose tolerance challenge. Once the animal
was injected with CTF, the plasma CTF levels rose 2035 ng/mL over 120 min. This
level of CTF was limited by the insolubility of the CTF peptides used. Unfortunately,
neither CTF 30 mer or 32 mer compounds tested in this study were soluble forms.
Comparison to a more soluble CTF 25 mer form is expected to allow higher plasma
CTF levels cause additional insulin loading in tissue and plasma. High activity of CTF
25 mer could also significantly increase IDE inhibition response.
104
5.3.4
M. Pugia and R. Ma
Disease Progression Study
A study was conducted with ZDF rats on a high fat diet separated into groups of
non-diabetic and rats that progressed to diabetes (See Tables 5.25.4). Groups of
rats that were diabetic for a short term where further separated from those who were
diabetic for a longer term. Blood glucose, insulin, HbA1c and free CTF were measure before and during a glucose tolerance test. Blood glucose and HbA1c values
increased as rats became diabetic (See Tables 5.3 and 5.4). Insulin secretion
decreased in longer term diabetes which also showed an impaired glucose
tolerance.
An increase in free CTF in blood was observed in early and late stage diabetic
rats. Normal rats had near zero values of free CTF until glucose challenge. Free
CTF in plasma increased during the glucose challenge, peaking at 60 min and still
elevated 120 min. Analytical validation showed the free CTF ELISA was suit-able
for animal models but not human clinical study. The Ig-CTF ELISA assay was
usable for human clinical but not the rat studies. In human studies, Ig-CTF was
unchanged during OGTT of 120 min (See Chap. 6). This suggested Ig-CTF is more
of a chronic marker while free CTF is more responsive in short term (acute marker).
In agreement tissue staining of long term rats from this study showed significant
increases in Ig-CTF in the liver, brains, pancreas and lymph nodes when compared
to normal and short term diabetics.
5.3.5
Suggested Mechanism of Action
Based on data on hand, it appears that CTF-IgG forms in the muscle by IgG coming into proximity of CTF during release from AdipoR1 by TACE (See Fig. 5.3).
While all the contributors to this mechanism are not known, it is likely that a reactive thioester of CTF is protected until the release is triggered. Here, adiponectin
could provide blocking role preventing insulin receptor desensitization by CTF,
however inhibition of formation of CTF-IgG has not been demonstrated to date.
It is clear that CTF is carried on antibodies and cells and transported into tissue
more so in disease (See Chap. 4). The attraction of Ig-CTF to certain tissue is
unknown. One possibility is this mechanism is used to stimulate immune cell
response to certain antigens. Another possibility is that absorption is part of
immunoglobulin clearance and inflammatory response. However in any case,
CTF Accumulation in tissue correlates with the progress of diabetes conditions.
As tissues absorb more Ig-CTF they would be expected to inhibit insulin degradation by IDE. The insulin inside the cell increases and which decreases the insulin
receptor response. The result is lower insulin receptor response and higher levels
of plasma insulin must be secreted to stimulate the insulin receptor in response to
glucose.
Animal #
Minutes after
dose
ZDSD rat 1
ZDSD rat 2
ZDSD rat 3
ZDSD rat 4
ZDSD rat 5
ZDSD rat 6
2
1
1
1
2
2
Weeks
Animal ID
815004013
815004014
815004015
815004016
815004017
815004018
Mean
St. Dev.
SEM
Body
weight (g)
534
536
569
550
517
497
533.83
25.07
10.24
HbA1c
%
9.64
7.77
6.98
7.81
8.29
9.26
7.46
2.79
1.14
pre
202.0
119.0
122.0
100.0
98.0
165.0
134.3
41.0
16.7
60
517.0
461.0
400.0
409.0
433.0
531.0
458.5
55.1
22.5
Glucose (mg/dl)
120
522.0
340.0
224.0
280.0
322.0
398.0
347.7
103.5
42.2
pre
345.3
879.7
466.1
545.3
852.2
558.8
607.9
214.0
87.4
60
370.2
1018
786.2
693.1
865.0
428.8
693.6
252.2
103.0
Insulin (pg/mL)
Table 5.3 Glucose, insulin, HbA1c and CTF response in normal and diabetic rats: short term diabetic rat group
120
254.3
835.7
398.3
663.6
682.3
427.9
543.7
217.8
88.9
pre
173.5
248.5
302.5
83.0
70.0
124.5
167.0
93.1
38.0
60
186.8
266.5
312.3
203.3
154.0
249.3
228.7
58.0
23.7
CTF (pg/mL)
120
437.0
431.5
340.0
111.0
363.0
195.0
312.9
132.2
54.0
5
105
Animal #
Minutes after
dose
ZDSD rat 1
ZDSD rat 2
ZDSD rat 3
ZDSD rat 4
ZDSD rat 5
ZDSD rat 6
8
9
9
9
9
7
Weeks
Animal ID
815004007
815004008
815004009
815004010
815004011
815004012
Mean
St. Dev.
SEM
Body
weight (g)
468
394
413
394
440
472
430.17
35.18
14.36
HbA1c
%
10.3
12.3
11.9
11.9
11.3
10.3
11.4
0.86
0.35
pre
306.0
512.0
471.0
431.0
249.0
234.0
367.2
119.4
48.7
60
610.0
678.0
672.0
668.0
607.0
566.0
633.5
45.7
18.7
Glucose (mg/dl)
120
507.0
534.0
583.0
581.0
499.0
525.0
538.2
36.2
14.8
pre
138.6
230.6
256.1
137.5
234.7
314.1
218.6
69.2
28.2
60
170.6
192.7
212.9
112.9
211.1
304.5
200.8
62.8
25.6
Insulin (pg/mL)
Table 5.4 Glucose, insulin, HbA1c and CTF response in normal and diabetic rats: long term diabetic rat group
120
196.0
140.8
137.7
105.0
226.5
299.5
184.2
71.5
29.2
pre
143.3
96.0
60.5
208.8
69.5
154.0
122.0
56.9
23.2
60
280.5
201.3
180.3
377.0
373.0
343.8
292.6
86.4
35.3
CTF (pg/mL)
120
81.8
227.0
79.5
519.3
290.0
155.5
225.5
165.7
67.7
106
M. Pugia and R. Ma
IgG
(blood)
Cell
bound
Ig-CTF
CTF transport
into
cell/tissue
107
Impact:
Cell
Growth
WBC
WBC
binding
Insulin
receptor
AdipoR
IDE
Circulating
Ig-CTF
Glucose
consumption
Proteolysis
Insulin
Antibody
binding
CTF-IgG
released from muscle
from AdipoR via TACE
Protein
Synthesis
Ig-CTF absorbed
into, liver, brain
pancreas
Key:
CTF
Antigen
IDE
IDE
Insulin
Fig. 5.3 Formation of Ig-CTF and impact on insulin response in disease. There are antibodies in
the human body with CTF attached. This attachment appears to occur in myoctyes with TACE
activity. As tissues absorb more Ig-CTF, they inhibit insulin degradation by IDE. The insulin inside
the cell increases and which decreases the insulin receptor response. The result is higher levels of
plasma insulin must be secreted to stimulate the insulin receptor. Insulin secretion is typically
determined by the level of blood sugar. This mechanism could cause a localized insulin resistance
5.4
Conclusion
The results of both cellular and animal studies provided new insights into the role of
AdipoR1 C -terminal fragment (CTF). Plasma levels of free CTF decrease with disease
progression from non-diabetic to diabetic states. As animal became more diabetic,
CTF is cleared from the plasma and absorbed into tissue inhibiting insulin degradation.
The result is CTF build up in liver, pancreas, brain, lymph nodes and other tissues leading to a reduced insulin response. Treatment with CTF can be used to cause insulin
levels to increase in animal models and human cells. Free CTF (acute marker) increases
with glucose over 120 min in a OGTT while Ig-CTF was unchanged (chronic marker)
The leptin resistant rats and cell models described allow determining new aspects
of this pathway which could stall the negative impacts of CTF during diabetes.
Screening of compound to block CTF inhibition of IDE or loading of with in tissues
with CTF appears possible through the animal and cell models described.
Acknowledgements We would like to acknowledge the animal work done by PreClinOmics Inc
(Indianapolis, IN) including Dr. Richard Peterson, Dr. WK Yeh, Troy Gobbett and others who
conducted animals trials, samples and performed on-site testing. We would like to acknowledge
the work done inside Bayer Healthcare Diagnostic on cell models by Dr. Manju Basu, Karen
Marfurt and Ronald Sommer.
108
M. Pugia and R. Ma
Supplements
IHC and ICC Cell Staining Methods
References
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Carling D (2005) AMP-activated protein kinase: balancing the scales. Biochimie 95:8791
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Clark J, Palmer CJ, Shaw WN (1983) The diabetic Zucker fatty rat. Proc Soc Exp Biol Med
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Copps KD, White MF (2012) Regulation of insulin sensitivity by serine/threonine phosphorylation
of insulin receptor substrate proteins IRS1 and IRS2. Diabetologia 55(10):25652582
de Artiano A, Miguel Castro M (2009) Experimental rat models to study the metabolic syndrome.
Br J Nutr 102(9):12461253
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Gorden P, Gavrilovay O (2003) The clinical uses of leptin. Curr Opin Pharmacol 3:655659
Hardie DG (2004) The AMP-activated protein kinase pathway new players upstream and downstream. J Cell Sci 117:54795487
Hayashi T et al (2000) metabolic stress and altered glucose transport activation of AMP-activated
protein kinase as a unifying coupling mechanism. Diabetes 49:15
Jessen N, Goodyear LJ (2005) Contraction signaling to glucose transport in skeletal muscle. J Appl
Physiol 99:330337
Kava R, Greenwood MRC, Johnson PR (1990) Zucker (fa/fa) rat. ILAR News 32(3):48
Ojuka EO (2004) Role of calcium and AMP kinase in the regulation of mitochondrial biogenesis
and GLUT4 levels in muscle. Proc Nutr Soc 63:275278
Pavn FJ, Serrano A, Prez-Valero V, Jagerovic N, Hernndez-Folgado L, Bermdez-Silva FJ,
Macas M, Goya P, de Fonseca FR (2008) Central versus peripheral antagonism of cannabinoid
CB1 receptor in obesity: effects of LH-21, a peripherally acting neutral cannabinoid receptor
antagonist, in Zucker rats. J Neuroendocrinol 20 Suppl 1:116123
Schmidt RE (2003) Analysis of the zucker diabetic fatty (ZDF) type 2 diabetic rat model suggests
a neurotrophic role for insulin/IGF-1 in diabetic autonomic neuropathy. Am J Pathol 163:
2128
Shibata R et al (2004) Adiponectin-mediated modulation of hypertrophic signals in the heart. Nat
Med 10(12):13841389
Shimizu H, Oh-I S, Okada S, Mori M (2009) Nesfatin-1: an overview and future clinical application. Endocr J 56(4):537543
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Shin JA et al (2013) Metabolic syndrome as a predictor of type 2 diabetes, and its clinical interpretations and usefulness. J Diabetes Investig 4(4):334343
Siddle K (2011) Signaling by insulin and IGF receptors: supporting acts and new players. J Mole
Endocrinol 47:R1R10
Tirabassi RS et al (2004) The BBZDR/Wor rat model for investigating the complications of type 2
diabetes mellitus. ILAR J 45(3):292302
Wang Y et al (2005) Adiponectin Inhibits cell proliferation by interacting with several growth factors in an oligomerization-dependent manner. J Biol Chem 280(18):1834118347
Yamauchi T et al (2002) Adiponectin stimulates glucose utilization and fatty-acid oxidation by
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Chapter 6
C-Terminal Fragment of Adiponectin

Receptor Clinical Correlations
Adrian Vella and Michael Pugia
Abstract The Ig-CTF immunoassay was tested in serum, plasma and whole
blood samples for healthy volunteers with normal glucose (n = 126), gestational
pre-diabetics (n = 48), long term diabetics (n = 63) and in a population undergoing
pre-diabetic assessment (n = 240). Patients with inflammation (n = 13), using
immunosuppressants (n = 20), anti TNF alpha therapies (n = 8) or with breast cancer (n = 5), liver disease (n = 23), or pancreatic cancer (n = 10) were also assessed.
The values for Ig-CTF were compared to Impaired fasting glucose (IFG) and
impaired glucose tolerance (IGT) detected by OGTT results and HbA1c test result.
The circulating Ig-CTF values are suppressed in diabetics and inflammation.
Patients using anti- TNF therapies (Remicade Infliximab) exhibited suppressed
Ig-CTF. The Ig-CTF values did not change following glucose administration or
over 45 days without significant weight changes and the within patient variability
was <10 %. The predictive value of Ig-CTF for an advanced diabetic disease to
healthy control diabetes was a 92 % sensitivity and 93 % specificity. Ig-CTF was
unable to resolve early diabetes from normals Ig-CTF did not meet any predictive
value criteria. Only 10 % of earth diabetics and pre-diabetics had abnormally low
Ig-CTF values.
A. Vella, MD (*)
Division of Endocrinology and Metabolism, Mayo Clinic,
200 First ST SW, Rochester, MN 55905, USA
M. Pugia
DOI 10.1007/978-3-319-21927-1_6
111
112
6.1
A. Vella and M. Pugia
Introduction
Diabetes is considered a disease that progresses through overlapping groups of disease phenotypes and pathologies (Brooks-Worrell and Palmer 2011). The measurement of the progression is of great epidemiological importance especially as there
are over 470 million pre-diabetics worldwide (Tabk et al. 2012). Diagnosis of the
on-set of diabetes in pre-diabetics is typically preformed by determination of glucose intolerance. The common clinical practice to detect on-set is by a combination
of testing including an oral glucose tolerance test (OGTT), fasting blood glucose
(FBG) and/or hemoglobin A1c (HbA1c) (Sacks et al. 2011). However predicting
which pre-diabetic will become diabetic is particularly difficult as on-set might not
occur for many years. Only small changes in beta-cell function are detectable in
pre-diabetes and arise from an interaction between insulin resistance and a decrease
in beta-cell responsivity to glucose (Sathananthan et al. 2012). The CDC estimates
application of common practices to the 72 million U.S pre-diabetics would add 4.52
mill newly diagnosed diabetics if the testing was done by OGTT, 2.25 mill if tested
by fasting blood glucose (FBG) or 1.58 million if tested by HbA1c (CDC.gov). Any
method used for prediction and early intervention of which patients would develop
diabetes would have to be highly specific. Otherwise, even a small % false positive
would result significant follow-up of the pre-diabetics population. At the same time
the diagnostic method must be sensitive, reproduce-able within an individual and
consistent with common practice. Any changes in diagnostic methodology could
cause significant numbers of patients to be impacted.
The OGTT is most sensitive and specific measure on onset, but least practical to
applay to every suspected case as timed sample collection is needed. Impaired fasting
glucose (IFG) and impaired glucose tolerance (IGT) are detected by OGTT and allow
placing individuals at higher risk of overt type 2 diabetes. The OGTT best correlates
with insulin resistance/sensitivity measures. The FBG measurement itself is a very
simple test, but very non-specific with false positives and has high inter-subject variance requiring repeat testing if discrete diagnostic cut-offs are applied. Fasting is
required for both and tests can suffer from glucose instability requiring specific sample tubes and storage consideration.
The HbA1c measurement is a highly specific measure of persistent diabetes with
a >95 % specificity at the >6.5 % HbA1c threshold. The HbA1c measurement has the
advantages of allowing non-fasting measurement with low inter individual variability
and has also been tied to retinopathy complications (Lopes-Virella et al. 2008).
However, the HbA1c measurement is insensitive to detection of on-set in pre-diabetes
and gestational diabetes and only detects persistent hyperglycemia. The HbA1c is not
very predictive of a positive OGTT, insulin resistance or -cell dysfunction (Dods
and Bolmey 1979).
Surrogate indexes of insulin sensitivity/resistance derived multiple time points
in the OGTT are available (e.g. QUICKI, HOMA, Log HOMA, 1/HOMA, Matusda
index and 1/Fasting insulin). While reported as useful methods for diagnosing
insulin resistance, and follow-up during the treatment of patients with diabetes
(Katsuki 2001), the correlations of most surrogates indexes to glucose clamp
C-Terminal Fragment of Adiponectin Receptor Clinical Correlations
113
testing are modest at best and do not offer much to clinical practice over direct
measurement (Lee et al. 2011).
Alternative approaches to sorting out pre-diabetic progression measure the
underlying insulin sensitivity/resistance (Muniyappa et al. 2008). These include
direct measurements based on hyperinsulinemic euglycemic glucose clamp or insulin suppression test. The glucose clamp is widely accepted as the reference standards for measuring insulin sensitivity/resistance (DeFronzo et al. 1979). Hepatic
catheterization allowing measurement of hepatic insulin extraction and systemic
insulin/c-peptide clearance does confirm the kinetics of altered insulin secretion and
absorption in obese and diabetic subjects (Tura et al. 2001). However these methods
can only done in highly controlled research studies where insulin is infused in a
complex time-consuming, labor-intensive, and invasive procedure.
Indirect measurements of insulin sensitivity/resistance are more commonly utilized as they require fasting followed by a bolus of glucose and plasma glucose,
c-peptide and insulin measurements. Indirect measurements by the frequently sampled intravenous glucose tolerance test are most practically used and methods for
estimating insulin sensitivity/resistance. Here model-derived estimates allow calculation of indices for insulin action and -cell responsiveness.
New diagnostic methodologies are often tested and compared to results obtained
in the screening process for glucose intolerance during gestation (Tieu et al. 2010).
This cohort allows a short duration for prediction of on-set based on patients who
progress to gestational diabetes. More pregnant women received glucose testing than
any other risk factor group, making this a good means to obtain samples for the study
of on-set of diabetes (Catalano 2010). This risk group also as an increasing number of
individuals classified as having either impaired fasting glucose (33.8 %) or impaired
glucose tolerance (15.4 %) which allows reasonable sampling numbers (CDC.gov).
Inhibition of insulin degradation by CTF suggests a potential to impact this correlation with insulin resistance and the possibility to further characterize diabetes
phenotypes. However, any new surrogate marker intended to replace or supplement
OGTT will need to achieve better clinical performance for detection of onset and for
monitoring of progression. Ability to flag at risk populations with glucose intolerance is also an attractive feature. The new marker must surpass the performance of
HbA1c or FBG. Our goal was to test the Iq-CTF clinical assay against an improved
clinical performance specification of a specificity of >95 % and a sensitivity of
>95 % for diabetes as determined OGTT. Additional clinical specificity of >95 %
and a sensitivity of >65 % for IGT would also be a significant improvement (3X).
As glucose is the primary treatment indicator for any surrogate marker would need
a correlation of at least an r >0.5 to OGTT for improved performance.
The Ig-CTF immunoassay was tested for patients with gestational diabetes, prediabetes, and diagnosed with diabetes. Reference range testing was also conducted
using healthy volunteers. The impact of sampling, sample type, fasting and days
between sample were determined. Within individual variability was measured.
Additionally the impact of patients having inflammation, cancer and liver disease
was determined. The impact of anti-inflammatory and immunosuppressant treatment
was assessed by measuring patients under treatment. Results were compared to a
combination of testing including hemoglobin A1c (HbA1c), fasting blood glucose
114
(FBG) and/or an oral glucose tolerance test (OGTT). As a new surrogate marker
intended to replace or supplement OGTT, higher better clinical performance criteria
were put forth and compared to HbA1c or FBG performance. Additional comparisons were made to indirect model-derived estimates of insulin sensitivity/resistance.
6.2
6.2.1
Methods
Reference Range Testing
Clinical specimens were collected and refrigerated (58 C) from time of collection
until frozen (20 C) shortly after they were poured off. Before additional testing,
specimens were thawed to 58 C and used to make dilutions for analysis. Matched
serum, plasma and whole blood samples were collected from 25 health volunteer
monthly for 45 days. All healthy controls maintained normal fasting blood glucose
(<99 mg/dL whole blood and <115 mg/dL plasma for venous samples). Whole
blood samples and plasma samples were obtained for patients outside of normal
range with either impaired fasting glucose (IFG), impaired glucose tolerance (IGT)
or diabetes. IFG was indicated with glucose of 115125 mg/dL at 0 min and IGT
was indicated with glucose of 140200 mg/dL at 120 min. Diabetes was indicated
with glucose of 126 mg/dL at 0 min or 200 mg/dL at 120 min or >6.5 % hbA1c.
The impact of cancer and liver disease was assessed by comparison of serum
samples from normal patients no history of cancer, liver disease, diabetes or metabolic syndrome, or inflammation to those with liver disease or cancer (Promeddx,
Norton MA). Equal numbers of male and female patients were used. Patients with
liver disease had either alcoholic hepatitis, nonalcoholic steatohepatitis (NASH),
autoimmune hepatitis or hepatocellular carcinoma, and cancer patients had either
pancreatic or breast cancer.
The impact of inflammation was also assessed by comparison of whole blood
samples from normal patient (n = 101) to patients (n = 13) with active proinflammatory response (CPR >5 mg/dl or CBC of >10 white blood cells/mL). Also
patients using anti-inflammatory therapies such as Remicade (Infliximab) were
compared. Infliximab is a monoclonal antibody against tumor necrosis factor alpha
(TNF-) used to treat autoimmune diseases. Transplant patients on immunosuppressant were also tested. These additional samples were obtained from
Bioreclamation LLC of Westbury NY and Promeddx of Norton MA.
6.2.2
Gestational Diabetic Collection
Whole blood fasting venous specimens were collected from gestational patients at
Methodist Medical Center of Illinois, Chicago IL from Nov 2011 to Feb 2012.
Patients were fasting for 8 hours prior to sample collection. Capillary fasting POC
glucose test (finger stick/glucose meter) was checked first, if >135 mg/dL then a
115
grey top tube collected and the OGTT given. The gestational diabetes OGTT procedure calls for 100 g glucose instead of 75 g glucose. Samples were collection at 0,
60, 120 or 180 min. Most physicians chose to collect a fasting, 60 min and 120 min
specimen. Whole blood fasting venous samples were sent immediately to chemistry
for laboratory glucose determination. Plasma glucose values for 0 and 2 h were
obtained.
Specimens for 48 female patients were flagged as pre-diabetic by glucose meter
were obtained for 0 and 120 min time points. Specimens for 101 female patients
with normal fasting plasma glucose were also obtained from a comprehensive metabolic panel performed with an EDTA tube. Fasting plasma blood glucose were
obtained and evaluated against the glucose limits for fasting venous samples
<115 mg/dL. Of the 48 diabetics patients flagged pre-diabetic, 28 were IGT, 2 were
diabetic and 18 were in the normal range
6.2.3
Pre-diabetic Collection
Whole blood and plasma fasting venous specimens were collected from normal,
pre-diabetic and diabetic at Mayo Clinic (Sathananthan et al. 2012). A total of 240
subjects (143 women, 97 men) gave informed written consent to participate in the
study. All subjects were in good health and were at a stable weight and did not
engage in regular vigorous exercise. All subjects were instructed to follow a weightmaintenance diet containing 55 % carbohydrate, 30 % fat and 15 % protein for at
least 3 days prior to the study. Samples were collected form patients at 0, 10, 20, 30,
60, 90 and 120 time points after an overnight fast and using a 75-g oral glucose
tolerance test (OGTT). Patients were characterized as normal glucose tolerance
(NGT), normal fasting glucose (NFG), impaired glucose tolerance (IGT), impaired
fasting glucose (IFG), and diabetes (DM) using analytical values for insulin and
glucose measurements modeled at 7 time points out to 2 h. Additional values for
c-peptide, pancreatic glucagon, human growth hormone, and cortisol were examined as well. Insulin action (Si) was estimated in this study using the oral glucose
minimal model, and -cell responsively indices (Phi total) were estimated using the
oral C-peptide minimal model. The disposition index (DI) for each individual was
calculated
Subjects with a prior diagnosis of diabetes or a fasting glucose >126 mg/dl
(>7 mm) at the time of screening were excluded, as were those taking medications
that could affect glucose metabolism (such as insulin, GLP, DPP-4 inhibitors, sulfonylureas, meglitinides, alpha-glucosidase inhibitors and thiazolidinediones (TZD))
or those taking medications which induce diabetic symptoms (glucosamine, rifampicin, isoniazid, olanzapine, risperidone, progestogens, corticosteroids, glucocorticoids, methadone many anti-retrovirals, D5W/glucagon and TPN-induced multiple
enteral feedings). Patients were excluded with infectious diseases (HIV, HCV, HBV
and immunodeficiency), certain cancers (leukemia, multiple myeloma, and lymphoma), certain liver disease (hepatitis & cirrhosis) those taking TNF alpha inhibitors (Infliximab and others).
116
The hbA1c values were measured on the thawed whole blood samples using the
DCA. Iq-CTF-Ig values were measured blinded on the thawed whole blood and
plasma samples using the ELISA method in duplicate for all patients in four separate run. Siemens results were sent to Mayo for examination prior to receiving Mayo
analytical data. Samples were placed in ice, centrifuged at 4 C, separated and stored
at 20 C until assayed. Samples were thawed to 4 C not more than three times.
6.2.4
Diabetic Collection
Whole blood venous specimens were also collected from 68 long term diabetic
undergoing regular HbA1c monitoring at Diabetes Diagnostic Laboratory,
University of Missouri School of Medicine, Columbia MS from Jan 2011 to Mar
2012. The patients were not presumed fasted and collected in either grey top or
EDTA tubes. The hbA1c values were measured on the thawed whole blood samples
using the DCA Advantage instrument in duplicate for all patients. Microscopic
examination of blood sample confirmed freezing caused complete blood lysis.
6.2.5
Ig-CTF and CTF Measurements
The Ig-CTF and CTF bioassay descriptions and analytical validation data for reproducibility & stability are shown in Chap. 3. The method calibration, precision, accuracy, interference and recovery were demonstrated. Variability of the Ig-CTF and
CTF determinations required use of preservatives in the blood tube and the dilution
buffer to allow an acceptable allowable total error. The addition of EDTA, citrate,
and BSA at pH 6.4 to the dilution buffer was required to allow to 20 C storage
freezing of diluted plasma. Deactivation of platelets in the sample was critical for
decreasing variance and the impact of freezing. Collections with EDTA tubes were
less variable than heparin and sodium fluoride tubes. Addition of heparin instead of
EDTA was required for freezing diluted whole blood. With the sample stabilized,
the average bias was 4.5 % CV for repeated plasma samples. Whole blood samples
were more variable 7 % CV. The data showed a spot measurement was possible.
Plasma, serum and whole blood use was possible.
6.3
6.3.1
Results
Normal Range and Individual Variability Testing
The values for Ig-CTF apparently healthy volunteers were measured for the three
sample types (See Table 6.1). Matching serum, plasma and whole blood samples
were collected from the same donor. Whole blood values were highest followed by
117
Table 6.1 Impact of inflammation on Ig-CTF R1 values

Specimen type
Serum
Serum
Serum
Serum
Plasma
Plasma
Plasma
Plasma
Plasma
Whole blood
Whole blood
Whole blood
Whole blood
Whole blood
Patient group
Normal
Liver disease
Breast cancer
Pancreatic cancer
Normal
Anti TNF therapy
Pre-diabetic (IGT)
Pre-diabetic (IFG)
Diabetic
Normal
Inflammation
Immunosuppressant
Pre-diabetic
Diabetic
n
25
23
5
10
25
8
119
100
24
101
13
20
48
63
Ig CTF ng/mL
Aver
sd
322.5
173.4
1141.2
1514.3
794.6
260.7
581.2
430.5
727.5
343.0
404.1
130.1
349.5
268.1
335.3
256.4
268.0
141.1
1234.1
651.9
402.1
173.6
566.0
361.1
527.0
195.6
222.3
97.3
min
55.5
293.8
340.7
293.2
290.2
194.1
95.1
99.0
91.3
178.6
145.2
160.7
250.9
51.6
max
778.8
6854.7
1140.0
1330.7
1723.4
601.0
2346.3
2346.3
645.7
4758.9
713.2
1427.1
1055.2
444.1
The values for Ig-CTF were measured in serum, plasma and whole blood for patients and health
donors. Patients were characterized by oral glucose tolerance test if sample was plasma. Donors
lacked inflammatory conditions (CBC & CRP normal) unless indicated. The HbA1c and FBG was
measured if the sample was whole blood. Matching samples for all three sample type were compared across 25 health donor and 48 pre-diabetics and produce values similar to those shown
Sample collection had to be optimized to reduce within patient variability to <10 % to suppress
coagulation. We found plasma with activated platelets caused 4-fold increase in Ig-CTF values
compared to the same plasma with deactivated platelets. Activation caused a visible cloudiness to
occured in diluted sample. The impact on the assay was explained by IgG binding to Fc receptors
on platelets and causing platelet aggregation artificially elevating values (Worth et al. 2006).
Complement components also bind to activated normal platelets and promote binding of immunoglobulin through C1 q (Hamad et al. 2010). As a control it was verified neither C1q nor Adpn
bound to Ig-CTF as tested by ELISA and platelet binding Ig-CTF is a likely through the immunoglobulin Fc receptor
plasma and then by serum values. The correlation between whole blood and plasma
for the same patients was R^2 of 0.63 and a slope of 1.17 for higher whole blood
values compared to plasma. Higher whole blood values were expected by white
blood cell binding of IgG (Dickler 1973). Platelet binding of Ig could explain higher
values in plasma than serum (Worth et al. 2006).
The values for Ig-CTF were next compared with patients undergoing glucose
and HbA1c testing. The Ig-CTF plasma values for patient with pre diabetes and
diabetes were significantly lower than patients with normal glucose tolerance
(P > 0.01). Overnight fasting did not impact Ig-CTF values in health donors or
diabetics with all values within 914 % of non-fasting values. The Ig-CTF values did not change during the 120 min following glucose administration. Values
did not change over 45 days. This is consistent with the 14 day plasma half-life
of immunoglobulin (Hinton et al. 2006). Values did changed in a patients who
118
underwent significant a weight change (>20 % drop) after 45 days. This contrasts the rapid changes of free CTF measured in animals during the 120 min
following glucose administration (See Chap. 5). Ig-CTF appears to be more a
chronic phase marker.
6.3.2
Correlation to Inflammation
The values for Ig-CTF measured shown for normal and diabetic donors were in
cases lacking inflammation. Inflammatory conditions were ruled out by a CPR
<5 mg/dl or CBC of <10 white blood cells/mL. These cut-off do not rule out mild
elevation of inflammation. The patients with significant inflammation, as indicated
a CRP and CBC positive, did exhibited a significantly lower Ig-CTF (P > 0.01) than
normal patients (Table 6.1). This decrease was of a similar magnitude to a decrease
in the Ig-CTF seen in diabetes.
Patients using anti-inflammatory therapies such as Remicade (Infliximab)
exhibited suppressed Ig-CTF values. Transplant patients on immunosuppressants
also had suppressed Ig-CTF values. The impact of anti-TNF alpha inhibitor drugs,
would be expected to prevent CTF formation by inhibition of TACE. Others drug
expected to impact the assay would include adalimumab (Humira), certolizumab
pegol (Cimzia), golimumab (Simponi), or etanercept (Enbrel). Immunosuppressants
would also be expected to lower immunoglobulin levels and suppress Ig-CTF
formation.
Patients who had diseases that would be expected to increase blood immunoglobulin levels, also had changes in the values for Ig-CTF. Liver diseases represent
a significant inflammatory disease group. Liver diseases (Hepatitis & cirrhosis)
elevate serum immunoglobulin. Values for Ig-CTF were significantly increased with
auto-immuno hepatitis patients having the highest values (Table 6.2). Patients with
leukemia, multiple myeloma, and lymphoma were excluded as these cancers would
clearly elevate immunoglobulin levels. However, cancer is generally associated
with greater blood immunoglobulin levels and many patients with breast and pancreatic carcinomas did exhibited increased Ig-CTF values.
6.3.3
Correlation in Gestational Diabetes
Whole blood fasting venous specimens were collected from gestational patients.
Patients were flagged by fasting POC glucose as normal (n = 101) or pre-diabetic
(n = 48). Additional whole blood fasting venous specimens were collected from
confirmed diabetics by HbA1c >6.5 % (n = 68). The diabetic and normal groups
are well separated using Ig-CTF values and cutoff of 500 ng/mL (Fig. 6.1). The
pre-diabetic group had Ig-CTF values in the middle of the normal and diabetic
groups
119
Table 6.2 The predictive

values of CTF-Ig for in
gestational study
TN
FN
FP
TP
Sens
Spec
PPV
NPV
Study 1
95
0
7
64
100.0 %
93 %
90 %
100 %
Study 2
110
10
10
84
89.4 %
92 %
89 %
92 %
Study 1 is a comparison of normal vs. diabetics with pre-diabetic considered normal and diagnosis based solely on FBG
and HbA1c values. Study 2 is a comparison of normal vs. diabetics with IGT pre-diabetics considered positive with diabetic and diagnosis based solely on OGTT and HbA1c values.
The efficiency of ruling in and out impaired glucose tolerance
(IGT) was also tested. Of the 48 patients flagged by FBG as
impaired, only 2 patients were confirmed by OGTT as a diabetic and 30 were classifieds as impaired glucose tolerance
(IGT). The Ig-CTF value eliminated 10 of the 30 follow ups
flagged by FBG and agreed with 13 of the 16 patients correctly rule out by FBG
Two patients were clearly Ig-CTF false negatives. Patient
A with fasting plasma glucose of 428 mg/dL, abnormal hbA1c
of 9.1 % and a normal Ig-CTF of 858 ng/mL and patient B
with abnormal hbA1c of 10.2 % and a very high normal
Ig-CTF of 2625 ng/mL. In both cases, elevated Ig-CTF values
could be due to generally elevated immunoglobulin. There
were seven patients with normal fasting glucose but false
positive by Ig-CTF value or <500 ng/mL. These specimens
were reanalyzed with new dilutions and plates. As the results
were repeated they were considered as clinical false positive
Correlations of Ig-CTF were made between non-diabetic, pre-diabetic, gestational diabetic and diabetic populations. Overnight fasting did not impact the
Ig-CTF results. Results for Ig-CTF were compared to diagnosis based on fast blood
glucose (FBG), oral glucose tolerance test (OGTT), and hemoglobin A1c (hbA1c).
The clinical correlation of Ig CTF was highest for a glucose tolerance test (OGTT).
The average variance between samples was 9 % with a minimum variance of 2 %
and maximum variance of 29 % in one sample. This reinforced that one CTF-Ig
measurement could be taken and a timed collection was not needed.
The correlation between values for Ig-CTF and glucose at 120 min is shown in
Fig. 6.2. In general, the higher Ig-CTF values were more likely it is to have lower glucose values which were in the normal range. However, the correlation to glucose was
poor, and Ig-CTF did not change with glucose administration. Ig-CTF values were
unchanged between the samples collected at 0 min and 120 min OGTT time points. In
contrast free CTF in animals had a higher correlation with glucose and does increase
sharply during the 120 min OGTT challenge after glucose administration (See Chap. 5).
120
Immunosuppressants
Inflammation
Anti-TNF therapies
60 %
50 %
% of patient in group
Diabetic
40 %
Prediabetic
Normals
30 %
20 %
Liver cirrhosis
& cancer
10 %
0%
Ig-CTF ng/mL
Fig. 6.1 Distribution of Ig-CTF in Diabetes and Inflammatory Diseases. Whole blood values of
Ig-CTF were measured for non-diabetic pregnancies, gestational diabetic and long term diabetic
populations characterized by fast blood glucose (FBG), oral glucose tolerance test (OGTT), and
hemoglobin A1c (hbA1c). Values for long term diabetics were lower than non-diabetics with prediabetics falling in-between. The patients with inflammation, using anti-inflammatory or immunosuppressant therapies exhibited a lower Ig-CTF. Liver diseases (Hepatitis & cirrhosis) and cancers
which elevate immunoglobulin levels exhibited a higher Ig-CTF
The predictive value of Ig-CTF for diabetes in a gestational population is shown

in Table 6.2. Both sensitivity and specificity were high for assessing diabetes
(100 % sens/93 % spec) and pre-diabetes or diabetes (89 % sens/92 % spec) (Study
1 and 2). The Ig-CTF measurements based on the Adiponectin receptor 2 produced
a similar correlation. This meets the target criteria of >95 % spec for diabetes in
combination with HbA1c with a >70 % sensitivity for diabetic detection. In the
second study, the fasting blood glucose (FBG) was not as predictive as CTF-Ig having poor specificity of 30 % but good sensitivity of 95 % for diagnosis of diabetes
based on OGTT. The hbA1c was also not as predictive as CTF-Ig having poor sensitivity. HbA1c values of 6.5 % only confirmed 68 of the 129 diabetes patients
(roughly half). HbA1c had a good specificity of 95 %. However none of the prediabetic population (0 of 48) had positive hbA1c and one of the normal population
(n = 102) had an hbA1c of 7.3 %.
121
1200.0
1000.0
CTF Ig ng/mL
800.0
y = 3.705x + 1090.7
R2= 0.218
600.0
400.0
200.0
0.0
100
120
140
160
180
200
220
240
260
Glucose at 120 min
Fig. 6.2 Correlation of Ig-CTF to the 120 min OGTT glucose value. Whole blood values of
Ig-CTF are plotted against the glucose value at 120 min in the oral glucose tolerance test (OGTT).
The liner correlation coefficient was not an r > .5 indicating a weak correlation
6.3.4
Correlation in Pre-diabetics
The predictive value of Ig-CTF for diabetes in a population with many pre-diabetic
and not well separated from normal is shown in Table 6.3. Whole blood and plasma
specimens were collected for a group of patients characterized by FBG, OGTT and
HbA1c. No patient was >6.5 % HbA1c. Patients were characterized into five groups.
Normal patients were normal glucose tolerance (NGT) & normal fasting glucose
(NFG) (n = 66, 27 % of population). Pre-diabetics (n = 150, 63 % of population)
were either impaired glucose tolerance (IGT) and NFG (n = 63) or impaired fasting
glucose (IFG) and NGT (n = 26) or IFG and IGT (n = 61). Diabetic patients (DM)
were positive for the OGTT (n = 24, 10 % of population).
The Ig CTF values did not separate pre- diabetics or diabetics in this population
(See Table 6.3). Ig-CTF did not meet any predictive value criteria separating prediabetic from normal and achieved only a ~50 % specificity. Only 10 % of diabetics
and pre-diabetics had abnormally low Ig-CTF values. The predictive value for
Ig-CTF also did not meet the ideal criteria achieving only a >50 % specificity for
pre-diabetes and clinical sensitivity of >56 % for IGT and of >75 % for DM (See
Table 6.3). While Ig-CTF was not predictive in this population, HbA1c was also
122
Table 6.3 The predictive values of Ig- CTF for pre-diabetic and diabetics
Patient group
Normal (NFG & NGT)
Pre-diabetics (NFG & IGT)
Pre-diabetics (IFG & NGT)
Pre-diabetics (IFG & IGT)
Diabetics (DM)
Total
Phenotype group
Normal
Flat curve (Cortisol high)
Pre-diabetic with normal insulin curve
Pre-diabetic with insulin resistance curve
Type 2 Diabetic -normal insulin curve
Type 2 Diabetic -insulin resistance curve
Total
IgG CTF
High Low
34
32
29
34
13
13
28
33
6
18
110
130
IgG CTF
High Low
46
43
1
0
26
27
27
39
3
7
3
13
106
129
TN
FN
FP
TP
sens
spec
PPV
NPV
TN
FN
FP
TP
sens
spec
PPV
NPV
Cat 1
104
6
112
18
75.0 %
48 %
14 %
95 %
Cat2
34
76
32
98
56.3 %
52 %
75 %
31 %
Cat 2
47
59
43
86
59.3 %
52 %
67 %
44 %
Plasma correlations shown were stronger than those observed in whole blood. However the impacts
observed in plasma were mirrored in whole blood. The clinical thresholds for an abnormal Ig-CTF
used were <300 ng/mL for plasma and <650 ng/mL for whole blood. A hemoglobin ratio helped
plasma and whole blood correlation, and is not shown. Categorization 1 (Cat 1) is a comparison of
normals vs. diabetics with pre-diabetics is considered normal. Categorization 2 (Cat 2) is a comparison of normals vs. diabetics and pre-diabetics
Despite the lack of diagnostic correlation, there were several notable trends which agreed with
biochemical model. Patient with lower plasma Ig-CTF were more likely to exhibit increased glucose of >125 mg/dL over time. Patient with lower plasma Ig-CTF were more likely to exhibit
smaller changes in insulin of <20 or C-peptide of <1. Greater changes in Phi total (>50), Si(>20) or
DI(>1000) were more likely with lower CTF. No correlation in pancreatic glucagon was observed
Several un-expected correlations were also found. Females had lower Ig-CTF (Avg (sd) of
269(150) ng/mL) compared to males (Avg(sd) of 373(266) ng/mL). This gender trend was in all
patient categories. Patients with high plasma Ig-CTF were more likely to exhibit with no changes
in growth hormone or high cortisone values. However, only a small percentage of normal and prediabetic patients exhibited high Ig-CTF values. Five patients had Ig-CTF of >2500 ng/mL. One
patient had Ig-CTF of ~6000 ng/mL. At the same time about 2.9 % of all patients exhibited
extremely low Ig-CTF values (<100 ng/mL)
completely insensitive for pre-diabetes and diabetes. Meanwhile this data demonstrated the false positive discordance of FBG for predicting IGT or OGTT.
Patients were also fully characterized by insulin action measures (DI, Si, Phi)
using timed measurement of insulin, glucagon, glucose, and c-peptide (Sathananthan
et al. 2012). Ig-CTF did not meet any predictive value criteria and was only ~50 %
specific and sensitive for predicting any difference in indices of insulin secretion and
123
action (data not shown). Insulin resistance phenotype was also defined by a hyperinsulinemia (fasting 60 min insulin of >48 IU/mL) and impaired glucose tolerance
(fasting 120 min glucose value from 140 to 200 mg/dL in OGTT). The plasma and
whole blood Ig CTF values could not separate this phenotype in either the pre-diabetic or diabetic groups (See Table 6.3).
6.4
Conclusions
The circulating Ig-CTF values are suppressed in diabetes in and inflammation. As a

diagnostic this marker was un-able to resolve early diabetes. Advanced diabetic
disease was resolved from healthy controls. The drop in circulating Ig-CTF is consistent with greater Ig-CTF absorption in liver, pancreas and other tissue during
diabetes. The circulating Ig-CTF values are also elevated during cancer and liver
disease with elevated immunoglobulins. The suppression by anti-TNF and antiimmune therapies suggests immune system modulation can impact the amount of
circulating Ig-CTF. As Ig-CTF is carried on activated white blood cells to tissue, it
is possible Ig-CTF could stimulate immune cell response to certain antigens. The
Ig-CTF assay could potential identify patients with immune mediated diabetic diseases (kidney, eye, liver, other) and impaired tissues loading of CTF (liver, pancreas, brain), and those with liver disease, cancer or other chronic inflammatory
diseases. Additional clinical investigations are required.
Acknowledgements We would like to acknowledge the samples collected by Sandra D. Pottorf,
CLS, ASCP (Methodist Medical Center of Illinois, Chicago IL); the samples removed from storage by Jessica Cray and Linda Sanders (Mayo Clinic Rochester, MN); the samples provided by
Randie R. Little, PhD (Diabetes Diagnostic Laboratory, University of Missouri School of Medicine,
Columbia MS); and the samples procurement help from Denise Cervelli of (Siemens Healthcare
Diagnostic Newark DE).
Supplements
ELISA Method for Ig-CTF assay
ELISA Method for free CTF assay
References
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Catalano PM (2010) Obesity, insulin resistance and pregnancy outcome. Reproduction 140(3):
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and resistance. Am J Physiol 237(3):E214E223
Dickler HB (1973) Lymphocyte binding of aggregated IgG and surface Ig staining in chronic lymphocytic leukaemia. Clin Exp Immunol 14:97106
Dods RF, Bolmey C (1979) Glycosylated hemoglobin assay and oral glucose tolerance test compared for detection of diabetes mellitus. Clin Chem 25(5):764768
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preceding proteolytic activation and promotes binding to complement receptor 1. J Immunol
184(5):26862692
Hinton PR et al (2006) An engineered human IgG1 antibody with longer serum half life. J Immunol
176:346356
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Lopes-Virella MF et al (2008) Diabetes control and complications trial/epidemiology of diabetes
intervention and complications cohort study group. Diabetes Care 31(10):20062012
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in vivo: advantages, limitations, and appropriate usage. Am J Physiol Endocrinol Metab
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Committee of the American Association for Clinical Chemistry (2011) Guidelines and
recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus. Diabetes Care 34(6):e61e99
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spectrum of fasting and postchallenge glucose concentrations. Clin Endocrinol (Oxf)
76(2):212219
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379(9833):22792290
Tieu J et al (2010) Screening and subsequent management for gestational diabetes for improving
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Tura A et al (2001) Insulin and C-peptide secretion and kinetics in OGTT humans: direct and
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Exp Hematol 34(11):14901495
Part IV
Uristatin Assay for Prediction of Renal

and Other Clinical Events
Chapter 7
Uristatin Immunoassay Usage in Glomerular

Nephritis Assessment
Abstract A novel immunoassays for Uristatin (Uri) was used for testing in patients
glomerular nephritis (GN) by using of an extensive clinical data and urine sample
bank from previous studies (n = 6292 patients). Proteinuira or hematuria was found
in 1407 of 6292 patients and used an indicator for potential cases needing glomerular nephritis follow up. Of these, 351 patients had asymptomatic GN without infection or Uri. The remaining 1194 patients had potential infections based on symptoms
of urinary tract infection, systemic infections, and/or a positive Uri value
>12 mg/L. Urine culture and CBC were used to characterize 419 patients as actually
having infections. Those remaining were sorted according to Uri value as 198 cases
of symptomatic GN with anti-inflammatory stress (Uri >12 mg/L) and 533 cases
without this stress (Uri <12 mg/L). The Uri method allowed sorting atypical antiinflammatory stress patient in glomerular nephritis.
7.1
Introduction
Diabetes leads to inflammation, higher chances of infection and impaired wound

healing (Larkin et al. 1985). During the progression of this disease, an innate
immune system response is necessary to prevent damage to normal tissue (Donath
et al. 2013). Urinary trypsin inhibitors (uTi) are a key part of the anti-inflammatory
S.A. Jortani
Department of Pathology, University of Louisville, School of Medicine,
511 S. Floyd Street (Room 227), Louisville, KY 40202, USA
M. Pugia (*)
Department of Pathology, University of Louisville, School of Medicine,
Louisville, KY 40202, USA
DOI 10.1007/978-3-319-21927-1_7
127
128
S.A. Jortani and M. Pugia
1. Release of trypsin
proteases
..from immune cell for
bacteria phagocytosis
Neutrophils,
Monocytes,
Eosinophils,
Natural killer cells,
Macrophages,
Mast cells
..from tissues for

cellular proliferation
Epithelial cell,
Endothelial cell,
Smooth muscle,
Fibroblast,
Platelets,
Neoplastic
Inter-a-Inhibitor
pro-Inhibitor always in blood
2. Release
of inhibitor
Bikunin
Inflammation
H1
H2
Urinary
trypsin
inhibitors
Elastase
3. Inhibition of
Cathepsin
Immune mediated
Tryptase
apoptosis
Trypsin
Kallikrein
Thrombin
Plasmin
Factors VII & X
4. Inhibition of
cellular proliferation
Bikunin
Uristatin
Additional
fragmentation
>99%cleared
into urine
Fig. 7.1 Urinary trypsin inhibitors are release from non-inhibitory pro-inhibitors. (Step 1) Serine
proteases of the trypsin family (Elastase, Cathepsin, Tryptase, Trypsin, Kallikrein, Thrombin,
Plasmin and Factors VII & X) are increased during inflammation by innate immune cells
(e.g. White Blood Cells; Neutrophils, Monocytes, Eosinophils, Natural killer cells, Macrophages,
Mast cells) and affected tissues (Epithelial, Endothelial, Smooth muscle, Fibroblast, Platelets and
Neoplastic). Step 2) These proteases produce inflammatory response of vascular dilation, coagulation, and leukocyte infiltration with destruction of pathogens. Step 3) Serine proteases (mainly
elastase) liberate plasma bikunin (Bik) the primary uTi from pro-inhibitor, inter--inhibitor (I--I),
activating the inhibitory response. Bik is rapidly excreted into urine. Step 4) Bik inhibits serine
proteases as an anti-inflammatory response controlling remodeling, smooth muscle contraction,
fluid/electrolyte balance and protecting tissue from leukocyte damage. Step 5) Bik signals cells to
reduce proliferation, inflammation cytokine mediator release, growth factor activation, uPA activation and intra-cellular Ca+ slowing cell growth. Step 6) Bik is rapidly metabolized and excreted
into urine avoiding a prolonged suppression of inflammatory system. Uristatin (Uri) is a primary
form in urine and lacks the O-linked glycoside
response functioning intrinsically as an inhibitor of the serine proteases causing

inflammation (Pugia et al. 2007b; Pugia and Lott 2005). The uTi suppress inflammation and then readily pass into urine (Fig. 7.1). Once uTi suppresses inflammation, they are readily filtered by the glomeruli (>99 %) through the glomerular
basement membrane and accumulating in the lysosomes of proximal tubular epithelial cells. Thus the kidney is exposed to the majority of uTi and their antiinflammatory properties, and urine is the primary sample for testing.
Urinary tract infections are the primary cause of glomerular nephritis as immune
cells responding to infection damage tissue and cause a pro-inflammatory response
(Hooton and Stamm 1997; Barnett and Stephens 1997; Saint et al. 1991). The
7 Uristatin Immunoassay Usage in Glomerular Nephritis Assessment
129
Blood
flow
Arteriole
Glomerular capillary
Mesangial cell
Endothial cell
Basement membrane
Epithelial cells (podoctye)
Proximal tubular
Bowmans capsule
Epithelial cell
Distal collecting tube
Brush border
Urine
Basement membrane
& blood capillary
Fig. 7.2 Kidney model. The kidney model is to explain the impact uristatin on kidney during
urinary tract infection and inflammation. Key kidney components for glomerular capillary and
proximal tubular are shown
kidney model shown in Figs. 7.2, 7.3 and 7.4 can be used to explain the impact of
inflammation and uTi on the kidney. The kidney components and blood and urine
flow are shown in Fig. 7.2. Pro- and anti-inflammatory processes involved in kidney
damage and regeneration can be explained by this model. In normal kidneys, only
low molecular weight (LMW) proteins (<30 kDa) pass the basement membrane of
the glomerular capillary into the distal collecting tube where proximal tubular cells
re-absorb this protein back into the blood leaving no proteinuria (Fig. 7.3). In abnormal kidneys, where the basement membrane has been damaged, large proteins
(> = 30 kDa) pass the basement membrane of the glomerular capillary into the distal
collecting tube and proximal tubular cells can fail to re-absorb high amounts of
protein into the circulation leaving gross proteinuria and loss of kidney function
(decline of glomerular filtration rate (GFR)) (Fig. 7.4).
As kidney tissue becomes inflamed, white blood cells (WBC) migrate into the
interstitial space to fight infection. Neutrophilic elastase is released from these
WBC. Elastase proteolysis of pro-inhibitors give rise to uTi. Interleukin--inhibitor
(I--I) and pre-interleukin--inhibitor (P--I) are the primary pro-inhibitor forms
(Fig. 7.1). More than 98 % of the uTi in blood is bound to these pro-inhibitor forms
and is inactive from inhibition of serine proteases. Bikunin (Bik) and Uristatin (Uri)
are two key forms of uTi. Uristatins (Uri) are small derivatives of Bikunin lacking
the O-linked glycoconjugates.
The uTi released during infections has a potent anti-inflammatory response
(Fig. 7.1). The presence of uTi correlates with febrile proteinuria and elevated white
130

Normal
Inflammation
Blood flow
Glomerular
capillary
Basement
membrane
Blood
Proximal
tubular
Urine
flow
Large proteins >30 kDa

Small proteins <30 kDa
Re absorption in
epithelial cell
Immune cells >5 uM

Urinary trypsin inhibitors <30 kDa
Small proteins <30 kDa
Fig. 7.3 Early kidney inflammation. In normal kidney, only the small proteins pass the basement
membrane of the glomerular capillary into the distal collecting tube and proximal tubular cells reabsorb this this protein into the blood leaving no proteinuria. In kidney where the basement membrane has been damaged, large proteins pass the basement membrane of the glomerular capillary
into the distal collecting and tube proximal tubular cells fail to re-absorb this protein into the blood
leaving proteinuria. Prior to iamass immune cells such as white blood cells response to infection
and migrate to inflamed tissue. The WBCs cause uTi to be released into the urine. The uTi correlates
low molecular weight proteinuria presumed to be due to shut down tubular re-absorption. Inflamed
tissue can also release serine proteases that are able to generate uTi from its pro-inhibitor
blood cells (WBC) during urinary tract and respiratory infections (Pugia et al. 2002,
2004). Low molecular weight (LMW) urinary proteins also increase during febrile
proteinuria. These LMW proteins are not reabsorbed by the proximal tubular.
Microalbuminuria (30300 mg/L of albumin) is also detected during fever (Pugia
et al. 2002). In cell models uTi has been shown to shut down the proteases need for
tubular re-absorption and potentially facilitating this excretion of LMW proteinuria
of during fever (See Chap. 10). Febrile proteinuria is gone once WBC returns to
normal and fever is gone. This temporary condition does not cause loss of renal
function or change glomerular filtration rates.
This potent anti-inflammatory agent has been used as an anti-shock treatment and
to protect tissues by suppressing vascular dilation, coagulation, and smooth muscle
contraction during wound healing (Pugia and Lott 2005). Studies have shown uTi to
protect kidney tissues from immune mediated apoptosis in animal models during
glomerulonephritis (Koizumi et al. 2000; Takahashi et al. 2001; Nakakuki et al.
1996). There are also uTi released in response to chronically inflamed tissue. The
benefits of these protective effects of uTi have been shown during disseminated intravascular coagulation, ischemia-reperfusion injury, endothelial injury and acute renal
131
Tissue damage
Glomerular leakage
Blood flow
Disease
progression
Tissue
recovery
Blood
Urine
Microalbuminuria & proteinuria
Blood
Urine
Tissue specific markers
Fig. 7.4 Tissue recovery or disease progression. White blood cells such as Neutrophils and
Macrophages lead to lesions in the basement membrane. Damage continues without a strong antiinflammatory response by urinary trypsin inhibitors (uTi) to shut down the immune mediated
apoptosis. Microalbuminuria is first detectable before the loss of renal function or of change in
glomerular filtration by blood markers. As tissue damages progresses and the glomerular leakage
of high molecular weight protein occurs and increases the amount of protein spilled into urine.
Regeneration of renal cells such as podocytes is possible at early stages and recovery of kidney
possible. In absence of regeneration, complement form and the disease will progress
failure (Eguchi 2003; Murakami 1988; Yamaguchi et al. 2000; Nakatani et al. 2001;
Onbe et al. 1991). uTi has also been shown to reduce release of growth factors and
cytokines, inhibit growth factor activation, and stop protease-activated receptor
(PAR) signaling in cell models (Fig. 7.1) (Pugia et al. 2007b).
Persistent white blood cells (WBC) will mediate cell death in tissues and lead to
glomerular nephritis causing lesions in basement membrane (Cochrane et al. 1965).
Glomerular nephritis is characterized by immune complex deposition in kidney and
is observed with hematuria and proteinuria (McCluskey 1970; Hatano 1984; Couser
1998). Damage can occur through an immune mediated apoptosis process
(Bonventre and Weinberg 2003). Untreated glomerular nephritis progresses to
chronic kidney disease (CKD). The inflammatory response will transition from the
innate immune response to complement and auto immune responses (McCluskey
1970; Pickering et al. 2013). Chronically inflamed tissue will develop glomerular
sclerosis with glomerular C3 fragment deposition (Pickering et al. 2013; Cameron
2003). As glomerular sclerosis develops, the kidney tissue fails to regenerate to
normal state. These chronically inflamed tissues promote proliferation by release of
serine proteases to activate protease-activated receptors (PAR) (Vesey et al. 2013;
132
Coelho et al. 2003; Tull et al. 2012). The key proteases causing this apoptosis and
proliferation are known to be inhibited by uTi (Pugia et al. 2007b). In the absence
of uTi protecting normal tissue, the damage increases and proliferation occurs leading to proteinuria (>300 mg/L) and decreased glomerular filtration rate (GFR)
(Fig. 7.4).
The kidneys can recover if protected from immune cells and allowed to regenerate normally (Yoshida and Honma 2014). The anti-inflammatory response appears
to be central to the ability of the kidney to regenerate and naturally reduce these
complications. Here, uTi is an ideal candidate for assessment of immune cell mediated kidney damage in chronically inflamed kidney tissue. However the role of uTi
in progression from a healthy kidney to damaged tissue is not completely known.
uTi offers value for identifying glomerular nephritis patients under anti-inflammatory stress.
Urinary trypsin inhibitors (uTi) in a urine specimen can be measured by inhibition of trypsin in a dipstick method based on a newly found enzyme substrate (Pugia
et al. 2002, 2004; Jortani et al. 2004). The dipstick is a urine test alternative to a
blood C-reactive protein (CRP), a complete blood count (CBC) or sedimentation
rate in patients with upper respiratory infections and other infections owing to bacteria and surpassed these measures in patients with urinary tract infections (UTI).
Clinical use of inhibition assays for urinary trypsin inhibitors were also demonstrated useful as acute phase reactant and for renal disease by other groups
(Kuwajima et al. 1989, 1992; Takemura et al. 1994; Mizon et al. 2002).
Immunoassays for uTi are typically unable to distinguish among the various
forms of urinary trypsin inhibitors from pro-inhibitors (I--I and P--I) (Pugia et al.
2002; Trefz et al. 1991). The cross-reactivity negates blood specimens where I--I
and P--I are present in healthy patients. Western blot analysis of urine identified
uristatin and bikunin were key forms of uTi (Pugia et al. 2002). A monoclonal antibody (IATI5) produced against I--I also recognized P--I, AMBK and bikunin
(Trefz et al. 1991). Others showed monoclonal antibodies raised against AMBK
were reactive to uristatin, AMBK, and bikunin (Bik) (Kobayashi et al. 2000).
New monoclonal immunoassays were developed that do not show an interfering
cross reactivity to pro-inhibitors in blood and urine (Pugia et al. 2007a). The antibodies uncovered an impact of glycoconjugation, measured certain glycoforms
could detect uristatin Uri and bikunin. The primary uTi remaining in blood is Bik
with two Kunitz inhibitor domains and O-linked and N-linked polysaccharides. In
urine, Uri are derivatives of Bik, lacking the O-linked glycoconjugates chondroitin
sulfate chains are often found along with Bik. The urine immunoassay has been
used to measure both Bik and Uri in urine.
The immunoassay detects forms that are more common in kidney disease, thus
we explored the application of this immunoassays for kidney diseases by making
use of an extensive clinical data and sample bank from previous studies (n = 6294
patients). It was possible that these new uTi assays could predict glomerular nephritis progressing to chronic kidney disease as an indicator of damaging inflammation
and infection. The goal was to test the role of Uri and Bik and to better understand
the involvement in the stages of nephritis.
7.2
7.2.1
133
Methods
Immunoassay for Clinical Analysis
Patients urine specimens were tested using a competitive ELISA method previously described with new antibodies (See Table 7.1) (Pugia et al. 2007a). The
immunoassay measures both Uri and Bik but does not cross-react with II or
pI. Calibration of the immunoassay used Uri isolated from human patients with
chronic renal failure (SciPac, Sittingbourne, Kent, UK, product code P392-1). All
lots were stable at 20 C to 70 C, either in solution or as lyophilized solids and
previously described (Pugia et al. 2007a).
The ELISA Method for Uri is initiated by coating each well with 0.1 ug/well Uri
in TBS (Tris buffered saline) overnight at 4 C followed by washing five times with
300 L of TBS per well (Elx 405 Auto Plate Washer, Biotek Instruments, Winooski,
VT). The plate is blocked with 300 L of SuperBlocker per well, incubated for
60 min at 37.5 C (JitterBug Microplate Shaker, Beokel Scientific, Feasterville,
PA), and finally washed the plates five times with 300 L of TBS-with 05 %
Tween-20 per well. Calibrators made with Uri are diluted with uristatin antibody
conjugated to alkaline phoshatase (ALP-Mab) conjugates in TBS at 0.084 g/L
(mg/L). A sample is made by diluting urine with the required a 100-fold dilution of
ALP-Mab. Diluted calibrator or specimen in 100 l volumes are loaded into the
coated wells, incubated at 35 C for 1 h, and washed five times with 300 LTBSTween per well. The antibody binding is measured at 37 C over 30 min after addition of PNPP 100 L/well (100 ng/mL) (1-Step solution, Pierce), by reading the
change in 410 nm absorbance on a FLx 800 microplate Reader (Biotek Instruments).
(See on-line supplement for ELISA Method for Uristatin for additional details at
www.extras.springer.com/2015/978-3-319-21926-4). A uristatin positive is considerd as 12 mg/L.
Table 7.1 Antibodies for Uristatin and Bikunin
Antibody
Uristatin/Bikunin N-link glycan binding
Raised against
Human Uristatin
Bikunin/Uristatin tyrpsin inhibitory peptide

domain binding
Human Uristatin
mAb for Uristatin/Bikunin conjugated to

ALP
pAb for Uristatin/Bikunin conjugated to
ALP
Human Uristatin
Urinary trypsin
inhibitors
Clone/lot
ATCC 420-5D11.5G8.1E4
PTA-7744
ATCC 421-3G5.4C5.3B6
PTA-7746
ATCC 421-5G8.1AB.5C1
PTA-7745
ATCC 421-3G5.4C5.3B6
Rabbit polyconal
Legend
Antibodies used for clinical analysis are shown. The urinary trypsin containing used to produce the
polyclonal contained both Uri and Bik. Monoclonal antibodies were produced with only purified
Uri lacking Bik. ATCC deposited cell lines are available from ATCC upon request a [email protected]
134
Some patients urine specimens were also tested using a western blot method
with rabbit polyclonal antibody conjugated to ALP A described (Table 7.1) (Pugia
et al. 2004). The antibody cross-reacted with Uri, Bik, P-a-I, I-a-I, and AMBP. Urine
specimens from diabetics were characterized using a commercial pre-cast SDSPAGE gel system (BioRad). In brief, urine from diabetic patients was concentrated
to about 20 g total protein and loaded onto the gel along with molecular-weight
markers and Bik standards. The SDS-PAGE gel was ran in 25 mM Tris, pH 8.3,
192 mM glycine, and 0.1 % SDS buffer at 110 V in for 11.5 h until complete. The
gel was transferred to the blotting cassette using filter paper. The gel soaked in
transfer buffer and proteins transfers to nitrocellulose paper at 30 V in a 4 C chamber overnight. The membrane was washed in TBS buffer plus 0.1 % Tween-20,
blocked with 5 % BSA (Sigma A3059) and reacted rabbit anti-uristatin antiserum at
a dilution of 1:250,000 at 4 C overnight with gentle agitation. The membrane was
incubated with the alkaline phosphate substrate and color developed and recorded.
(See on-line supplement for Western Blot Methods for additional details at www.
extras.springer.com/2015/978-3-319-21926-4)
7.2.2
Sample and Data Bank
All clinical and diagnostic data was collated into one database from four previous
studies. The combined population (n = 6294) was intended to mimic the natural
prevalence of renal disease (3 %), urinary tract infection (3 %), nephritis (15 %) and
systemic infection (2 %) expected in a general population without exclusion or
selection criteria. Clinical samples stored at 70 C were thawed and used once for
additional testing for uTi by the Uri ELISA immunoassay methods. Uri values by
ELISA of <12 mg/L was considered normal. Test strips for urinary tract infection
had been tested in original studies. Here a Uri by dipstick of < = 7.5 mg uristatin/g
creatinine or <12.5 mg/L Uri was considered normal.
The retested samples from the original cohorts are summarized in Table 7.2
along with the diagnosis of sub-groups within each and criteria used. Urinalysis
strip testing included pads for detection of albumin, protein, creatinine, occult
blood, leukocytes, glucose, and ketone (MULTISTIX PRO, Siemens Healthcare
Diagnostics). Potential nephritis was indicated with as a urinalysis strip with a positive for albumin, protein or occult blood.
7.2.3
Health Screen Collection
Random urine specimens were collected from a Japanese health screening study as
part of an annual physical examination by a General Practice physician (Pugia et al.
2002). Of the original study samples, 4022 were available. Of these, 3665 were
135
Table 7.2 Clinical sample and data bank used for uTi immunoassay testing
Study
Health screen
(n = 4022)
Diagnosisa
Normal (n = 3665)
Asymptomatic GN(n = 292)
Bacteriuria (n = 3)
Systemic infection (n = 62)
Urinary tract
infection
(n = 1743)
Hospital
infections
(n = 341)
Normal (n = 788)
Symptomatic GN (n = 731)
Bacteriuria (n = 224)
Normal (n = 137)
Asymptomatic GN (n = 67)
Bacteriuria (n = 77)
Hospital
patients
(n = 188)
Normal (n = 137)
Asymptomatic GN (n = 46)
Bacteriuria (n = 3)
Urinalysis Criteriab
Urinalysis strip
negative
Urinalysis strip
positive
Urinalysis strip
positive
Not used
Urinalysis strip
negative
Urinalysis strip
positive
Not used
Urinalysis strip
negative
Urinalysis strip
positive
Not used
Not used
Urinalysis strip
negative
Urinalysis strip
positive
Not used
Not used
Additional criteria usedc

none
Neg. BUN, CRE
Pos. urine culture
Pos. CBC, ESR, CRP
or fever
Neg. urine culture
Neg. urine culture
Pos. urine culture
Neg. CBC, CRP
& bloodculture
Neg. CBC, CRP, blood
culture
Pos. urine culture
Pos CBC, CRP or blood
culture
Neg CBC
Neg CBC
Pos. urine culture
Pos CBC
Legend
Diagnosis follows typical standard of practice with appropriate follow up testing and physician
diagnostic judgment based on these tests
b
Urinalysis strips were run on all samples, and used as first test result and additional follow up
testing used for diagnosis . For indication of nephritis, only the protein and occult blood result were
used. For normal, all results were considered
c
In cases of pre-existing renal disease, the strip result was not used for diagnosis and blood results
are used. In the case of infection the follow up urine culture and blood testing were used and strip
result was not used for diagnosis
a
urine samples were from apparently healthy patients, 292 were urine samples from
patients with potential renal disease with previous abnormal blood urea nitrogen
(BUN) or serum creatinine and were follow-up visits, and 205 urine samples patients
with diagnoses of potential infects and/or fever (> = 38 o C) were followed up
erythrocyte sediment rate (ESR) or abnormal serum C-reactive protein (CRP).
136
7.2.4
Urinary Tract Infection Collection
Uri Immunoassays were run on 1743 of the clean catch urine samples were collected patients with suspected urinary tract infection (Pugia et al. 2004). These
samples were consecutive samples sent for urine culture. Of the 1743 specimens,
244 had bacteruria with positive cultures for gram-positive bacteria and/or gramnegative bacteria at 105 organisms/ml. Urine sediments were centrifuged and
examined by microscopic to estimate of the counts of erythrocytes, leukocytes,
number and types of casts, epithelial cells, crystals, clarity, color, and mucus
(Freeman and Beeler 1983; Schumann 1996). A finding of yeast was considered as
a negative result for bacterial infection. Of the 1743 specimens, 19 had renal disease
based on urine microscopic (renal cells cast) and gross proteinuria.
7.2.5
Hospital Infection Collection
Immunoassays for Uri were run on 341 collected patients with presenting with
upper respiratory infection and several other systemic types of infection or presumed healthy (Jortani et al. 2004). Matched blood samples were collected and all
samples test by CBC, CRP, and erythrocyte sedimentation rate (ESR). Patient with
infections were diagnosed as bacterial, probable bacteria, viral and probable viral
by two reviewers (board-certified microbiologist/pathologist and a clinical chemist)
in a double blind procedure. Blood culture and urine culture were run for all patients
with suspected infection. Patients having been diagnosed with HIV, leukemia, parasitic or yeast infections were excluded from this collection.
7.2.6
Hospital Patient Collection
Immunoassays for Uri were run on 188 patients presenting with cardiovascular disease or presumed healthy (See Chap. 9). Cardiology work up was provided for all
patients and match blood and urines samples were collected. All samples were test
by CBC and CRP and systemic infections identified (n = 2). Chart review only indicated 1 with pre-existing renal disease patients.
7.3
Results
Uri and Bik were commonly found by western blot in urines collected from diabetics with glomerular nephritis (See on-line supplement for example data at www.
extras.springer.com/2015/978-3-319-21926-4). No detectable urinary P-a-I or I-a-I
137

Table 7.3 Uri immunoassay results for glomerular nephritis follow up
Diagnosis
Normal (n = 4747)
Asymptomatic Glomerular nephritis
(n = 385)
Symptomatic Glomerular nephritis
(n = 731)
Urinary Tract Infection (n = 307)
Systemic bacterial infection (n = 122)
total
Uristatin
<12 mg/L
4544
351
Uristatin
> = 12 mg/L
203
34
Uristatin
<25 mg/L
4683
378
Uristatin
> = 25 mg/L
64
7
533
198
648
83
200
53
5681
107
69
611
257
86
6052
50
36
240
Table 7.4 Expected Uri during infection follow up

Condition
Normal
Viral Infection
Bacterial Infection
Urinary tract infection
Glomerular nephritis
Uristatin
neg
neg
pos
pos
pos
Urine culture
neg
neg
neg
pos
neg
Blood test CBC

neg
pos
pos
neg
neg
Blood test CRP

neg
pos
pos
neg
neg
was found in any urine by western blots. Smaller Uri fragments were found most
diabetics with chronic kidney disease (CKD) or urinary tract infection. The Uri
immunoassay agreed with western blot and allowed measurements of both Uri and
Bik to be made on large sample banks.
Uri immunoassay results for 6292 urine samples were used to test for correlation
to glomerular nephritis (Table 7.3). Urine samples from health patients (n = 4747)
lacked Uri, with 95.7 % of patients sample having less than 12 mg/L of Uri and
98.7 % less than <25 mg/L. These patients all were negative for glomerular nephritis by the criteria of lacking proteinuria or hematuria or CKD. Hospital patients had
other conditions such as cardiovascular diseases, cancers, and metabolic diseases
(e.g. diabetes, etc.) but lacked known infections. Cases with suspected urinary tract
infections were considered normal if negative by urine bacteria culture (<104 cell/
mL). Cases with suspected systemic infections were considered normal by CBC
and CRP. The high % of true negatives by Uri immunoassay makes it suitable for
follow up of proteinuria or hematuria for general population and gives a very high
negative predictive value.
Suspected infections are followed-up regardless of the Uri immunoassay result. In
the presence of systemic bacterial infection or UTI, the Uri can be positive due to the
innate anti-inflammatory response to the infection. Uri and Bik abnormally elevate
when white blood cells in blood and urine are clearly abnormally elevated. Elevated
WBCs are characteristic of acute systemic bacterial and urinary tract infections and
an infection work up is required (Table 7.4). Uri immunoassay results were 12 mg/L
in 35 % of UTI patients and 25 mg/L in 12 %. Of these UTI patients, 84 % were
138

All patients (n=6292)
No
Yes
Proteinuria or
Hematuria ?
Normal (n=4887)
Gomerular
Nephritis (n=1407)
Yes
No
Infection or
uristatin
Indicated? Asympotmatic
Gomerular
Nephritis (n=351)
No
Uristatin
Still positive ?
Symptomatic
Glomerular
Nephritis(n=533)
Potential
infection (n=1194)
Follow up for
infection
Yes
Symptomatic
Glomerular
Nephritis with Antiinflammatory
response (n=198)
Resolved
infection
(n=419)
Fig. 7.5 Flow chart. Proteinuira or hematuria was an indicator of glomerular nephritis in 1407 of
6292 patients tested. Of these, only 351 patients had asymptomatic GN. The remaining 1194
patients had potential infections based on symptoms of urinary tract infection, systemic infections,
and/or a positive Uri value >12 mg/L. These were all followed by urine culture and CBC. This
resulted in 419 patients as needing to be treated for infections. Those remaining were sorted
according to Uri value as 198 cases of symptomatic GN with anti-inflammatory stress and 533
cases without this stress. Proteinuria or hematuria is often resolved with anti-inflammatories, however persistent proteinuria or hematuria in absence of infection continues in progressing glomerular nephritis. Patients with positive Uristatin indicate with atypical anti-inflammatory stress
either uTi, WBC, or NIT strip positive. In systemic bacterial infection patients, Uri
values were 12 mg/L in 57 % of patients and 25 mg/L in 41 % of patients. CRP
and CBC did detect systemic infection but did not detect UTI. Follow up for infection
with any positive Uri value is recommended (Fig. 7.5). Uristatins ability to detect
infections helps kidney disease management, as these infections are known to damage
kidneys. Management of infections is part of typical standards of care for diabetics.
Uri immunoassay results, unlike CRP, were not elevated by viral infections.
Common viral infections (CBC negative for granulocytosis) did not elevate uTi, such
as viral tonsillitis (Adenovirus), pneumonia, enterocolitis, gastroentritis, viral meningitis & bronchiolitis (viral respiratory), mumps (Rubulavirus) varicella (chickenpox),
rotavirus, influenza (A/B), measles (rubeola) mononucleosis (EBV), hepatitis, and
herpes zoster. Uri was also not elevated in conditions such as asthma, allergies or autoimmune diseases like systemic lupus erythematosus or amyloidosis. However several
conditions do interfere with Uri usage. AIDS/HIV patients can be falsely negative for
Uri. Uri is elevated in cancers which increase white blood cells like multiple myeloma.
139
Predicting progression of patients with glomerular nephritis (GN) to CKD is an

important unmet need. An initial finding of GN based on proteinuria or hematuria
in urinalysis requires significant follow up (Pugia et al. 2000). In diabetes, the prevalence of diabetic hematuria or proteinuria is as high as 40 %. However, progression
to CKD occurs in only <13 % of cases and end stage renal disease (ESRD) occurs
in <6 % of population (Park 2004). As inflammation becomes more chronic, the
anti-inflammatory response increases and Uri values become abnormally elevated.
Patients with asymptomatic GN who have hematuria or proteinuria start to report
the classical symptoms of chronic inflammation with pain and swelling. Uri immunoassay results were 12 mg/L in 9 % of asymptomatic GN patients and 25 mg/L
in 2 %. Patients with symptomatic GN were Uri positives with 12 mg/L in 27 %
of cases and 25 mg/L in 11 %.
The anti-inflammatory status in these 27 % of patients with elevated Uri is
expected to impact the prognosis. While Uri is renal protective, a population with
chronic exposure to inflammation is expected to have reduced kidney regeneration
due to anti-proliferative cell signaling of Uri (Chap. 10). Uri does protect kidney
damage by the white blood cells until tissue could clear the WBCs after infection is
addressed. The immune response often continues in the absence of detectable infection. Uri also inhibits proteolysis in the renal tubular cell, which correlates with
reduced re-absorption and adds additional proteinuria. Controlling the action of this
cascade would seem necessary to prevent further damage to the normal tissues.
Patients with symptoms of chronic urinary inflammation without overt infection are
difficult to resolve. Here, Uri allows identification of patients under antiinflammatory stress (Fig. 7.5).
7.4
Conclusion
In this report, we have shown the value of urinary trypsin inhibitors (uTi) as a measure of anti-inflammatory stress during glomerular nephritis (GN). In management
of diabetic GN today, yearly urinalysis screening is recommended with identified
positives follow up regularly with repeat testing. Once infection is ruled out, patients
in the chronic inflammatory phase without active infection are not further assessed.
Use of Uri as a measure of chronic anti-inflammatory stress is a possible tool to
assess the risk but requires follow up studies to determine the impact on progression
to CKD. The Uri immunoassay could aid in the development of drugs for glomerular nephritis by allowing screening of patients for renal regeneration therapies,
immune cell suppression therapies and to allow identification of non-responders.
Acknowledgements We would like to thank Ronald Sommer, Dr Hai-Hang Kuo, and Dr. Manju
Basu for their many hours of effort in performing uristatin ELISA testing. We would also like to
thank, Dr. Roland Valdes, Mr. Glen Renfrew, Dr. Darryl L. Gopual, Dr. John Lott and Dr. Chris
Price for their helpful guidance to build this patient sample bank, with helpful advice over many
years and the various studies.
140
Supplements
ELISA Method for uristatin
Western Blot methods
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Saint S, Scholes D, Fihn SD, Farrell RG, Stamm WE (1991) The effectiveness of a clinical practice
guideline for the management of presumed uncomplicated urinary tract infection in women.
Am J Med 106:635641
Schumann GB (1996) Comparing slide systems for microscopic analysis. Lab Med 27:15
Takahashi M, Sawaguchi T, Sawaguchi A, Suzuki T (2001) The cytoprotective effect of protease
inhibitor on programmed cell death of endothelial cell. Tokyo Joshi Ika Daigaku Zasshi
71:669678
Takemura T et al (1994) A clinical study of urinary trypsin inhibitor, an acute phase reactant in
urine, in acute pediatric infectious diseases. J Inflamm 14:5357
Trefz G et al (1991) Establishment of an enzyme-linked immunosorbent assay for urinary trypsin
inhibitor by using a monoclonal antibody. J Immunoassay 12:347369
Tull SP et al (2012) PR3 and elastase alter PAR1 signaling and trigger vWF release via a calciumindependent mechanism from glomerular endothelial cells. PLoS One 7(8):e43916
Vesey DA et al (2013) PAR2-induced inflammatory responses in human kidney tubular epithelial
cells. Am J Physiol Renal Physiol 304(6):F737F750
Yamaguchi Y, Ohshiro H, Nagao Y, Odawara K, Okabe K, Hidaka H et al (2000) Urinary trypsin
inhibitor reduces C-X-C chemokine production in rat liver ischemia/reperfusion. J Surg Res
94:107115
Yoshida M, Honma S (2014) Regeneration of injured renal tubules. J Pharmacol Sci 124(2):
117122
Chapter 8
Acute Response of Uristatin in Surgery

Mitchell H. Rosner and Michael Pugia
Abstract Prediction of those patients at highest risk for the development of acute
kidney injury (AKI) allows for institution of specific kidney protective strategies.
Novel urinary biomarkers could serve as important pre-exposure risk stratification
tools. In this study, we identify a novel urinary biomarker, uristatin, that has the ability to aid in the determination of the risk of AKI in patients undergoing cardiac
bypass (CPB) surgery in a single center. Thirty-six patients were enrolled and
underwent CPB surgery. Fifteen patients developed AKI and pre-operative predictors of AKI on univariate analysis included: male gender (OR 6.88 (CI 1.56, 30.36);
ejection fraction <35 % (OR 3.18 (CI 1.37, 7.38)) and CPB time >85 min (OR 2.54
(CI 1.183, 5, 47). On multivariate analysis, those patients with a urinary uristatin
level <40 mg/L had an OR for developing AKI of 3.41 (CI 1.23, 9.44). There was
an inverse correlation between pre-operative uristatin levels and the risk of AKI
with those patients having a baseline level >80 mg/L having at least a fivefold lower
risk of AKI than those patients with baseline levels <40 mg/L. Urine uristatin as a
pre-operative predictive marker for development of AKI post-operatively had an
area under the curve (AUC) of 0.855. In this cohort, pre-operative levels of either
urine or serum IL-6 were not predictive for the development of AKI. Urine uristatin
levels may allow for better discrimination of pre-operative AKI risk and allow for
targeted preventative strategies
M.H. Rosner, MD, FACP (*)

Division of Nephrology, Department of Medicine, University of Virginia HSC,
Charlottesville, VA 22908, USA
M. Pugia
DOI 10.1007/978-3-319-21927-1_8
143
144
8.1
M.H. Rosner and M. Pugia
Introduction
Acute kidney injury (AKI) can occur in critically ill patients (Uchino 2005), after
surgery (Rosner and Okusa 2006) and due to drug or contrast agent nephrotoxicity
(Park et al. 2010; Alexopoulos et al. 2010; Nadim 2008). Defining patients at high
risk for AKI has typically relied on risk stratification tools that utilize a combination
of clinical and laboratory parameters to define a risk score. However, these scoring
systems typically have good negative predictive power but suffer from poor positive
predictive power. Biomarker-based risk profiles have sought to improve predictive
power (Rosner 2009).
An ideal setting for testing such risk profiling may be in those patients undergoing cardiopulmonary bypass surgery (CPB). Various risk assessment tools have
been developed and validated for specific clinical scenarios: for instance, precardiac catheterization or pre-CPB surgery (Thakar et al. 2005; Chertow et al.
1997). AKI occurs in about 18 % of CPB patients and 26 % require hemodialysis
(Khilji and Khan 2004). In all settings, AKI is associated with significant morbidity
and mortality and efforts to find therapeutic agents to prevent or hasten resolution of
AKI have been largely unsuccessful (Bagshaw et al. 2005; Hoste et al. 2003).
Reliance on serum creatinine for the diagnosis of AKI has hampered the use of
therapeutics (Jo et al. 2007; Star 1998; Liu and Glidden 2009). Serum creatinine
assessments in the first 24 h do not predict the glomerular filtration rate (GFR) (Soni
et al. 2009; Moore 2010). Serum creatinine tends to rise 2448 h after surgery and
too late for therapeutic intervention.
Recently, several pro-inflammatory biomarkers have shown promise for the early
detection of AKI (Kinsey et al. 2008). Pro-inflammatory cytokines such as interleukin (IL) play a role in inflammatory processes involved in the development of AKI
when measured post-surgically but not pre surgically (Liu 2009; Parikh et al. 2004).
Neutrophil Gelatinase-Associated Lipocalin (NGAL) released from immune cells
during inflammation precedes the rise in serum creatinine hours after surgery in
patients progressing to AKI (Haase et al. 2009; Bennett et al. 2008; Mishra et al.
2005). Acute tubular necrosis markers such as Adenosine Deaminase Binding
Protein (ABP-26) and liver type Fatty Acid Binding Protein (L-FABP) are useful
measures of renal injury from nephrotoxin exposure (Nakamura et al. 2006; Doi
et al. 2010; Mueller et al. 1990; Thompson et al. 1985; Moore 2010). Cystatin C and
Kidney Injury Molecule 1 (KIM) have shown benefits for detection of pre-existing
GFR dysfunction over creatinine (Koyner et al. 2008; Bonventre 2008).
Anti-inflammatory proteins such as Adiponectin (Adpn) and have also been correlated to renal status (Shen et al. 2008). Urinary trypsin inhibitors (uTi) such as
uristatin (Uri) and bikunin (Bik) play a key role in AKI by halting immune mediated inflammatory processes (Pugia et al. 2007a). These serine protease inhibitors
reduce cytokine and growth factor release during stresses such as renal ischemia by
shutting down the trypsin family of serine proteases such as trypsin, elastase, kallikrein, chymotrypsin, plasmin and cathepsin (Pugia et al. 2007b; Fries and Blom
2000). uTi are readily filtered by the glomeruli and are increased in the urine of
8 Acute Response of Uristatin in Surgery
145
patients with most inflammatory disorders (Piette et al. 1992),bacterial infections

(Pugia et al. 2002), neoplasia (Takubo et al. 2001), and kidney stones (Moriyama
et al. 2001).
The release of elastase and cathepsin G from immune cells is implicated in basement membrane damage (Johnson et al. 1998; Takahashi et al. 2001). uTi have been
shown to protect endothelial cells during pre-eclampsia (Maradny et al. 1994), suppresses neutrophile elastase and protects respiratory function in CPB patients
(Sugita et al. 2002), have a protective effect against disseminated intravascular
coagulation (DIC) (Murakami 1988) and hepatic microvascular injury (Itabashi
et al. 2003). As such, uTi represent a potential target for biomarker development
based upon a putative pathophysiological role. As an acute-phase indicator, uTi are
generated rapidly owing to the pro-inhibitor inter--inhibitor being available in
blood prior to the time of injury and before surgery due to innate response to organ
damage. Patients with impaired innate response have lower uTi values (Masaki
et al. 2003). Surgery induces an increase in uTi which parallels tissue damage (Endo
2003; Masaki et al. 2003). uTi usually continue to rise during the course of trauma.
The amount of urine uTi excreted typically increases 24 after the surgery to a peak
on about the third day. By the seventh day, uTi usually decreases to its basal value.
Our goal was to test the Uri immunoassay which detected Uri and Bik in urine
samples using a CPB patient cohort for risk prediction for the development of AKI
with pre and post-operative samples. An improved prediction model for the probability of developing AKI after cardiopulmonary bypass was tested. Comparisons
were made between several biomarkers.
8.2
8.2.1
Methods
Patient Population
The patient population, was enrolled prospectively, and is described in Table 8.1.
The protocol was reviewed and approved by the Institutional Review Board of the
University of Virginia. We screened 78 patients and enrolled 36 patients undergoing
CPB at a single institution. Reasons for screening failure included: failure to obtain
consent (16 patients), baseline serum creatinine >2.0 mg/dL (12 patients), WBC
>10,000/mm3 (4 patients), recent IV contrast exposure (4 patients), and postoperative anuria (1 patient). AKI as defined as a persistent (>48 h) rise in serum
creatinine >0.3 mg/dL occurred in 15 patients (42 %) within the 48 h postCPB. Based upon the RIFLE criteria (using serum creatinine criteria), 6 patients
were in the Risk, 7 patients in the Injury and 3 patients in the Failure categories. The
characteristics of the patients who developed AKI and those who did not are displayed in Table 8.1. While the baseline serum creatinine and eGFR were higher in
the group that developed AKI, there was no statistically significant difference
between baseline renal function between the two groups.
146
Table 8.1 Characteristics of the patient population

Variable
Age in years
Gender (male (%))
Diabetes (%)
Baseline sCr (mg/dL)
sCr at 36 h post-op (mg/dL)
Baseline eGFR (ml/min/1.73 m2)
Left ventricular ejection fraction <40 %
(%)
Peripheral vascular disease (%)
Baseline WBC (cells/mm3)
Surgery type:
CABG (%)
Valve only (%)
Combined (%)
CPB time (min)
Cross clamp time (min)
Use of pressors for >24 h postoperatively (%)
CCF risk score
Need for post-operative dialysis (%)
In-hospital death (%)
Patients with AKI

(n = 15)
63.8 7.88
11 (73)
11 (73)
1.69 0.44
2.26 0.54
53 15
5 (33)
Patients without
AKI (n = 21)
67.4 7.18
6 (29)
15 (7)
1.25 0.35
1.43 0.36
74 18
8 (38)
3 (20)
8.7 3.1
7 (47)
5 (33)
3 (20)
4 (19)
7.66 2.6
12 (57)
7 (33)
2 (10)
0.89
0.54
0.56
118 33
80 12
5 (33)
103 29
70 9
5 (24)
0.64
0.10
0.21
6.9 2.1
3 (20)
2 (13)
4.87 1.9
0
1 (5)
0.06
0.07
0.4
P value
0.87
0.20
0.76
0.10
0.04
0.08
0.91
Legend: sCr serum creatinine, eGFR estimated glomerular filtration rate, CABG coronary artery
bypass grafting, CPB cardiopulmonary bypass, AKI acute kidney injury, CCF Cleveland Clinic
Foundation
A preoperative risk assessment for the occurrence of AKI was performed using
both the renal risk stratification system of Chertow as well as the Cleveland Clinic
Foundation (CCF) scoring system (excludes systemic infection) (Thakar et al. 2005;
Chertow et al. 1997). Based upon these scoring system which utilize clinical variables and baseline renal function, there was no statistically significant difference in
baseline risk between those patients who developed AKI and those who did not.
However, there was a trend toward higher CCF scores in the group that developed
AKI (see Table 8.1). Most patients who developed AKI had white blood cells in
urine (n = 13) indicating an innate immune mediated inflammatory reaction. Patients
who did not develop AKI did not have WBC in their urine.
Exclusion criteria included: age <18 years, failure to obtain consent, recent
(within 3 month) development of AKI (defined as a rise in serum creatinine greater
than 0.3 mg/dL from a stable baseline creatinine if this data was available), recent
infectious disease (defined as a documented fever >38 C within 1 week of surgery,
use of antibiotics within 7 days of surgery with the exclusion of pre-operative or
intra-operative prophylactic antibiotics), or a white blood cell count (WBC)
>10,000/mm3, end-stage renal disease, baseline serum creatinine >2.0 mg/dL, use
147
of intra-aortic balloon pump, pre-operative use of inotropic agents or vasopressors,

pre-operative cardiogenic shock, post-operative anuria and pre-operative use of
intravenous contrast within 7 days of the surgery. At total of 78 patients were
screened and 36 enrolled. Post-operatively all patient physiological parameters
(blood pressure, pulse, temperature, urine output, use of inotropes/vasopressors)
were recorded.
8.2.2
Sample Collection
Time matched blood and urine specimens were collected immediately pre-operatively,
and then at 13 h, 57 h and 911 h on the day of surgery and every day thereafter
out to 6 days post-operatively. Blood was analyzed for creatinine, electrolytes, white
blood cell count, hemoglobin and biomarkers as described below. Duplicate urine
and blood specimens were stored at 70 C until analyzed for biomarkers. A protease
inhibitor cocktail (contains 0.1 mg/mL Pefabloc SC (Centerchem Inc, Norwalk,
CT), 0.1 mg/mL leupeptin (SigmaAldrich, St. Louis MO), and 1 mM sodium azide
(SigmaAldrich, St. Louis MO)) was added to each urine sample. Ejection fraction
was estimated visually by surgeon. Surgical time for the bypass (min) and urinary
output (ml/h) were recorded. Granular casts were measured on urine sample at time
of collection by spinning the urine and viewing under high power light microscopy.
Serum creatinine values and a complete white blood cell count (WBC) was obtained
using specimens sent to the hospital laboratory. Duplicate specimens were stored at
70 C until analyzer for remaining biomarkers.
8.2.3
Biomarker Analysis
Quantitative urinalysis for albumin and creatinine were measured using the DCA
Vantage instrument (Siemens Healthcare Diagnostics, Tarrytown, New York).
Urinalysis strip results for occult blood, creatinine, protein, albumin, nitrite, ketone,
glucose, pH and luekocytes were measured using the CLINITEK Status instrument
(Siemens Healthcare Diagnostics, Tarrytown, New York) with the MULTISTIX
PRO 10LS and CLINITEK Microalbumin. Casts are determined by spinning the
urine and viewing under high power light microscopy
Enzyme-linked immunosorbent assay (ELISA) measurements were made with
commercially available kits. Urine and serum IL-6 used kits from E-Bioscience,
San Diego, CA (Cat No. 88706). Control and standards were preformed according
to manufacturers instructions. Serum measurement of Cystatin C used kits from
Biovendor Laboratory Medicine Inc, Czech Republic (Cat No RD191009100).
Serum measurement of Adpn used kits from Panomics Inc, Fremont, CA (Cat No
K1001-1). Urine measurement of NGAL used kits from Affinity BioReagents Inc,
Colden, CO (Cat No 037). All kits used monoclonal antibodies directed against the
148
human peptide in a sandwich assay format with enzyme labels. Urine assays for
Uristatin were performed by Brendan Bioscience (Hopewell MA) following literature competitive antibody ELISA method (Pugia et al. 2007b) (see Chap. 8 and
Supplement Materials). Control and standards were preformed according to manufactures instructions.
Urine assays for ABP-26 was performed by Brendan Bioscience (Hopewell,
MA) following the literature ELISA Nephroscreen method (Thompson et al. 1985).
Briefly, ELISA plates (Corning, 9018) were coated overnight at 48 C with antiABP-26 (Uro 4 antibody, 804099, Covance) in bicarbonate buffer, pH 9.6 at 8 ug/
ml. The plates were washed 2X with coating buffer then blocked with bicarbonate
buffer containing 0.2 % bovine serum albumin (Sigma, A8022) for 2 h at 48 C. The
block was decanted and the plates were stabilized with 10 % sucrose for 1 h at room
temperature (RT). The stabilizer was decanted and the plates were dried in vaco
then sealed in a mylar pouch with desiccant. The detection conjugate was made by
conjugating a second anti-ABP26 antibody (Uro-4a, 804599, Covance Inc) to
HRP (Uro4a-HRP). The standard curve was constructed by diluting ABP26 (R&D
Systems, 1180-SE) from 500 to 7.81 ng/ml in wash/sample diluent (W/SD) prior to
adding it to the plate along with the samples which were diluted 1:100 in W/SD.
Urine samples and standards were diluted and 100 L was added to each well
and incubated for 4 h at RT. The fluid was decanted and the plate was washed
3X. The Uro4a-HRP was added to each well and incubated for 2 h at RT. The conjugate was decanted and the plate was washed 3X. The OPD substrate (Sigma,
P-8412, 4 mg/ml in 100 mM citrate, pH 5.0) was added and incubated at RT for
2030 min. The reaction was stopped using 3 N H2SO4 and the absorbance was read
at A492. A linear regression was performed using the data from the standard curve.
The concentration of ABP-26 was calculated for each sample using the regression
values derived from the standard curve.
8.2.4
Statistical Analysis
AKI was defined as a rise in serum creatinine >0.3 mg/dL occurring within 48 h
after CPB surgery and sustained for >48 h. Patients were parsed into two groups
(AKI v. no AKI) based upon meeting this criteria of AKI. SAS version 8.2 was used
for analyses. Values for groups are shown in Tables 8.2 and 8.3. To compare continuous variables, we used a two-sample t test or MannWhitney rank sum test, and
to compare categorical variables we used the 2 or Fishers exact test, as indicated.
To measure the sensitivity and specificity for urine uristatin and IL-6 at different
cutoff values, a conventional receiver-operating characteristic (ROC) curve was
generated for preoperative uristatin and IL-6. We calculated the area under the curve
to ascertain the quality of uristatin as a biomarker. An area of 0.5 is no better than
expected by chance, whereas a value of 1.0 signifies a perfect biomarker. Univariate
and multivariate stepwise multiple logistic regression analyses were undertaken to
assess predictors of acute kidney injury. Potential independent predictor variables
WBC
Urine output
Urine bypass
time
Ejection
fraction
Clinical
variables
Age
ARF
0
1
0
1
0
1
0
1
0
1
n
22
15
22
15
22
15
22
15
22
15
Mean
63.82
67.40
40.45
28.67
70.05
80.13
96.36
87.33
7.66
8.69
SD
7.88
7.18
8.99
11.41
8.38
12.08
23.96
32.94
2.61
3.07
SE
1.68
1.85
1.92
2.95
1.79
3.12
5.11
8.51
0.56
0.79
Median
64.00
68.00
37.50
25.00
68.50
78.00
95.00
80.00
7.35
8.00
Table 8.2 Summary statistics for the demographic and clinical variables
GM
63.36
67.04
39.50
26.89
69.60
79.27
93.62
76.94
7.25
8.27
Min
51.00
56.00
25.00
15.00
60.00
60.00
60.00
10.00
4.30
5.60
Max
79.00
78.00
55.00
55.00
94.00
98.00
150.00
125.00
13.40
16.80
Q25
58.25
63.50
35.00
20.00
65.00
72.00
80.00
75.00
5.48
6.55
Q75
67.00
72.50
45.00
30.00
73.50
89.50
118.75
120.00
8.98
9.65

149
Albumin/creatine
Urine creatinine
Urine albumin
Urine ABP-26
Urine IL6
Serum IL6
Response
Serum creatinine
ARF
0
1
0
1
0
1
0
1
0
1
0
1
0
1
n
22
15
22
15
22
15
22
15
22
15
22
15
22
15
Mean
1.43
2.26
749.68
836.35
567.32
150.26
689.02
503.88
135.49
140.09
140.64
96.83
182.18
340.47
SD
0.36
0.54
949.41
1021.92
2321.93
230.66
481.11
565.47
107.77
124.96
40.87
54.43
183.69
430.72
SE
0.08
0.14
202.41
263.86
495.04
59.56
102.57
146.00
22.98
32.26
8.71
14.05
39.16
111.21
Median
1.45
2.10
313.00
327.00
58.10
43.20
574.45
379.00
79.40
67.80
121.50
85.30
127.50
169.00
GM
1.39
2.20
418.21
425.07
57.31
53.51
520.87
297.86
99.66
82.63
135.56
80.31
129.21
163.26
Min
0.80
1.50
68.90
83.20
10.80
3.20
61.10
23.40
21.10
13.60
89.00
20.90
30.00
23.00
Max
2.50
3.30
3751.00
2909.00
10954.00
685.00
1721.80
2352.00
300.00
300.00
230.00
204.00
873.00
1350.00
Q25
1.23
1.95
190.50
167.00
18.23
18.85
337.85
212.50
53.28
29.65
109.25
73.20
73.25
66.00
Q75
1.50
2.40
709.75
865.00
82.18
150.00
933.30
605.20
267.25
300.00
158.75
128.50
211.50
440.50
Table 8.3 Summary statistics for the mean of the patients observations up to and including the time point when the patient was determine to be ARF
150
151
included age, sex, ethnic origin, cardiopulmonary bypass time, ejection fraction,
urine albumin/creatinine, urine and serum IL-6, and urine uristatin. We judged
p < 0 05 significant
8.3
8.3.1
Results
Pre-operative Predictors of AKI
Thirty-six patients were enrolled and underwent CPB surgery. Fifteen patients
developed AKI and pre-operative predictors of AKI on univariate analysis included:
male gender (odds ratio (OR) 6.88 (CI 1.56, 30.36)); ejection fraction <35 % (OR
3.18 (CI 1.37, 7.38)) and CPB time >85 min (OR 2.54 (CI 1.183, 5, 47). The unadjusted odds of developing AKI based upon pre-operative clinical and laboratory
features are shown in Table 8.4. The pre-operative risk factors identified are similar
to other studies in this population (Thiele et al. 2014). On multivariate analysis,
those patients with a urinary Uri level <40 mg/L had an odds ratio (OR) for developing AKI of 3.41 (95 % confidence interval 1.239.44).
Of the 36 patients 15 had abnormally high Uri prior to surgery (>12 mg/L) indicating pre-existing acute anti-inflammatory response. Analysis of pre-surgical values for other biomarkers tested including IL-6 (serum and urine), neutrophil
gelatinase lipocalin (NGAL), dipeptidyl peptidase-4 (adenosine deaminase binding
protein ADP-26), cystatin C, protienuria, microabluminuria, hematuria and urinary
adiponectin, had no predictive value for the development of AKI.
Table 8.4 Odds of developing AKI according to baseline clinical and laboratory characteristics
Variable
Age (per decade)
Gender (male)
Race (African American)
Ejection Fraction (less than <35 %)
Cardiac Bypass Time (>85 min)
Urine Albumin/Creatinine >200
Serum IL-6 >500 pg/ml
Urine IL-6 >90 pg/ml
Urine Uristatin <40 mg/L
Variable
Urine Albumin/Creatinine >200
Serum IL-6 >500 pg/ml
Urine IL-6 >90 pg/ml
Urine Uristatin <40 mg/L
Unadjusted odds ratio

1.812 (0.73, 4.52)
6.875 (1.56, 30.36)
0.458 (0.09, 2.45)
3.179 (1.37, 7.38)
2.543 (1.183, 5.47)
1.193 (0.94, 1.52)
1.021 (0.94, 1.11)
0.99 (0.99, 1.02)
3.41 (1.23, 9.44)
Odds ratio adjusted for age, gender, ejection fraction,

bypass time
1.159 (0.85, 1.58)
1.039 (0.78, 1.38)
0.99 (0.93, 1.042)
1.262 (0.96, 1.624)
152
Pre-operative levels of Uri did not correlate with the level of RIFLE criteria,
however the numbers of individuals in higher levels of RIFLE AKI were small and
thus this analysis is limited. This small number of patients limited the utility of any
analysis. In an attempt to account for potential confounders in the relationship
between baseline laboratory parameters and the development of AKI, a step-wise
logistic regression analysis was performed utilizing the significant clinical features
found in the unadjusted analysis (see Table 8.4). This regression analysis does
reduce the potential impact of pre-operative Uri and without additional sample sized
the true predictive value cannot be uncovered.
8.3.2
Post-operative Prediction of AKI with Urinary

Biomarkers
Urinary biomarkers were measured serially after CPB surgery. Neither urine uristatin
nor serum and urine IL-6 measured at time periods immediately post-operative were
predictive of rises in serum creatinine of greater than 0.3 mg/dL (data not shown). Uri
continued to rise in patients after surgery to two to six times higher than pre-surgical
values. A rise in Uri occurred in days 16 for all patients except 2 that failed to produce Uri. At day 6, Uri values still did not return to normal but appeared to peak for
most patients. This agrees with previous findings that Uri is part of the acute inflammatory response in wound healing increase during the first week after major surgery.
Of the other biomarkers tested, neutrophil gelatinase lipocalin (NGAL) a measure of immune cell activation and Cystatin C a measure of glomerular filtration
response had the highest areas under the curve (AUC) of 0.60.7. Urinary Ig complement, a measure of auto-immune response, was not predictive. Adiponectin was
more likely to be lower in the failure group but was not sensitive. There were only
two failure patients with low adiponectin. Dipeptidyl peptidase-4 (adenosine
deaminase binding protein ADP-26) a well-studied marker of tubular necrosis
marker (cell disruption) was not sensitive for failure due to inflammation. There
were only five failure patients with acute tubular necrosis. Protienuria, microabluminuria, and hematuria were too sensitive and not specific.
8.3.3
Uristatin and Pre-operative Prediction of AKI
The performance characteristics of Uri for the pre-operative prediction of AKI are
shown in Figs. 8.1 and 8.2. Urine Uri as a pre-operative predictive marker for
development of AKI post-operatively had an area under the curve (AUC) of 0.855
(see Fig. 8.1). In patients undergoing CPB-surgery there was an inverse correlation
between pre-operative uristatin levels. The probability of AKI is shown in Fig. 8.2
along with the 95 % confidence intervals. There is an inverse relationship between
pre-operative urine uristatin levels and the risk for post-operative AKI and the

Fig. 8.1 Receiver operator
curve analysis of preoperative urine uristatin
levels for prediction of
AKI
153
ROC Curve
1.0
0.9
0.8
Sensitivity
0.7
0.6
0.5
0.4
0.3
Model ROC Curve
Random discrimination
0.2
0.1
0.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
A
1 - Specificity
1.0
Probability of APF
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
100
Maximum Uristatin (mg/L)

Model ROC = 0.855
Fig. 8.2 Relationship between the probability of AKI and baseline uristatin level
subsequent development of AKI. Those patients with urine uristatin levels

>80 mg/L had at least a fivefold lower risk of AKI as compared to those patients
with baseline uristatin levels <40 mg/L. At urinary uristatin levels <40 mg/dL there
is nearly a 50 % or greater risk of developing AKI, independent of inclusion of any
154
clinical variables. The predictive ability of this biomarker compares well to risk
stratification based upon clinical variables alone (for instance the AUC for the
prediction of AKI for the CCF scoring system is 0.81 and 0.68 for the Chertow
scoring system).
Based upon the known effects of inflammation to cause AKI, it is not surprising
that a urinary anti-inflammatory protein may have either have protective effects or
be a marker for renal protection (Bennett et al. 2008). It is interesting that another
urinary marker that has been found to predict pre-operative risk (-defensin-1) is
also an anti-inflammatory protein. Importantly, uristatin levels post-CPB surgery
were not predictive of later AKI nor were serum or urine IL-6 levels. Thus, preoperative urine uristatin levels may be helpful in identifying those patients at high
risk of AKI and allow for targeted strategies of renal protection.
This study is limited by the size of the cohort and that the study occurred at a
single-center. It should also be cautioned that this cohort of patients was selected
carefully based upon stringent inclusion and exclusion criteria. Further studies in a
larger multi-center cohort representative of the group of patients undergoing CPB
surgery are required to validate these findings as well as determine how a uristatinbased predictive model would operate in clinical practice and whether interventions
based upon a pre-operative uristatin level would lead to renal protection. Furthermore,
it is intriguing to speculate that recombinant uristatin or other protease-inhibitor
therapies may offer therapeutic benefit in the prevention of AKI. Future animal
studies and trials will need to address these possibilities.
8.4
Conclusion
In this study, we show uristatin provides pre-operative risk assessment for the development of AKI post-CPB. The presence of uristatin in the urine and its potent antiinflammatory effects made it an ideal candidate biomarker for the assessment of
pre-operative risk for AKI.
This study supports the premise that uTi protects patients from renal failure and
decreases the likelihood of immune-mediated kidney injury. As uTi is induced to
clinically significant levels during acute events, it is an important finding for assessment of patient. Monitoring in an acute setting presurgically and after event for
return to baseline could be helpful for predicting patient recovery. If uTi continues
to be induced in chronically inflamed tissue, the prolonged protective state could
slow renal regeneration (see Chap. 10).
Acknowledgements We thank Brent Dorval of Brendan BioScience (Hopedale, MA) for his
contributions of Uristatin testing that made this study possible. We thank Dr Manju Basu who
participated in the analysis of the urine and serum samples. We thank Glenn Fung of Siemens
Healthcare Image and Knowledge Management, Malvern, Pennsylvania for additional statistical
analysis.
155
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Chapter 9
Cardiovascular and Metabolic Syndrome

Impact on Uristatin
Abstract Urinary trypsin inhibitors (uTi) were modestly elevated in cardiovascular

disease patients (CVD), coronary artery disease (CAD) or metabolic syndrome
patients (MetSyn). Urinary and plasma immunoassay values for Uristatin (Uri) and
Bikunin (Bik) were within their normal ranges for 94 % of CVD patients and 97 %
of MetSyn patients. Urinary and plasma Bik and Uri values were also only slightly
elevate with metabolic syndrome risk factors such as diabetes (DM), lipidemia
(LIP), hypertension (HTN), and obesity (OBS). Bik and Uri were typically elevated
in CVAD patients with clinically significant events such as acute myocardial infarction (AMI) or congestive heart failure (CHF). Here urinary trypsin inhibitors (uTi)
are indicative of a white blood cell (WBC) response, as acute events also elevated
granulocytes in blood. Granulocytes values significantly correlated (P < .005) with
the degree of coronary artery stenosis and coronary angiography. Generally patients
with cardiovascular and metabolic conditions and without renal disease or infection
had uTi values below the acute phase level (<12 mg/L).
9.1
Introduction
Cardiovascular disease (CVD) is associated with elevated lipids which are deposited
in the arteries thereby causing atherosclerosis. Cardiovascular disease is also characterized by a number of metabolic syndrome (MetSyn) risk factors (Libby et al. 2002).
S.A. Jortani
Department of Pathology and Laboratory Medicine, University of Louisville,
School of Medicine, 511 S. Floyd Street (Room 227), Louisville, KY 40202, USA
M. Pugia (*)
Department of Pathology and Laboratory Medicine, University of Louisville, School of
Medicine, Louisville, KY USA
Strategic Innovation, Siemens Healthcare Diagnostics, 3400 Middlebury Street, Elkhart, IN
46530, USA
DOI 10.1007/978-3-319-21927-1_9
157
158
These indicators include; smoking, diabetes, sedentary lifestyle, hyperlipidemia,

homocysteine levels and total cholesterol to HDL ratio. Patients with metabolic syndrome have an a higher likelihood of finding cardiovascular disease (CVD), coronary artery disease (CAD), acute coronary syndrome (ACS). MetSyn also increases
the chance of vascular damage, atherosclerosis, cardiomyopath, congestive heart
failure (CHF) and acute myocardial infarction (AMI).
Accumulating epidemiological evidence currently indicates that inflammation also
plays a significant role in the initiation and progression of CVD and CAD (Libby et al.
2002; Ridker et al. 2002; Danesh et al. 1998). Inflammation plays a significant role in
the initiation and progression of all these conditions. As CVD and CAD progresses
from MetSyn risk factors to full disease, the inflammatory response further impacts
the vascular system. The likelihood of finding CVD, CAD, and ACS is associated
with increased basal concentrations of acute phase reactants, cytokines and adhesion
molecules (Blake and Ridker 2003; Avanzas et al. 2004; Szmitko et al. 2003).
Atherosclerosis occurs after lipid accumulation causes the endothelial cells in
the artery wall to express molecules that attract various classes of leukocytes leading to plaque destabilization and rupture (Libby et al. 2002). The immune cell
response mediates platelet activation, coagulation, and endothelial damage (Szmitko
et al. 2003). Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been linked
to the oxidized low-density lipoprotein (oxLDL) activation of macrophages
(Avanzas et al. 2004). Acute phase reactants, such as myeloperoxidase, placental
growth factor, gelatinases and tissue factor pathway inhibitor have been linked to
oxLDL uptake by macrophages and monocyte release of cytokines and chemokines
(Heeschen et al. 2004; Brennan et al. 2003; Nguyen et al. 2001; Kato 2002). These
markers have been used to estimate the immune cell recruitment, adhesion and activation. The end result is formation of macrophage foam cells and ruptured plaques
triggering thrombus potentially followed by ischemia, endothelial degradation or
acute myocardial infarction (AMI).
Inflammation increases the risk of AMI and stroke. Acute phase reactants such
as high sensitivity C-reactive protein (CRP) have been used for cardiovascular risk
assessment (Ridker et al. 1998a, b, 2000). The highest quartile of CRP has twice the
risk of future stroke and three times the risk of future myocardial infarction (Ridker
et al. 1997; Pasceri et al. 2000). Inflammation markers are present in 1015 % of
events but provide increased hazard risk ratings of 24. Other complications such as
arrhythmias and heart failure can follow these processes as well. While the amount
and nature of inflammation in a plaque is quite variable, persistently high or increasing concentrations of many acute phase biomarkers are indicators of a poor prognosis and increase risk of development of an event.
Anti-inflammatory responses by urinary trypsin inhibitors (uTi) such as Bikunin
(Bik) and Uristatin (Uri) are typically present during inflammation (Pugia and Lott
2005; Pugia et al. 2007a). Serine protease inhibition by uTi impacts cell signaling
and many of the processes known to arise in CVD and CAD. Inhibition serine proteases prevent signaling of Protease Activated Receptors (PAR). This subgroup of
the G-protein-coupled receptors uses proteases to initiate cell signaling via specific
Cardiovascular and Metabolic Syndrome Impact on Uristatin
159
cleavage sites for thrombin, trypsin and possibly other serine proteases (Olear and
Nouza 1999). PAR activation has been proposed as a key part of the vascular inflammation mechanism (Szmitko et al. 2003; Miike et al. 2001). PAR contribute to cellular changes as part of the chronic inflammatory defense and precedes abnormal
function (Cottrell 2002; Coelho et al. 2003). PAR also regulate vascular tone and
participate in the response to vascular injury (Bono et al. 1997). uTi is shown to
inhibit PAR activation during coronary artery bypass grafting surgery (CABG)
(Shibutani and Kunihiro 1986). uTi also inhibits blood coagulation through action
on plasmin, Factors IXa, Xa, XIa and XIIa (Nii et al. 1994, 1995; Egeblad and
Astrup 1966). Inhibition of plasmin by uTi prevents activation of Urokinase-type
Plasminogen Activator and slows tissue remodeling. Finally, inhibition of serine
proteases from white blood cells (WBC) prevents release of cytokines and growth
factors, and signaling of apoptosis.
Urinary trypsin inhibitors have not been studied in progression of CVD or
CAD. The impact of MetSyn risk factors is also unknown. Our goal was to measure
the impact of vascular inflammation and associated risk factors on the uTi measurements in blood and urine. We wanted to compare immunoassays for Bik, Uri and
inter--inhibitor (I--I) to CRP, WBC and uTi inhibition assays in patients with and
without vascular inflammation and associated risk. Secondarily, we wanted to retrospectively compare the inflammation markers with the degree of arterial blockage and by presence of AMI or CHF. Correlations between inflammatory markers
were tested to determine synergic and independent relationships. As a final objective, we also wanted to establish the normal ranges for the Bik, Uri and I--I immunoassay in apparently healthy adults.
9.2
9.2.1
Methods
Patient Assessment
We collected matched urine, plasma and serum from 188 patients from several hospital settings including the cardiac unit. Patients were risk-stratified. Those without
angina pectoris (chest pain within the last 48 h), with no more than one cardiovascular disease risk factor, and no existing cardiovascular disease were considered
normal and without coronary artery disease (n = 101). The remaining patients
(n = 87) underwent coronary artery angiograms. Angiographic measurements were
made of the degree of stenosis in the left anterior descending, left coronary artery,
right coronary artery, ramus intermedius, graft-immediate distal vessel, and the circumflex artery. Two board-certified cardiologists reviewed the angiographs and
grouped the patients according to their risk findings. Angiogram patients were considered normal (n = 32) when they uniformly had <30 % stenosis in all the coronary
arteries and as affected with acute coronary syndrome when having at least >50 %
stenosis in any artery (n = 55).
160
We followed the IRB practices of our institutions. The analysts were blinded to
all test results. Specimens were collected during the initial assessment and prior to
the angiographic evaluations. Most of the patients undergoing angiography had
multiple cardiovascular risk factors. Data on age, gender, BMI, blood pressure, and
plasma lipids were collected along with medical histories over the last 5 years. A
systolic pressure of >140 mmHg, a diastolic pressure of >90 mmHg, or oral use of
anti-hypertensive agents were used as an indication of hypertension (n = 57). We
used a BMI cutoff of >30 ((weight in lb/(height in inches^2))*703) to define overweight (n = 60). Patients with hyperlipidemia (n = 100) were those with a ratio of
cholesterol/ HDL >5 or if they were getting lipid-lowering agents such as a statin
drug. Patients with diabetes mellitus (n = 28), and (or) smoker status (n = 69) were
identified and counted. While patients were not using anti-inflammatory agents during the angiography and sample collection period, their history defined regular use,
anti-inflammatory medicines, anti-thrombotic or nitroglycerine.
Patients with infection(s), HIV, AIDS, or leukemia were excluded. One patient
had an elevated CRP >10 mg/L and a WBC of >12.4 cell/nL indicating possible
unknown infection. No patient had abnormal kidney function as judged by a creatinine clearance of >100 mL/min. Clinical evaluation, and diagnosis of CHF (n = 7)
was made by echocardiographic and B-type natriuretic peptide (BNP) of >100 ng/L
assays (Remme 2002). A diagnosis of AMI (n = 10) was based on abnormal EKG
findings, troponin I >1.5 g/L and clinical evaluation by our cardiologists. A diagnosis of cardiovascular disease (AMI nor CHF) was not considered abnormal for
CVD unless the angiogram was positive.
9.2.2
Sample Collection and Marker Analysis
We obtained clean-catch midstream urines specimens from all patients and controls. We stored the specimens at 4 C for testing within 24 h. Storage was at
20 C for samples used in assays performed after 24 h. We collected plasma and
serum from all patients and controls in Vacutainer tubes (BD, Franklin Lakes,
NJ). We separated the serum or plasma (EDTA) immediately after collection. We
performed urine dipstick tests for urinary trypsin inhibitors by enzyme-inhibition
assays on a Clinitek 50 strip reader to obtain concentrations of 0 (undetectable),
<6.25, 12.5, 25, and 50 mg/L by a method described elsewhere (Siemens Healthcare
Diagnostics) (Pugia 2007b). We measured for uristatin (Uri) and bikunin in blood
and urine and I--I in blood by ELISA methods (Siemens Healthcare Diagnostics)
(Pugia et al. 2007b). Enzyme linked immunosorbent assays (ELISA) for adiponectin were performed with plasma according to manufacturers instructions (B-Bridge
International, Inc Mountain View, CA). The MULTISTIX PRO strip was used to
measure blood, nitrite, leukocyte, protein, and creatinine ratios (Siemens Healthcare
Diagnostics). The percent granulocytes and lymphocytes were determined on a
Cell-Dyne 4000 cell counter, (Abbott Diagnostics, Santa Clara, CA). All CBCs
were performed within 1 h of collection. We determined the total cholesterol, HDL,
LDL, and triglycerides on a Vitros 950 analyzer (Ortho Clinical Diagnostics,
161
Raritan, NJ) along with a high sensitivity CRP test (BN II nephelometer Dade
Behring, Deerfield IL). Analysis of plasma for B-type natriuretic peptide (BNP) and
troponin I (TnI) were performed by immunoassay (ADVIA Centaur, Siemens
Healthcare Diagnostics).
9.2.3
Patient Characterization
Comparisons of the cardiovascular risk factors, demographics, and medications are

shown for the control (unaffected) population and the affected group with risk of
CVD or CAD in Table 9.1. Comparisons of the standard diagnostic tests are shown
in Table 9.2. The control population was characterized as either having no risk of
CVD or CAD, if they had more than one risk factor, they had no evidence of stenosis by angiogram. The affected group included at risk patients who underwent angiography with abnormal outcome. The affected group consisted of patients
characterized at least >50 % stenosis in one artery and the expected differences in
risk factors. We found that any chest pain did not significantly predict the degree of
vascular stenosis. All confirmed patients with AMI had stenosis while most patients
with CHF had stenosis. Medication usage was significantly higher for the disease
group than the control group regardless of gender.
9.2.4
Statistics Methods
Comparisons (as P-values) between these two groups, again for each sex (and both
together) are included. For the qualitative factors (e.g., smoking status) the number,
percent and the Clopper-Pearson 95 % confidence interval for the percent are tabulated. The P-value is that for the difference in proportions between the control and
affected groups calculated using the normal approximation to the binomial distribution with continuity correction. For the quantitative factors (e.g. age and diagnostic
test), the mean and standard deviations (SDs) are tabulated. The P-values are for the
difference between each groups mean assuming a t distribution. Before calculating
the t value, the two groups SDs are compared using the F distribution. If they were
not statistically different, (two-sided, 95 % confidence), the SDs were pooled and
the SD of the difference is estimated with the pooled SD. Otherwise, the SD of the
difference is estimated from the two separate SDs and its number of degrees of
freedom estimated using Satterthwaites approximation (rounded down). All
P-values are two-sided. Differences between the control and affected groups are
deemed statistically significant if the corresponding P-values are less than 0.05. For
Bik and the cell counts, exact non-parametric P-values are calculated using the
Wilcox on scores (equivalent to the Mann-Whitney U test). For the cell counts, the
exact P-values are estimated using a Monte Carlo approximation with 10,000 samples and a random (system clock generated) seed. Again, all P-values are
two-sided.
Male
n = 54
(n, %)
22 (40.7 %)
13 (24.1 %)
21 (38.9 %)
3 (5.6 %)
11 (20.4 %)
2 (3.7 %)
0 (0.0 %)
14 (25.9 %)
10 (18.5 %)
5 (9.3 %)
2 (3.7 %)
6 (11.1 %)
Control group
All
n = 133
(n, %)
34 (25.6 %)
27 (20.3 %)
55 (41.4 %)
9 (6.8 %)
30 (22.6 %)
3 (2.3 %)
1 (0.8 %)
26 (19.5 %)
20 (15.0 %)
10 (7.5 %)
2 (1.5 %)
10 (7.5 %)
4 (5.1 %)
0 (0.0 %)
5 (6.3 %)
10 (12.7 %)
Female
n = 79
(n, %)
12 (15.2 %)
14 (17.7 %)
34 (43.0 %)
6 (7.6 %)
19 (24.1 %)
1 (1.3 %)
1 (1.3 %)
12 (15.2 %)
23 (41.8 %)
12 (21.8 %)
26 (47.3 %)
37 (67.3 %)
Affect group
All
n = 55
(n, %)
35 (63.6 %)
50 (90.9 %)
45 (81.8 %)
17 (30.9 %)
30 (54.5 %)
3 (2.3 %)
6 (10.9 %)
34 (61.8 %)
10 (40.0 %)
6 (24.0 %)
9 (36.0 %)
13 (52.0 %)
Male
n = 25
(n, %)
17 (68.0 %)
22 (88.0 %)
19 (76.0 %)
7 (28.0 %)
11 (44.0 %)
2 (3.7 %)
4 (16.0 %)
16 (64.0 %)
13 (43.3 %)
6 (20.0 %)
17 (56.7 %)
24 (80.0 %)
Female
n = 30
18 (60.0 %)
28 (93.3 %)
26 (86.7 %)
10 (33.3 %)
19 (63.3 %)
1 (1.3 %)
2 (6.7 %)
18 (60.0 %)
<0.005
<0.005
<0.005
<0.005
<0.005
0.01
<0.005
<0.005
<0.005
<0.005
<0.005
<0.005
P-value, affected vs. controla,f

All
Male
Female
<0.005
0.02
<0.005
<0.005
<0.005
<0.005
<0.005
<0.005
<0.005
<0.005
0.01
<0.005
<0.005
0.03
<0.005
<0.005
0.01
0.48
<0.005
<0.005
0.13
<0.005
<0.005
<0.005
Comparisons were made for control (unaffected) population and the affected group with risk of CVD or CAD The affected group had at least 50 % stenosis in
at least one artery. The number of patients with the parameter and the % of population with the parameter are shown
b
A systolic BP > 140 mmHg, a diastolic BP > 90 mmHg, and/or the use of anti-hypertensives defines a hypertensive patient
c
Patients with a value >5 and/or using lipid reducers are defined as hyperlipidemic
d
Obesity defined as a body mass index (BMI) > 30
e
Anti-inflammatories include aspirin (ASA) and non-steroidal anti-inflammatories (NSAIDs). Lipid-reducing medications are statins. Anti-hypertensives are
ACE inhibitors and/or beta blockers
f
P-values are for difference between the proportion in the affected and in the control groups
Parameter
Smokers,
Hypertensiveb
Hyperlipidemicc
Diabetic
Obesityd
CHF patients
Have had AMI
Anti-imflammatory
medicationse
Anti-hypertensive
medicationsb, e
Lipid-reducing
medicationsc, e
Anti-thrombotic
medications
Uses nitroglycerine
Table 9.1 Cohort demographic characteristics
162
Male
n = 54
X (sd)
51.4 (9.4)
187.9 (45.8)
69.6 (2.9)
27.2 (5.8)
131.0 (12.2)
74.2 (10.7)
198.9 (45.5)
133.1 (75.7)
44.6 (14.5)
124.1 (56.2)
4.8 (1.7)
(n, %)
8 (14.8 %)
Control group
All
n = 133
X (sd)
50.2 (8.9)
171.4 (51.4)
66.7 (4.1)
27.0 (7.2)
131.1 (10.1)
72.9 (9.4)
202.0 (45.2)
129.2 (58.8)
46.5 (16.6)
126.7 (45.5)
4.7 (1.6)
(n, %)
13 (9.8 %)
4.7 (1.5)
n, (%)
5 (6.3 %)
128.5 (36.7)
47.8 (17.8)
126.6 (44.1)
Female
n = 79
X (sd)
49.4 (8.4)
160.1 (52.3)
64.7 (3.5)
26.8 (8.1)
131.2 (8.4)
72.0 (8.3)
204.2 (45.2)
5.1 (1.8)
(n, %)
14 (25.5 %)
126.3 (41.8)
42.5 (13.3)
133.6 (56.1)
Affect group
All
n = 55
X (sd)
56.1 (9.2)
193.2 (42.0)
65.7 (4.5)
31.4 (5.7)
151.1 (27.5)
84.4 (14.9)
202.0 (45.2)
5.0 (2.0)
(n, %)
10 (40.0 %)
123.2 (39.9)
43.5 (15.9)
127.8 (62.9)
Male
n = 25
X (sd)
53.9 (9.1)
211.0 (47.6)
69.3 (3.9)
30.7 (5.9)
152.1 (30.0)
87.9 (14.1)
194.1 (44.1)
5.1 (1.6)
(n, %)
4 (13.3 %)
129.0 (43.9)
41.6 (10.9)
138.4 (50.4)
Female
n = 30
X (sd)
57.9 (9.0)
178.4 (30.3)
62.7 (2.1)
31.9 (5.6)
150.2 (25.7)
81.5 (15.1)
204.5 (54.8)
<0.005
0.19
0.96
0.11
0.64
<0.005
0.60
0.94
0.76
0.76
<0.005
0.20
0.96
0.03
0.23
P-value, affected vs. controla

All
Male
Female
<0.005
0.02
<0.005
0.01
0.04
0.03
0.15
0.70
<0.005
<0.005
0.01
<0.005
<0.005
<0.005
<0.005
0.005
<0.005
<0.005
0.76
0.67
0.98
Comparisons were made for control (unaffected) population and the affected group with risk of CVD or CAD The affected group had at least 50 % stenosis in
at least one artery
b
Body Mass Index (BMI) is calculated 703 weight (lb)/height (in)2. A BMI >30 indicates overweight (obese patient)
c
The lipid ratio is that of total to HDL cholesterol
d
Urine protein to creatinine ratio is estimated with a strip indicating Normal or Dilute Normal or numerical values. An elevated ratio is indicated by any
numerical value
Proteiunuriad
Parameter
Age years
Weight lbs
Height in
BMIb
Systolic BP mmHg
Diastolic BP mmHg
Total cholesterol
(mmol/L)
Triglycerides
(mmol/L)
HDL cholesterol
(mmol/L)
LDL cholesterol
(mmol/L)
Lipid ratioc
Table 9.2 Cohort diagnostic risk factor characteristics
9
163
164
9.3
9.3.1
Results
Correlation to Risk Factors
In Tables 9.1 and 9.2, we compared the demographic and diagnostic risk factor for the
controls to patients with stenosis. Patients with stenosis were more likely to be smokers, to be obese by weight or BMI, and to have high blood pressure, be older or diabetics. Of the diagnostic tests evaluated, proteinuria was the most significantly different
between the control and affected groups. Proteins in urine are independent risk factor
of CVD in essential hypertension (Pontremoli et al. 1997). Lipids were not significantly different as most of the affected population was on lipid-reducing medications.
The usage of nitroglycerines, anti-hypertension, anti-inflammatory and anti-thrombotic medications were also significantly more used in the affected population.
9.3.2
Correlation of Tested Markers
In Table 9.3, we compared the values of the inflammation tests between controls and
affected patients with stenosis. As expected, the mean values of most inflammatory
tests were slightly higher in the stenosis patient groups. The exception was I--I
where mean values decreased. Of the tests evaluated, granuloctye counts were the
most significantly difference (P < .005) between the control and affected groups.
While Uri and uTi values were elevated, 98 % were below 12 mg/L and below.
Predictive value of inflammatory response in vascular stenosis of these markers
was measured. The thresholds for a positive result are shown above in Table 9.4. For
CRP, WBC, granulocytes, and lymphocytes these thresholds were obtained from the
literature. We used the high sensitivity threshold for CRP. We found that a threshold
of 36 mg/L for I--I was best for reporting the control group (total n = 133) as
normal. For the Uri immunoassay and the uTi strip we used a normal urine
concentration limit of <7.8 mg/L based on previous studies with health individual.
In practice, only results 12 mg/L are considered abnormal. In this case, 98 % of
Table 9.3 Impact of vascular stenosis on inflammation markers
Assay
Uri (mg/dL)
uTi Strip (mg/dL)
Bik (mg/L)
I--I (mg/L)
CRP (mg/L)
Total WBC (cell/nL)
Granulocytes (cell/nL)
Lymphocytes (cell/nL)
Control group
3.6 (5.9)
1.9 (6.4)
0.9 (1.0)
87.5 (26.4)
0.4 (0.5)
6.2 (2.0)
3.6 (1.6)
1.9 (0.6)
Affected
group
6.6 (15.0)
5.9 (12.8)
1.1 (1.5)
82.6 (30.7)
1.4 (3.6)
8.6 (2.5)
5.5 (2.4)
2.2 (0.9)
P value for control vs affected

.16
.02
.34
.27
.04
<.005
<.005
.19
165
results were normal. The strip method for Bik gave more false positives with proteinuria and was less specific. Here, only 90 % of the controls were reported as
normal. For blood values, we used a threshold of 2.5 mg/L for Bik based on
reporting a comparable number of control group as normal.
Table 9.4 Predictive value of inflammatory response in vascular stenosis

Positive
cutoff
>7.8
True
neg
125
False
pos
8
True
pos
11
False
neg
44
Bik strip
(mg/dL)
Bik (mg/L)
>7.8
120
13
13
42
>2.5
128
46
I--I (mg/L)
<36
129
50
CRP (mg/L)
>2
129
49
Total WBC
(cell/nL)b
Granulocytes
(cell/nL)b
Lymphocytes
(cell/nL)b
Bik or Uric
>10
124
15
40
>7
127
14
41
>3
125
10
45
121
12
19
36
Bik or Uri or
CRPc
Bik or Uri or
I--Ic
Bik or Uri or
granulocytesc
Bik or Uri or
granulocytes
or CRPc
120
13
21
34
117
16
23
32
116
17
29
26
15
18
30
25
Assay(s)a
Uri (mg/dL)
%Specificityd, e
(95 % Conf)
94.0
(88.597.4)
90.2
(83.994.7)
96.2
(91.498.8)
97.0
(92.599.2)
97.0
(92.599.2)
93.2
(87.596.9)
95.5
(90.498.3)
94.0
(88.597.4)
91.0
(84.895.3)
90.2
(83.994.7)
88.0
(81.293.0)
87.2
(80.392.4)
86.5
(79.591.8)
%Sensitivitye, f
(95 % Conf)
20.0
(10.433.0)
23.6
(13.237.0)
16.4 (7.828.8)
9.1 (3.020.0)
10.9 (4.122.2)
27.3
(16.141.0)
25.5
(14.739.0)
18.2 (9.130.9)
34.5
(22.248.6)
38.2
(25.452.3)
41.8
(28.755.9)
52.7
(38.866.3)
54.5
(40.668.0)
Legend:
Uri, Bik, I--I and CRP were measured by immunoassay. In this table, Uri is a urine value, and
Bik a plasma value. Note that both measure all inhibitory fragments in specimens (e.g., both Uri
and Bik). I--I is a plasma immunoassay for the family of I--I pro-inhibitors. Bik is a strip assay
performed on a CLINITEK analyzer and based on trypsin enzyme inhibition. Values above the
cutoff (except for I--I) are taken as positive
b
1 Cell/nL = 103 cell/mL
c
The combination tests are positive if any one of the assays being combined is positive. Compared
non-parametrically with the Mann-Whitney U test and exact, two-sided P value calculations using
the SAS NPAR1WAY procedure. The P values are two sided. A value less than .05 is considered
as statistically significant
d
Specificity = 100 True Negatives/(True Negatives + False Positives)
e
95 % confidence intervals are Clopper-Pearson intervals
f
Sensitivity = 100 True Positives/(True Positives + False Negatives)
a
166
The specificities and sensitivities for the various inflammation markers for
detecting vascular stenosis (>50 %) were calculated as shown in Table 9.4. For all
the markers, at least 9397 % of the controls were considered normal and only
927 % of the stenosis group were considered positive. The WBC test was most
sensitive with 27 % of the affected groups with vascular stenosis reported. The combinations of pro-inflammatory (CRP, granulocytes) and anti-inflammatory (Bik,
Uri) biomarkers as a panel indication of inflammation increased the sensitivity to
stenosis to 55 %. But not all patients with vascular stenosis had a positive inflammation test. Moreover, increasing the number of tests in a panel increased the number
of false positives and reduced the specificity from 93 to 87 %. The immunoassay Uri
and Bik values were typically within the normal ranges (see thresholds in Table 9.4)
except when patient had elevated granuloctye counts. Also half of the CHF and MI
patients (n = 13) had elevated Uri or Bik and this contributes significantly to the
positives observed.
9.3.3
Correlation to Metabolic Syndrome
In Table 9.5, we compared the values of the inflammation tests between controls and
patients meeting metabolic syndrome (MetSyn) criteria or only a single risk factor such
as lipidemia (LPD), hypertension (HTN), diabetes (DM) or obesity (OB) (Deen 2004).
The average patient ages in all groups was 5152 years and not significantly different
between groups. Males and females were evenly distributed. As expected, the mean
values of most inflammatory tests were slightly higher in the stenosis patient groups.
In addition, we tested all samples for plasma adiponectin (Adpn). Adpn is significantly lowered in patients with metabolic syndrome or diabetes (Daimon et al.
2003; Kadowaki et al. 2006). This adipocytokine is released by adipose tissue to
slow liver production of glucose and to initiate muscle to oxidize fatty acids and
glucose (Maeda et al. 1996). MetSyn patients tend to have inflammation and have
difficultly controlling insulin and obesity (Hotamisligil et al. 1995; Kahn et al.
2006). Lipid accumulation and inflammation in the adipose is thought to lower
Adpn by inhibiting expression by the Peroxisome Proliferator-Activated Receptor
(PPAR). Hypertrophic adipose requires more insulin to activate PPAR, thus
increasing insulin resistance. Activating PPAR with thiazolidinediones (TZD) is
therapeutic intervention for hyperglycemia and hypoadiponectinemia (Tsuchida
et al. 2006; Kubota et al. 2005).
9.3.4
Pro-inflammatory Response
The pro-inflammatory response measured by White Blood Cell counts (WBC)

and CRP was increased in metabolic syndrome and risk groups compared to the
control group in a sub group of patients with all metabolic factors measured (see
Normal
84
avg
51.9
10.9
2.6
5.6
3.1
1.9
0.3
sd
9.1
6.9
2.0
1.2
1.0
0.5
0.3
sd
6.4
2.4
6.0
2.4
1.8
0.8
0.6
Hypertension (HTN)
36
avg
sd
51.7
7.1
6.2
2.7
4.7
6.1
7.2
2.4
4.1
1.8
2.3
0.7
0.6
0.7
Obesity (OB)
34
avg
sd
50.9
6.9
6.9
4.3
4.9
6.3
7.5
2.2
4.4
1.8
2.3
0.7
0.6
0.6
Lipidemia (LPD)
25
avg
sd
51.0
7.6
8.1
4.5
2.9
2.7
6.7
2.4
3.9
1.8
2.2
0.7
0.5
0.5
Diabetes (DM)
15
avg
sd
50.9
6.4
6.0
2.6
5.3
7.1
7.0
2.4
4.1
2.0
2.0
0.8
0.4
0.4
Legend:
a
Urine, plasma and serum were collected from patients diagnosed with (n = 40) and without (n = 84) metabolic syndrome according to AFP definition (Deen
2004) as either a diagnosis of insulin resistance or having more than two metabolic risk factors. Patients (n = 124) with at least one risk factor: hypertension
(HTN), obesity (OB), lipidemia (LPD) or diabetes (DM) are also shown
b
In this cohort, all patients lacked kidney disease and infections including urinary tract infection
c
1 Cell/nL = 103 cell/mL
Counta, b
Age (year)
Plasma Adiponectin (mg/L)
Uri (mg/L)
Total WBC (cell/nL)c
Granulocytes (cell/nL)c
Lymphocytes (cell/nL)c
CRP (mg/L)
Parameter
Metabolic
syndrome
(MetSyn)
40
avg
50.3
5.8
4.6
7.4
4.3
2.3
0.6
Table 9.5 Comparison of metabolic syndrome factors
9
167
168
Table 9.3). Values for WBC increased by 32 % and values for CRP increased by
149 % for patients meeting metabolic syndrome criteria (see Table 9.3). The WBC
differences were the more significant: cardiovascular (P = 0.017), HTN (P = 0.030),
OB (P = 0.014), LPD (P = 0.086) and DM (P = 0.075). Differential counts showed
granulocytes correlated strongly while lymphocytes changes were less significant.
Serum CRP differences were significant for cardiovascular (P = 0.033), HTN
(P = 0.045), OB (P = 0.036) but not for LPD or DM (P > 0.1). Most values (<99 %)
were well within normal reference ranges of <12.4 cell/nL for WBC and <2.0 mg/L
for high sensitivity threshold for a negative CRP. A few patients were above the
WBC (n = 1) or CRP (n = 4) clinical thresholds.
9.3.5
Adiponectin Response
Plasma Adpn was significantly reduced in patients with cardiovascular (P = 0.019),

HTN (P = 0.034), OB (P = 0.064) and DM (P = 0.054) but not those with LPD
(P = 0.195). The mean Adpn value for the cardiovascular group was 55 % lower than
for the group without cardiovascular. A clinical threshold of <5 mg/L for a positive
result gave a sensitivity of 50 % and specificity of 87 % for the detection of MetSyn
patients. Urinary levels of Adpn were not correlated to the presence cardiovascular
or risk factors.
9.3.6
Uristatin Response
Uri values were higher in 79 % metabolic syndrome (MetSyn) patients and those
with lipidemia (LPD), hypertension (HTN), diabetes (DM) or obesity (OB). Normal
reference ranges for Uri values were <7.8 mg/L for urine. Most (>99 %) patients
and controls had values below this threshold. Therefore the clinical sensitivity for
metabolic factors was only 515 %. A few patients had values above these clinical
thresholds in either urine (n = 7) or plasma (n = 3) and this was likely due to elevated
WBC.
9.4
Conclusion
Urisatin can be used for assessment of kidney disease and infection in patients with
cardiovascular disease patients (CVD), coronary artery disease (CAD) or metabolic
syndrome patients (MetSyn) without significant interference from underlying conditions. This allows potential applications for diabetic nephropathy and other
nephropathy.
169
Acknowledgements We would like to acknowledge the work done by Department of Pathology

and Laboratory Medicine, University of Louisville, School of Medicine. We thank Dr. Manju Basu
and Ron Sommer for running ELISA as needed. We also thank Dr. Robert Dwyer, Paul W. Dillon
PhD, Prakash Tewari, Laura Tinker, and Peter Connolly for the ADVIA Centaur results, and
assistance of Dr. Sumanth Prabhu (University of Louisville Cardiology) in recruiting some of the
patients into this study and Mr. Edward P. Womack from Diagnostic Reference Laboratory at
University of Louisville is acknowledged for technical assistance.
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Chapter 10
Uristatin Anti-inflammatory Cellular

Signaling
Manju Basu, Subhash Basu, and Michael Pugia
Abstract Bikunin (Bik) and uristatin (Uri) impacted cellular signaling of kidney
proximal tubular, kidney gluomerlar, muscle, and carcinoma cells. Cell cultures
were induced with Bik and Uri at 1240 mg/L and compared to unexposed cell
cultures. The balance in serine protease trypsin activity was shifted to complete
trypsin inhibition. Cell proliferation was slowed to 50 %. Cell death by apoptosis
signaling though caspases, was not activated by the presence of Bik or Uri.
Proliferation signaling though the mitogen-activated protein kinases (MAPK) was
active. Phosphatidylinositol 3-kinase (PIK3) and protein kinase B (Akt) signaling
were activated. Cell death occurs through a non-programmed cell death. Exposed
cells showed increased release of a membrane-bound protein, dipeptidyl peptidase
4 (DPP-4). Membrane disruption was also evidenced by phosphatidylserine (PS)
externalization measured by binding a fluorescence dye (PSS-380). Exposed cells
were depleted of intercellular calcium as shown by using calcium- binding fluorescence dye (Fluo-4). Blockage of the Ca2+ sensitive potassium (KCa) channels (or
calcium-activated potassium (BK) channels) is thought to lower the intracellular
Ca2+. All of the information was supported by a kidney cell model, of Bik/Uri being
protective of immune mediated apoptosis and proliferation during the inflammatory
process, but causing cell loss by decreased renal regeneration by Akt-PIK3
signaling.
M. Basu (*) S. Basu

Notre Dame, IN 46556, USA
M. Pugia
Notre Dame, IN 46530, USA
DOI 10.1007/978-3-319-21927-1_10
171
172
10.1
M. Basu et al.
Introduction
Inflammatory processes promote the release of trypsin serine proteases. Proper control of inflammation is necessary to prevent further damage to normal tissue. Urinary
trypsin inhibitors (uTi) are small proteins serving as Kunitz type inhibitors of serine
proteases that can be detected in human urine during inflammation (Pugia and Lott
2005; Pugia et al. 2007b). Bikunin (Bik) is one of the key forms of uTi with N- and
O- linked complex glycans. Uristatins (Uri) are another key form lacking the
O-linked glycan. Bik binds to renal cell surfaces not only through the peptide
Kunitz-type domains but also through the large complex glycans. The O-glycoside
chain binding increases the affinity to cells, calcium and TSG-6 (Hirashima et al.
2001; Kanayama et al. 1997; Milner and Day 2003). The sequences of the carbohydrate chains are known to change during disease progression (Hochstrasser et al.
1967; Zhuo et al. 2002; Lindstroem et al. 1997).
Many researchers have shown uTi plays a key role in the human anti-inflammatory
system to protect tissues and suppress vascular dilation, coagulation, and smooth
muscle contraction (Pugia et al. 2007b). Bik inhibits trypsin, chymotrypsin, kallikrein and plasmin, elastase, cathepsin Factors IXa, Xa, XIa and XIIa through binding with either of two Kunitz binding domains. uTi reduces release of growth factors
and cytokines inhibiting growth factor activation (Huang et al. 2001). uTi has been
measured in multiple patient groups and proved to be a sensitive diagnostic marker
for infections (Pugia et al. 2004; Jortani et al. 2004). uTi is almost entirely excreted
into urine to prevent its extended negative impact on the anti-inflammatory processes. Rapid renal clearance is due to a glomerular filtration rate 80-fold higher
than that of albumin (Lindstroem et al. 1997; Joberg et al. 1995; Ohlson et al. 2001).
Thus the kidney is exposed to the majority of uTi. This interaction likely impacts the
inflammatory repair and healing processes of the kidney. However, the cellular
impact of Bik and Uri exposure is not fully understood.
Kunitz-type protease inhibitors such as aprotinin have been shown to slow cell
proliferation, protect renal cells, and significantly decreased the rise in serum creatinine and apoptosis caused by kidney ischemia-reperfusion injury (Bono et al.
1997; Yamaski et al. 1996; Kher et al. 2005). Similarly, Uri and Bik have been
shown to protect against renal failure induced in animal models and programmed
cell death (Nakakuki et al. 1996; Takahashi et al. 2001) Aprotinin is shown to
prevent protease-activated receptor (PAR) signaling of proliferation through the
p38 mitogen-activated protein kinase activation (MAPK) and protein kinase A
and B (PKA, PKB) (Bono et al. 1997). Aprotinin also blocks calcium-sensitive
potassium channels (Moczydlowski et al. 1992). uTi have also been shown to
inhibition of Ca2+ influx in vascular smooth muscle (Kanayama et al. 1998). The
Kunitz-type peptide domains Bik and Ur are homologous to aprotinin (Pugia et al.
2007b). A key difference is aprotinin lacks the added N- and O- linked complex
glycans of Bik.
In this study, we explored the responses in cultured cells to pathological levels of
Bik and Uri to evaluate which signal transduction pathway is involved in the innercellular signaling. Additional we want to compare Bik to Uri to uncover any
10
Uristatin Anti-inflammatory Cellular Signaling
173
potential impact of glyco-conjugation. Therefore active site on Bik was also characterized to understand its the distinct functions of Bik protein domains.
10.2
10.2.1
Methods
Bikunin and Uristatin
Urinary trypsin inhibitors were isolated from chronic renal failure patients (SciPac,
Sittingbourne, Kent, UK) (Sumi et al. 1981). Two isolations of >90 % purity were
obtained: Bik (~30 kDa) and Uristatin (Uri) (17 kDa).
10.2.2
Cell Culturing Procedure
Normal cells used were African green monkey proximal tubular kidney cells (Vero),
mouse myocytes (C2C12) (American Type Cell Culture, ATCC, Manassa VA) and
human mesangial gluomerlar cells (NHMC) (Cambrex Inc, Cambridge MA).
Carcinoma cells used were human breast cancer lines SKBR-3 and MDA-468
(ATCC). Carcinoma cells were maintained in culture using RPMI-1640 media,
while normal cells were maintained with modified Eagles medium (MEM). Both
media contained 2 mM glutamine, penicillin (50 units/ml), streptomycin (50 g/
ml), and 10 % fetal bovine serum (Invitrogen, Carlsbad, CA).
Cells were grown at 37 C in an atmosphere of 95 % air and 5 % CO2, and p
propagated by sub-culturing at confluence by asceptically treating with trypsinEDTA, and replenishing with media. Cell confluence was about 5 million cells per
T-75 cm2 flask. Cells were grown to 7080 % and synchronized by adding hydroxyurea (0.5 mM, sterile, Sigma-Aldrich, St. Louis, MO) 2 times for 24 h each.
Synchronized cells were induced with various concentrations of Bik (5, 50, 500 g/
mL) in full media under sterile conditions. After induction, the cell media was discarded and aliquots taken for cell counts.
Cells were harvested at 4, 24, and 48 h from start of induction. The cells were
harvested in PBS/EDTA and suspended in 2 ml PBS. Cell counts were taken immediately using Trypan Blue staining. Harvested packed cells were suspended in two
volumes of HEPES buffer, 0.05 M and pH 7.2. The cells were sonicated at 4 C for
15 s with 30 s rest intervals three times. The homogenates were centrifuged at
12,000 rpm (50,000 g) for 15 min at 4 C. The resulting supernates were saved in
small aliquots at 20 C. The lysate residues (pellets) were suspended again in
twice the volume of HEPES with 0.1 % Triton X-100 stored at 20 C until tested.
The protein content in cell lysates was measured in triplicate by the Bradford dyebinding procedure (BioRad Laboratories Hercules, CA). Exposures and analysis
were repeated on three separate trials. Cells for kinase phosphorylation assays were
added to the mircotiter plate wells and fixed with 100 uL of 4 % formaldehyde in
174
M. Basu et al.
PBS. The plates were kept for an additional 20 min at room temperature and then
covered with parafilm, and stored at 4 C until assayed.
10.2.3
Serine Protease Activity and Inhibition Assays
Serine protease inhibitor concentrations were determined by the inhibition of trypsin hydrolysis of a chromogenic arginine peptide substrate (3-(N-tosyl-N-nitro
arginyloxy)-5-phenylpyrrole) placed in a dipstick (Siemens Healthcare Diagnostics,
IN). The rate of substrate disappearance was measured by the rate of color formation with 2-methoxy-4-morpholino-benzenediazonium on a Clinitek 50 strip
reader (Siemens Healthcare Diagnostics, IN), as previously published. The samples
were read in duplicate and the value determined from a standard curve obtained
with Bik standard solutions containing 0, 12.5, 25, and 50 mg/l (SciPac,
Sittingbourne, Kent, UK, product code P205-1). Serine protease activity was also
measured using another trypsin specific chromogenic substrate BAPNA
(N-Benzoyl-DL-arginine 4-nitroanilide, Sigma-Aldrich, St. Louis, MO) and monitoring trypsin hydrolysis at 405 nm. BAPNA at 18.3 mM was prepared in 20 %
dimethyl formamide: 80 % water. Triethanolamine reaction buffer was prepared at
250 mM and pH 7.8. Samples of cell extracts (6 L) were diluted into 75 L water.
Trypsin calibration samples included TPCK treated from bovine pancreas:
7300 unit/mg was from Sigma-Aldrich, St. Louis, MO. At time zero, 15 L of water
and 25 L of BAPNA were added to 100 L of reaction buffer and 75 L sample
under light-proof conditions. Samples were read in triplicate every 10 min for 1 h at
37 C. The results were plotted to determine the serine protease amount in mg/L in
cell extracts.
10.2.4
DPP-4 Peptidase Activity Assay
Serine peptidase activity was measured using the chromogenic substrate GPN
(H-Gly-Pro-p-nitroanilide, Sigma-Aldrich, St. Louis, MO) specific for dipeptidyl
peptidase 4 (DPPIV porcine, Sigma- Aldrich, St. Louis, MO). Cell extracts
(100 uL) or DPP-4 at 0, 3.6, 7.2, and 14.4 mg/L were added to 100 uL of 100 mM
Tris buffer (pH 8.0). At time zero, 25 uL of GPN (0.76 mM in Tris) was added and
triplicate samples read every 1 min for 20 min at 37 C. The hydrolysis rates were
monitored at 405 nm and plotted to determine peptidase amount in mg/L. One
miligram equals 0.91 units of DPP-4 activity. Bik did not inhibit DPP-4 and was
not an interferent.
10.2.4.1
Enzyme Linked Immunosorbent Assays (ELISA)
ELISA for DPP-4 was performed with cell extracts according to manufacturers
instructions (Nephroscreen kits, Brendan Bioscience, LLC of Hopedale, MA).
10
10.2.5
175
Western-Blot Analysis
Western blots were performed against anti-caspase antibodies (3/8/9) and MAPK antibody (phosporylated p38 and ERK) (Cell Signaling Technology, Inc. Danvers, MA)
and the monoclonal antibodies against Bik/Uri (421-3G5) and DPP-4 (Anti-URO-4
mAb S27, Abcam, Cambridge, MA). Proteins separated on SDS-PAGE electrophoresis were transferred to a nitrocellulose membrane in 1x Tris-Glycine buffer (Bio-rad,
Hercules, CA) and 20 % methanol. The transferred proteins reacted with the specific
first and ALP-conjugated second antibodies at a desired concentration after 1 h blocking with 5 % BSA blocker in TBS-0.1 % Tween-20. The target binds were visualized
using an ALP substrate BCIP/NBT tablet dissolved in ddH2O (Sigma, St. Louis, MO).
10.2.6
Akt and PIK3 Activation Measurements
Measurements of the relative increase in phosphoralyation of Akt and PIK3 were

performed according to the ELISA manufacturers instruction (Superarray,
Frederick, MD). Results were repeated in 3 independent experiments with new cultures. Harvesting of packed cells was accomplished by suspending cells in 2X volume of HEPES buffer, 0.05 M and pH 7.2. The cells were sonicated at 4 C for 15 s
with 30 s rest intervals three times. The homogenates were centrifuged at 12,000 rpm
(50,000 g) for 15 min at 4 C. The resulting supernates were saved in small aliquots
at 20 C. The membrane pellets were suspended again in twice the volume of
HEPES with 0.1 % Triton X-100 stored at 20 C until tested. Cells for kinase
phosphorylation assays were added to the mircotiter plate wells and fixed with
100 uL of 4 % formaldehyde in PBS. The plates were kept for an additional 20 min
at room temp and then covered with parafilm and stored at 4 C until assayed
Kinase phosphorylation assays can generally be conducted by adding cells to the
mircotiter plate wells and fixing with 100 uL of 4 % formaldehyde in PBS. The
plates were kept for an additional 20 min at room temperature and then covered with
parafilm and stored at 4 C until assayed. ELISA measurements of the relative
increase in phosphoralyation of Akt and PIK3 were performed according to the
ELISA manufacturers instruction (Superarray, Frederick, MD). Measurements of
the relative increase in MAPK ERK phosphoralyation were preformed according to
the ELISA manufacturers instruction (Assay Designs, Ann Arbor MI). Results
were repeated in 3 independent experiments with new cultures. Cells were fixed to
the micro-titer plate followed by removal of the fixing buffer and cell quenching
performed with H2O2 and NaN3. The quenching buffer was removed and the plate
was heated, washed, and blocked. This was followed by 1 h incubation with primary
antibodies for Akt, Phos-Akt, PI3K, and Phos-PI3K (1:100 dilution). Each antibody
and induction condition was tested in separate triplicate wells. The unbound primary antibodies were removed by washing and the plate was then incubated for 1 h
with a secondary antibody conjugated to HRP (1:16 dilution). The plate was washed
twice and developing solution was added. After incubation, the resultant dark blue
color formed after the addition of stop solution was read at 450 nm.
176
10.2.7
M. Basu et al.
Apoptosis Assays
Apoptosis assays by caspase activation are generally conducted by using various cell
extracts and supernatants. Western blots were performed against anti-caspase antibodies (3/8/9) (Cell Signaling Technology, Inc. Danvers, MA). The electrophoresis
was run under standard conditions using Tris-Glycine-SDS buffer at 1820 mAmp
with 3 g BioRad prestained standards. After electrophoresis, the proteins were
transferred from the gel onto a nitrocellulose membrane at 80 V for 1 h or 40 V overnight at 4 C. The transferred proteins were reacted with the specific antibodies
(1 mg/mL) mentioned above after 1 h blocking with casein blocker. After washing
off the unbound antibody and blocking with TBS containing Tween20, the blotted
membranes were incubated with ALP-conjugated corresponding second antibody
for another hour. Finally, the cross-reactivity of the samples with the antibodies was
monitored by the addition of 66 ul of nitro blue tetrazolium (50 mg/ml 70 % DMF)
and 35 ul of bromo chloro indolyl phosphate (50 mg/ml DMF, 100 %) in 10 mL TBS.
10.2.8
Intra-cellular Analysis
Measurements of cytosolic calcium were done by infusing cells with the membrane
permeable calcium sensitive Fluo-4 AM dye as previously described in Chap. 5.
Apoptosis assays using phospatidyl serine analysis were conducted by using viable
cells. Cells induced with CTF or Adpn were treated with PSS-380 dye to recognize
phosphatidyl serine on the outer leaflet of the apoptotic cells (Molecular Targeting
Technologies Inc). Cells were plated onto a Falcon Microslide System (Fisher) and
washed two times with TES buffer (5 mM N-tris [Hydroxymethyl]-2-aminoethanesulfonic acid: TES, 150 mM NaCl, pH7.4) followed by incubation with 200 L new
TES buffer containing 25 M PSS-380 at 37 C for 10 min. The buffer was removed
after staining, and the cells were washed with TES buffer once before observation
for fluorescence. Phase contrast images were used to locate regions of fluorescent
signals and to calculate the percentage of affected cells. Vero cells induced with Bik
at 10, 25, and 50 mg/L were treated with PSS-380 dye to recognize phosphatidyl
serine externalization as previously described (Smith et al. 2003).
10.2.9
Active Site Studies
Trypsin inhibition by different Bik peptides (AE), Bikunin, Uristatin, and aprotinin on trypsin inhibitory was measured in the presence of antibody to Bik (mAb
421.3G5) using measurement by trypsin-specific chromogenic substrate (N-benzoyl-DL-arginine 4-nitroanilide, BAPNA, Sigma-Aldrich) at multiple trypsin
and inhibitor levels (10500 mg/L). Hydrolysis rates are plotted to determine the
average inhibition expressed as mg trypsin to mg inhibitor. Five peptides (AE)
10
177
represent different regions in Bikunin sequence were chemically synthesized

without the attached glycoconjugates. Trypsin inhibition was observed by Bik
(0.76 mg/mg), Uri (0.89 mg/mg) and aprotinin (0.58 mg/mg) but not synthetic peptides AE even if these overlapped the predicted active sites. Competitive inhibition
experiments were preformed by adding peptide (AE) or protein (Bik, Uri or aprotinin) to the sample of Bikunin and mAb.
The hydrolysis rates were plotted to determine the average percentage of inhibition w/w and the amount of restored inhibition calculated from the Bikunin inhibition rate without mAb. Inhibition of trypsin by Bik or Uri (0.4 mg/L) was reduced
by 6070 % when either were incubated for 1 h at 37 C with Uristatin monoclonal
antibody (mAb 3G5) (1.6 mg/L). Only peptide B restored any trypsin inhibition
when added in 50-fold excess as a competitive binder. This supported an active site
inhibition, and mAb 3G5 binding was in peptide B region. Trypsin inhibition by
BAPNA assay was plotted to determine the average percentage of inhibition for
equal weight of trypsin to inhibitor (mg/mg). Binding of the Bikunin proteins and
peptide analogs by mAb (421.3G5) was measured by ELISA with the full protein or
peptide sequence (11000 ng/well).
10.3
10.3.1
Results
Protease and Inhibitor Balance
The levels of serine proteases (Trypsin type) and inhibitor, and DPP-4 were measured
in normal proximal tubular kidney cells (Vero): primary human kidney mesangial
gluomerlar cells (NHMC); mouse C1C12 myoctyes cells; and human breast carcinoma cells SKBR-3 and MDA-468. All regular growing cells exhibited significant
levels of trypsin enzymatic activities as shown in Table 10.1. Proximal tubular kidney
and carcinoma cells had the most activity. Glomerular mesangial cells and myocytes
had the least trypsin activity. Trypsin levels were higher in membrane pellets than in
soluble cellular supernate. Trypsin inhibitors were detected by inhibition assays.
However, the balance of trypsin/inhibitor in these cells favored greater trypsin than
inhibitor by 870 times (ug/ug). All cells were shown to lack the Bik inhibitor in both
ELISA and western blot immunoassays. Exposure of cells to 50 mg/L Bik shifted the
protease to inhibitor balance in favor of greater inhibitor by a 1.5 to 10-fold (ug/ug).
10.3.2
Cellular Signal Response Due to Bik
Vero cells were induced with sterile Bik in serum-growth media at the G0 phase.
Cell numbers declined by approximately half of initial cell counts at 24 h after Bik
exposure, as shown in Fig. 10.1 (closed circles). Control cell cultures were revived
with serum growth media and continued to double during the same time period.
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M. Basu et al.
Table 10.1 Protease and inhibitor balance

Cell typea
Proximal tubular
Glomerular
mesangial
Myoctyes
Carcinoma
Supernatant
Pellet
Pellet
Trypsinb (g/
mg protein)
132
243
17
Trypsin Inhibitorsc, d
(g/mg protein)
8.9
15.2
2.4
DPP-4e (ng/mg
protein)
2.2
19.3
32.4
Pellet
Supernatant
Pellet
20
213
445
1.9
3
6.3
nil
467
22.5
450
20
400
17.5
350
15
300
12.5
250
10
200
7.5
150
100
DPP-4 (ng/mL)
Kidney cell x106/mL
Legend:
a
Normal proximal tubular kidney cells were African green monkey cells (Vero). A primary human
kidney cell line was used for mesangial gluomerlar cells (NHMC). Myoctyes were mouse C1C12
cells. Human breast carcinoma cells were SKBR-3 and MDA-468. Cells were lyzed and homogenates separated in membrane-bound pellet residues and supernate
b
Trypsin protease activity was measured using trypsin specific chromogenic substrate calibrated to
trypsin standards
c
Trypsin inhibitors were measured by the inhibition of trypsin substrate hydrolysis
d
Bikunin (Bik) levels were 0.1 mg/L for all cell extracts measured by ELISA and western blot
immunoassays
e
Dipeptidyl Peptidase 4 (DPP-4), was measured by ELISA
50
2.5
0
0
10
20
30
40
50
60
Bik (mg/L)
Fig. 10.1 Normal cell Loss and DPP-4 release upon exposure to Bik. Live cell count was performed with Trypan Blue staining. Cell cultures exposed to 25 mg/L Bik lost 50 % of cells within
a 24 h period. Kidney cell (Vero) counts (closed circles) decreased with increasing Bik. In addition, Vero released DPP-4 (open circles) upon increased Bik exposure (units on right hand axis).
Exposures and analysis were repeated in three separate trials
10
179
Phosphorylation of MAPK-ERK and MAPK-p38 was unchanged between control

and exposed cells (data not shown here).
10.3.3
Membrane Disruption Due to Bik
Membrane disruption after Bik exposure was detected by membrane phosphatidylserine (PS)-binding dyes PSS-380 (see Table 10.2). PSS-380 binds PS on outer
leaflet of cell membrane which presents only when the cell undergoes the dying
process (Ma et al. 2004) Phosphatidylserine externalization was detected after 4 h
of Bik treatment and increased with Bik amounts from 10 to 50 mg/L, as shown by
the increased blue PSS-380 staining. Incorporation of the propidium iodide into the
cell nucleus DNA was observed at a lesser extent after 24 h (data not shown here).
Apoptosis, or programmed cell death, was measured by western blot using antibodies for the intra-cellular caspase proteases 3, 8, and 9 (see Table 10.2). The absence
of apoptosis signaling was confirmed in all cell models before and after exposure to
Bik. Western blots showed no cleavage of caspase 3 into active forms (see Table 10.2).
Inactive caspases 9 confirmed no intrinsic apoptosis signaling occurred. Caspases 8
remained inactivate which supported no extrinsic apoptosis signaling. There was no
activation of apoptosis at extreme Bik exposure levels of 500 mg/L.
10.3.4
Calcium In-flux Blocking by Bik
The cellular calcium (Ca2+) was measured by fluorescent microscopy using a Ca2+
sensitive dye (Fluo-4 AM) (as previously described in Chap. 7). Harvested myocytes were treated with Bik or Uri. Muscle, and kidney cell cultures typically maintained intra-cellular Ca2 in the range of 0.080.12 M. Cells exposed to Bik
exhibited significantly reduced intra-cellular Ca2 concentration. Intra-cellular Ca2
decreased to 0.01 M with increasing Bik concentrations. Cell lysates showed Bik
was maintained on cell surfaces after washing.
10.3.5
Release of DPP-4 Due to Bik
We found kidney cell cultures did not release DPP-4 into the cell supernate
(<10 ng/mL) unless cell cultures were exposed to Bik (see Table 10.2 and Fig. 10.1).
This release increased with concentrations of Bik. Western blots showed released
DPP-4 from membrane as the expected 110 kDa monomeric form along with bands
77, 50, and 45 kDa for fragments. Release of DPP-4 occurred within 4 h of Bik
exposures and was reproducible in separate experiments.
Dipeptidyl Peptidase 4 (DPP-4) was detected more in cell membranes compared to cell supernate. In agreement with literature, DPP-4 was detected in kidney
Active
(17 kDa)
Caspase 8b
Inactive
(57 kDa)
+
+
+
+
+
Active
(43/18 kDa)
Caspase 9b
Inactive
(47 kDa)
+
+
+
+
+
Active
(37 kDa)
At 4 h
0.93
0.47
0.09
0.01
0.01
Intracellular
calcium
(M)c
At 4 h
0%
17 %
NM
26 %
NM
At 24 h
2%
42 %
NM
57 %
NM
% Cells with outer

membrane
disruptiond
At 4 h
0%
0%
NM
0%
NM
At 24 h
0%
5%
NM
14 %
NM
% Cells with
inner membrane
disruptiond
a
Synchronized cultures for normal kidney cells (Vero) and myocytes (C2C12) were induced with Bik at 0, 12.5, 25, 50, and 500 mg/L. Cells were harvested
after 4 and 24 h. Exposures were repeated in three separate trials
b
Apoptotic caspase cleavages were measured by western blots using cell extracts with antibodies for caspase proteases (3/8/9). Inactive and active bands are
identified by molecular weights as shown above and are indicated (+) for present and () for absent
c
Intra-cellular calcium was measured by incubating the Vero cells with membrane permeable calcium sensitive Fluo-4 AM for 30 min at 36 C to allow intracellular hydrolysis to cause fluorescence in the presence of calcium. Cells were washed and centrifuged to remove extra-cellular dye, and fluorescent images were
captured. Microscopic images of Bik treated kidney cells were collected by phase contrast and fluorescence microscope. The % of cells with fluorescence was
determined by area comparisons
d
Measurements of outer membrane disruption were done with cell membrane-binding dye PPS-380 while inner membrane disruption was monitored with
DNA-binding dye propidium iodide. (NM = not measured)
Bik mg/
La
0
12.5
25
50
500
Caspase 3b
Inactive
(32 kDa)
+
+
+
+
+
Table 10.2 Stress response in kidney and myocytes cells exposure to Bik
180
M. Basu et al.
10
181
and carcinoma cells but not myocytes (see Table 10.1) (Mentlein 2004). The high
concentration of DPP-4 in proximal tubular cells was expected as the peptidase is
part of the brush border membrane and participates in protein absorption by hydrolysis of proline containing peptides (Mueller et al. 1990; Thompson et al. 1985).
However the presence of DPP-4 was not previously reported for glomerular
mesangial cells.
10.3.6
Proliferation
The high level of Trypsin and Trypsin inhibitor expression in the proximal tubular
cells reflect their dynamic living status. Proximal tubular epithelial cells play a pivotal role in kidney disease. Most renal cell carcinoma and kidney cancer arises from
this region. During inflammation, endothelial and epithelial cellular proliferation is
signaled by trypsin-type serine proteases which generate wound healing. Trypsin
and thrombin cleave Protease Activated Receptors (PAR) which increases phospholipase C (PLC), adenylcyclase (AC), and phosphatidylinositol 3-kinase (PIK3) activation of ERK 1/2 mitogen-activated protein kinase (MAPK/ERK). MAPK/ERK
activation plays a key role in cell growth and differentiation through transcriptional
activation. Active PAR also increases cytosolic phospholipase A2 (PLA2) synthesis
of prostaglandins causing nuclear receptor signaling of transcriptions.
10.3.7
Activation of PI3K-Akt Pathway by Bik
Phosphorylated and non-phosphorylated forms of PI3K were measured by ELISA

with cell cultures before and after exposure to Bik and Uri (see Fig. 10.2). Activated
PI3K was increased in cells when exposed to Bik and Uri. Increased activity would
reduce immobilization of calcium from storage organelles by inositol 1,4,5 triphosphate. Phosphorylation of PI3K increased more with the exposure to Uri. Protein
kinase B (Akt) is an important target of increased PI3K phosphorylation.
Phosphorylated and non-phosphorylated forms of Akt were measured at the same
time points using ELISA. Cell exposed to Bik did not increase in Akt phosphorylation. Cells exposed to Uri showed Akt activation in a dose-dependent manner (see
Fig. 10.2). The highest level of Akt phosphorylation occurred at 500 mg/L of Uri. A
drop in PI3K activation was noted at 500 mg/L.
10.3.8
Bik Active Site Study
The structure indicates the two Kunitz domains and two oligosaccharide chains.
Colored peptides were labeled with letters in parentheses (A) to (E) (see Fig. 10.3).
182
M. Basu et al.
1.60
Relative increase from basal activity
1.50
1.40
1.30
1.20
1.10
1.00
0.90
0.80
Control
Bik
(5 mg/L)
Bik
Bik
Uri
(50 mg/L) (500 mg/L) (5 mg/L)
Uri
Uri
(50 mg/L) (500 mg/L)
Fig. 10.2 Activation of PI3K/Akt with Bikunin and Uristatin. The relative increases in phosphoralyation of phosphatidylinosinol 3 kinase (PI3K) (solid bars) and Akt (open bars) are shown for
epitheilal (Vero) cell cultures simulated with 0, 5, 50, and 500 mg/L of Bik or Uri. Measurements
were performed using immunoassays for both phosphoralyation and non- phosphoralyation forms
of PI3K and Akt. Exposures and analysis were repeated in three separate trials
The mAb 3G5 responded well to Bikunin and Uristatin at ~1 ng/well and showed
no cross-reactivity to aprotinin even at an excess of 10,000 ng/well. The mAb 3G5
also responded to peptide B at 362 ng/well, that supporting the epitope in this
region. The affinity for purified Bikunin standard was compared to that for native
Bikunin in diabetic urine specimens. The antibody affinity of ~2.9 1011 was
reduced for many diabetics when the urine was not diluted. Diluting urine (5 L) in
PBS (45 L) eliminated the urine sample matrix affect. Undiluted urine inhibiting
mAb binding in some patients supports additional associating molecules.
Trypsin inhibition was observed with Bik, Uri, and aprotinin (see Table 10.3).
The loss of the O-linked glycan in Uri did not impact inhibitory strength. Synthetic
peptides AE were not inhibitory to trypsin. The results suggested that a secondary
structure for the bikunin molecule is needed for trypsin inhibition. The mAb 421.3G5
was able to bind only fragment B, which is the region for N-linked polysaccharide.
The affinity is much lower than for the purified Bik standard (362 ng/well vs. 1 ng/
well), indicating that this mAb recognizes both the amino acids and N-linked sugars
of fragment B. Inhibition of trypsin was prevented when Bikunin was bound to mAb
3G5 in peptide region B (see Table 10.3 an Fig. 10.3). This supports the explanation
that mAb was binding the region B of Bik where is active sitde of inhibition.
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183
O-linked glycan with chondroitin sulphate
N-terminal tail
Xyl-Gal-Gal-GlcUA-(NeuAc-GlcUA)5-(GalNAc-GlcUA)20-GalNAc-GlcUA
AVLPQEEEGS GGGQLVTEVT KK
A
N-linked glycan
GlcNAc-GlcNAc-Man-(Man-GlcNAc-Gal-NeuAc)2
Kunitz domain 1
DSCQLGYSAG PCMGMTSRYF YNGTSMACET FQYGGCMGNG NNFVTEKECL QTCRTV

B
Trypsin
inhibition site
Kunitz domain 2
AACNLPIVRG PCRAFIQLWA FDAVKGKCVL FPYGGCQGNG NKFYSEKECR EYCGVP
D
C-terminal tail
GDGD EELLRFSN
Fig. 10.3 Peptide analogs and aprotinin comparison to Bik predicted structure. The predicted full
length Bikunin (Bik) peptide and carbohydrate sequence is compared to five synthetic peptides and
aprotinin. Peptides in bold are a match to the aprotinin sequence. Underlined peptides are the predicted contact points between trypsin- aproptinin based on X-ray structure. This single Kunitz
binding domain has matching peptides (darkened circles) to both inhibitor domains of Bik. The
location of the predicted active site (Underlined circles), an arginine (R) or lysine (K) peptide
which can be locked into the trypsin pocket is shown for Bik domain 1 and 2. The likely antibody
binding site is the arginine peptide in the middle of peptide region B. The region B arginine is connected to N-linked glycan (RYFYN) and an example of a glycoconjugated protease inhibitor. For
Bik domain II, the predicted trypsin binding site is an arginine (R) peptide. The other peptides of
the domain II active site also match the preferred and strongest trypsin binding sequences of GPCR
and are examples of a peptide-only protease inhibitor This active site was not mimicked by peptide
D as the native sequence must be in a distorted conformation to cause trypsin inhibition
Bikunin active sites are often studied by comparison to aprotinins single Kunitz
binding domain. For Bikunin Kunitz domain 1 (see Fig. 10.3), however, the location
of the predicted active site lacks an arginine (R) or lysine (K) residue which can be
locked into the trypsin pocket. The likely binding site is the arginine peptide in the
middle of peptide region B. The region B arginine is connected to N-linked glycan
(RYFYN), an atypical structure of a glycoconjugated protease inhibitor (Otlewski
et al. 2001). For Bikunin Kunitz domain 2, the predicted trypsin binding site is an
arginine (R)-containing peptide. In our study, we characterized the mAb 3G5 binding site as the active site for serine protease inhibition, located in the N-linked oligosaccharide region.
184
M. Basu et al.
Table 10.3 Bikunin active site study for trypsin inhibition and antibody binding
Inhibitora
Peptide A
Peptide B
Peptide C
Peptide D
Peptide E
Bik
Uri
Aprotinin
% Trypsin inhibitionb
Average (standard
deviation)
0%
0%
0%
0%
0%
76 % (3 %)
89 % (5 %)
58 % (2 %)
Antibody binding in ng/

wellc
Average (standard
deviation)
NB
362 (0.66)
NB
NB
NB
1.25 (0.43)
1.03 (0.12)
NB
Restored of trypsin
inhibitiond
Average (standard
deviation)
0%
35 % (16 %)
0%
0%
0%
89 % (7 %)
72 % (12 %)
0%
a
Synthetic bikunin peptide fragments A to E, native Bik, Uri and aprotinin standards were used to
study the trypsin inhibitory effect
b
(column 2), antibody binding ability to mAb 421.3G5
c
(column 3), and restoration of trypsin inhibition activity
d
(column 4). NB = No Binding
10.4
Discussion
Bik readily passes through the glomerular basement membrane and accumulates in
the lysosomes of proximal tubular epithelial cells, and is not reabsorbed in the condensing vesicles. As Bik inhibits the proteolytic process in proximal tubular epithelial cells and shifts the protease activity to inhibitor balance, it is reasonable that Bik
would inhibit the proximal tubular from proteolysis and protein reabsorption in the
kidney. Inhibition of tubular reabsorption would result in LMW proteinuria during
periods of inflammation. This is in agreement with clinical observation that Bik correlates well to proteinuria during fever and infection (Pugia et al. 2002; Pugia et al.
2004; Jortani et al. 2004).
Glomerulonephritis is the lead cause of renal injury leading to failure and is typically associated with infections (Hotta et al. 1999; Huang et al. 2001). Tissue damage by White Blood Cell (Neutrophilic polymorphonuclear leukocytes and
macrophages) does cause capillary wall injury in glomerulonephritis mediated by
the release of proteinases (Nakakuki et al. 2001). Elastase and cathespin are known
to damage the basement membrane leading to proteinuria and enlargement of the
network structure the basement membrane and charge barrier (Cochrane et al.
1965). Serine proteases such as elastase, proteinase 3, and cathespin cause the
release of apoptotic cytokines such as tumor necrosis factor- (TNF), interleukin-1
(IL-1), interleukin-18 (IL-18), and transforming growth factor - (TGF )
(Mania-Pramanik et al. 2004; Johnson et al. 1988; Nakatani and Takeshita 1999)
The intrinsic and extrinsic caspase pathways were not activated after Bik exposure in this study (Basu et al. 2014). Bik inhibitors, like aprotinin, have been shown
to prevent apoptosis in animal models of ischemia-mediated kidney damage
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185
Inhibited extrinsic Reduced

Reduced
death factor
cytokine &
K + influx
& NF-KB
growth factor
signaling
release
KCa Channel Ca Channel
K+ K+
K+ +
Ca+
Bik
Ca+ Ca+
K
CD44
Bik
Bik
Bik-R
Ca+
Inhibited
Inhibits
growth factor
PAR
signaling
activation
Bik
NF-B
Activation
MAPK/ERK
MAPK p38
Slows cell
Inflammatory proliferation
(~50% cell/24h)
Akt&PI3K
Activation
No Apoptosis
(Caspases 3, 8, 9)
Ca+
Out-flux
Reduced
uPAR
activation
uPA
uPAR
Bik
LP
Membrane
disruption via
osmotic changes
Bik
Granzyme B
inhibition
Fig. 10.4 Impact of Bik on cell signaling. Kunitz inhibitor are known to stop PAR activation
reducing cell proliferation through MAPK/ERK. Additionally, activation of MAPK is inhibited
through CD44 binding to growth factors (EGF, TGF-, and TGF-) upon Bikunin (Bik) has been
shown vis interaction with its receptor (Bik-R). Bik is also known to inhibits release of growth
factors and cytokines, subsequently reducing extrinsic death factor (FasL) and NF-KB signaling
(TNFR). Bik reduces expression of uPA and inhibits membrane-bound plasmin, impacting membrane stabilization through binding to link protein (LP). Bik is also known to inhibit Granzyme B
from activating apoptosis. We here report, no activation of apoptosis via Caspase 3, 8, and 9 was
observed after Bik exposure. There was no release of DNA in fluorescence microscopic cell
images. No activation of MAPK p38 was observed after Bik exposure. PI3K-Akt phosphorylation
was observed after Bik exposure. Bik reduced intracellular Ca2+ in the fluorescence microscope
cell images which could explain PIK3-Akt activation. Membrane disruption and cell death was
observed by phosphatidyl serine membrane-changes in fluorescence microscope cell images and
release of membrane bound ADBP-26 increased. Exposure causes cell proliferation slows to half
the rate seen with non-exposed cells (~50 % cell/24 h)
(Kher et al. 2005). Elastase, proteinase 3, and cathespin, are all inhibited preventing
subsequent inflammatory cytokine and growth factors release. Our results in normal
cell models, without inflammatory cytokine stimulation, did show that Bik does not
activate apoptosis supporting the anti-apoptotic action of Bik on leukocytes proteases. Additionally, Bik activation of PI3K-Akt could activate NF-B independently
of TNF- which prevents Caspase 3 activation (Vivanco and Sawyers 2002).
Necrosis is a distinguished from of cell death where membrane disruption occurs
without the programmed process that can be observed in apoptosis (Skulachev
2006). Osmotic changes within the cell cause outer membranes to expand and rupture during necrosis. Necrotic cell death, not apoptosis, occurred in normal cells
exposed to Bik. As Fig. 10.4 shows, during this process, Bik continued to protect
against immune mediated apoptosis and inflammatory signaling of cell proliferation. However, Bik exposure induced stress on the cell. Membrane disruption was
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M. Basu et al.
clearly evident by phosphatidylserine externalization along with decreases in microscopic cells counts. This change could be partially due to the blocking the KCa channels with the changes of membrane permeability. Additionally, DPP-4 was released
from the membrane. This is consistent with DPP-4 used as a diagnostic marker of
acute renal necrosis (Mueller et al. 1990; Thompson et al. 1985).
Proximal tubular epithelial cells play a pivotal role in kidney disease. The high
level of Trypsin and Trypsin-inhibitor expression in the proximal tubular cells
reflect their dynamic living status. Most renal cell carcinoma and kidney cancer
arise from this region (Tomita 2006). Normal cellular proliferation is central to kidney regeneration and repair. During inflammation, endothelial and epithelial cellular proliferation is signaled by trypsin-type serine proteases which generate wound
healing. Trypsin and thrombin cleave Protease Activated Receptors (PAR) which
increases phospholipase C (PLC), adenylcyclase (AC), and phosphatidylinositol
3-kinase (PIK3) activation of ERK 1/2 mitogen-activated protein kinase (MAPK/
ERK) (Bono et al. 1997). MAPK/ERK activation plays a key role in cell growth and
differentiation through transcriptional activation. Active PAR also increases
cytosolic phospholipase A2 (PLA2) synthesis of prostaglandins causing nuclear
receptor signaling of transcriptions.
Previous groups have shown Bik inhibits cell proliferation in a carcinoma cell
model (Kobayashi 2001). In carcinoma, inflammatory signaling is an important
component of proliferation. Bik inhibition of trypsin and thrombin prevents activation of PAR. Bik inhibition of plasmin prevents activation of urokinase-type plasminogen activator which breaks down the basement membrane during tissue
remodeling. Inflammatory MAPK activation is also inhibited through Bik interaction with CD44 binding (Suzuki et al. 2002). Our studies with normal cells, showed
Bik does not completely stop cell proliferation and that non-inflammatory activation of MAPK is not impacted. Activation of MAPK ERK through the tyrosinespecific protein receptors or the G-protein-coupled receptors is not impacted with
Bik. This is consistent with Bik only inhibiting proliferation due to inflammation
signaling through PAR and uPAR.
Bik exposure caused significant loss of intra-cellular Ca2+ favoring osmotic stress
and cell lysis. Reduction of intracellular Ca2+ is consistent with suppression of vascular smooth muscle contraction by Bik (Moczydlowski et al. 1992). Kunitz-type
protease inhibitors are known to bind Ca2+ activated K + channels causing modulation (Moczydlowski et al. 1992). Bik is similar in peptide sequence to aprotinin and
might be acting through same mechanism. The binding causes a blockage of the
channel whether by partial occlusion, repulsion or allosteric interaction. Blockage
of the Ca2+ sensitive potassium (KCa) channels (or calcium-activated potassium (BK)
channels) is thought to lower the intracellular Ca2+. The ability of K+ to exit the cell
is responsible for the resting membrane potential and controls the Ca2+ in-flux
through the voltage sensitive Ca2+ channels (Jensen et al. 2001). A reduced potassium in-flux is counterbalanced by the calcium channels (Kanayama et al. 1997).
This change in intracellular Ca2+ would be expected to impact the calcium
dependent pathways The calcium channel potentially explains the observed activation of PI3K and Akt signaling. Other groups have recently confirmed uTi that
10
187
activate PI3K-Akt and ERK signal transduction and inhibit of p38 MAPK and JNK
(Kim et al. 2009). The uTi affect is of great interest as the PI3K-Akt -mTOR pathway is an important topic in cancer where cells have reduced apoptosis and increased
proliferation (Vivanco and Sawyers 2002). Protein kinase C and phospholipase C
could also be impacted by decreased intra-cellular calcium. The difference in PI3K
and Akt signaling observed between the Uri and Bik could be due to Uri lacking the
O-linked chondrotin sulfate chain which promotes calcium binding. Previous studies have shown the chondroitin sulfate chains of Bik impact muscle contraction
(Kanayama et al. 1997).
We also found adiponectin (Adpn) signaling of G-protein coupled receptor
(AipoR1) restored cytosolic calcium in the presence of Bik. Therefore Adpn and Bik
activate the anti-inflammatory responses through separate but dependent cell signal
pathways of metabolic importance. Adpn initiates glycolysis through AMPK (Mao
et al. 2006; Deepa and Dong 2009). Bik activates PIK3-Akt and could interfere with
insulin receptor signaling (Deepa and Dong 2009; Kobayashi et al. 2005; Bertrand
et al. 2008). Bik inhibits proliferation without apoptosis and decreases intra-cellular
Ca2. Adpn counteracts the impact of Bik on the intra-cellular Ca2+ and could be
modulating intra-cellular Ca2+ through PI3K (Marcantoni et al. 2006; Lu et al. 2005).
10.5
Conclusion
Our results suggest that kidney exposure to Bik during abnormal stress might contribute to reduced renal regeneration with the signal transduction shown below. Bik/
Uri slowed kidney cell proliferation by more than half with exposure at 25 mg/L.
Bik/Uri did not activate caspases or mitogen-activated protein kinases (MAPK).
Natural cell death increased with over exposure of cells to Bik/Uri.
Bik/Uri caused a release of membrane-bound proteins, Dipeptidyl Peptidase 4
(DPP-4) and phosphatidylserine externalization.
Bik/Uri activated Phosphatidylinositol 3-kinase (PIK3) and protein kinase B
(Akt)
Bik/Uri reduced intra-cellular calcium in treated cells
The N-Glycan structure of the Uri and Bik is needed for trypsin inhibition.
The mAb 421.3G5 was able to bind the inhibitor region of Uri and Bik.
Under inflammatory stress condition the amounts Bik inhibitors overwhelm the
proteases involved in proliferation and proteolysis. While these biochemical cell
models support that Bik does not cause apoptosis and could protect tissues damaging immune cells, endothelial and epithelial proliferation is also slowed cell growth
which would reduce regeneration and repair (during wound healing) and cell proliferation (PKI3-Akt).
All of the information discussed here may provide new drug target design clues
for treatment of kidney inflammatory diseases. For example, the Bik site could be
blocked. Previously, a report showed a synthetic trypsin inhibitor could reduce the
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urinary albumin excretion in diabetic rats and human patients during the diabetic
renal hypertrophy (Ikeda and Hoshino 1996; Ikeda et al. 1999).
Acknowledgements We would like to acknowledge the laboratory work of the Notre Dame
undergraduate researchers in enzyme studies and cell models done by: Vesta Anilus, Yoonie Cho,
Kunal Saxena, Kimberly St. Jean, and Gabriel J. Crawford. We would like thank Dr. Sipra Banerjee
of the Cleveland Clinic Foundation for the gift of cancer cell lines. We would like thank Dr. Rui
Ma for his helpful efforts with advise to the students. Part of this research was supported by the
Jacobs Javits Research Grant Award from NIH-NINDS (NS-18005) and NCI R01 Grant (CA14764) awarded to SB and a grant-in-aid from Colman Cancer Foundation to MB.
Supplements
ELISA Method for Uristatin
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Part V
Summary
Chapter 11
Progress in New Markers for Diabetes

Inflammation
Michael Pugia
11.1
Introduction
The impact of hyperglycemia, obesity, and inflammation add significant tissue

stress during diabetes. These stress factors cause damaging pathologies in the key
affected tissues, namely the liver, brain, pancreas, muscle, adipose, and vascular
system (Table 11.1).
11.2
Hyperglycemia Stress Markers
It has been clearly shown that hyperglycemia correlates to diabetic inflammation,

complications, and worsened progression to end stage disease. Under normal conditions, hyperglycemia induces insulin secretion by the pancreas (Table 11.1). Insulin
reduces production of glucose and fatty acid oxidation by the liver as well as glucose stored by muscle (Akt/PIK3 pathway), and it decreases adipose energy storage
and lipolysis. Glucose consumption by the brain and red blood cells is stimulated by
glucose transport (GLUT1/4), and appetite suppression is signaled by the brain.
Insulin resistance is an early indicator of diabetic conditions, and this impairment leads to hyperglycemia. As such, glucose measurement remains the primary
tool relied upon for diabetes diagnosis and monitoring. During fasting, the liver is
the sole source of glucose production with the major route being gluconeogenesis
(production of new glucose from noncarbohydrate precursors) and the minor route
being glycogenolysis (break down of glycogen) Adipose is the major energy store
M. Pugia, PhD
DOI 10.1007/978-3-319-21927-1_11
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M. Pugia
Table 11.1 Impact of stress factor on key tissue

Liver
Brain
Pancreas
Adipose
Muscle
Vascular
system
Hyperglycemia
Glucose
production
Insulin
ROS
AGE
Fatty acid
oxidation
Glucose
consumption
Appetite
suppression
ROS
Insulin production
Obesity
Hyperglycemia
Insulin resistance
Cytokines/chemokines
Fatty acids (fatty liver)
Innate immune cells
Adipokine
response
Energy storage
Lipolysis
FFA/TG
Glucose uptake
Mitochondria
Fatty acid
oxidation
ROS
AGE
Kallikrein
(Kinins)
RAGE
Vascular
dysfunction
NO (ROS)
NO (eNOS by
insulin)
Kinins
AGE (HbA1c)
glyCD59
(complement)
RAGE
Adipokine response
Cytokines/chemokines
Hypoxia
Lipid loading
Lipolysis
Glucose uptake
IgG-CTF generation
Appetite suppression
Insulin production
Vascular dysfunction
NO (cytokines)
Apidose AgnII
Immune cell adhesion
eNOS (AMBK)
Inflammation
Glucose production
Insulin degradation/
sens.
Apoptosis ( damage)
Fibrosis ( response)
IgG & complement
Glucose consumption
sens.
IgG deposits
Amyloid deposits
Insulin production
Glucose sens.
Apoptosis ( damage)
IgG & complement
Auto-immune response
Insulin sens.
Apoptosis ( damage)
IgG & complement
Glucose consumption
sens.
Apoptosis ( damage)
Vascular dysfunction
Fibrosis (elasticity)
Membrane thickening
Apoptosis
Coagulation
Complement
of noncarbohydrate precursors (e.g. triglycerides lipolysis to free fatty acids and

glycerol) for gluconeogenesis, and the brain and red blood cells are the major consumers of glucose. These organs normally act in concert, and impaired insulin
metabolism leads to hyperglycemia stress across all organs along with reduced fatty
11 Progress in New Markers for Diabetes Inflammation
195
Table 11.2 Markers of glycation stress

Measurement
Insulin resistance/sensitivity
Glycemic control
Glycemic control & complement
stress
Markers
Glucose
Insulin
C peptide
HbA1c
Glycated CD59
Key cellular pathways

AKT-PIK3
GLUT1/4
IDE
Membrane glycation
MAC (complement activation)
acid metabolism. Hyperglycemia stress increases reactive oxidative species (ROS),

advanced glycation end products (AGE), vascular dysfunction, and complement
activation in the key tissues.
Measurements of glucose, insulin, and glycated hemoglobin (HbA1c) are essential tools to manage hyperglycemia (Table 11.2) where the goal of hyperglycemic
management is to use diet, exercise, and medications to obtain glucose values in a
normal range. Insulin resistance is often the underlying cause of an inability to manage hyperglycemia. As reviewed in Chap. 1, many factors impact and correlate with
insulin sensitivity. Insulin degradation is providing new understandings of insulin
sensitivity. The direct or indirect measurement of insulin resistance is difficult to
obtain and is best estimated using the traditional methods. However, the oral glucose tolerance test (OGTT) is not a spot test, and HbA1c is a late marker for prediction of diabetes in a group of prediabetes patients, and diabetes cannot be predicted
from a single new marker. This reduces the ability to identify patients who are likely
to progress to diabetes and respond to therapy. Additionally, hormones such as
growth hormone growth hormone (GH), insulin-like growth factors (IGF), IGF
binding proteins (IGFBP), epinephrine, glucagon, and cortisone impact the glucose
metabolic pathways.
Advanced glycation end products (AGE) such as HbA1c have shown value for
measurement of glycemic control over periods of 34 months. Whereas, other nontraditional AGE assays, such as glycated albumin, fructosamine, and
1,5-anhydroglucitol (1,5-AG), which measure glycemic control over a shorter
period, have not proved more valuable than HbA1c (Beck et al. 2011). Correlations
of complications with nontraditional AGE assays are typically less significant.
All current AGE assays lack a direct mechanistic link to complications, and the
receptors for advanced glycation end products (RAGE) have not provided that
mechanistic link. While insulin increases the receptors for AGE (RAGE), activation
of RAGE requires immune cell activation, and sRAGE levels do not correlate with
glycemic control or complications (Lam et al. 2013; Motawi et al. 2013). The formation of AGE on glomerular basement membrane (GBM) directly correlates with
HbA1c but is not linked to tissue damage or cellular proliferation without immune
cell involvement. Next generation AGE markers such as glycated CD59 offer hope
of a true mechanistic link (Chap. 2). Glycated CD59 could offer improvement over
HbA1c by its correlation with glucose intolerance in short term glycemic control
and by offering a molecular connection that is tied to a direct cell death pathway, as
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M. Pugia
exemplified in the literature by an increased interest in the formation of membrane

attack complex (MAC) in the compliment cascade (Qin et al. 2004).
Many therapeutic approaches have been allied to lowering hyperglycemia
(Mazzola 2012), and insulin is by far the most effective means used. Other
approaches include sulfonylureas and glinides, which lower glucose by enhancing
insulin secretion; metformin which suppresses glucose production in the liver;
-glucosidase inhibitors, which reduce the rate of digestion of polysaccharides in
the small intestine; thiazolidinediones (TZD), which activate PPARs (peroxisome
proliferator-activated receptors) to increase insulin sensitivity of muscle, fat, and
liver; incretins of the secretin family of hormones like Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), which improve
hyperglycemia by indirectly stimulating insulin secretion and suppressing glucagon
secretion; dipeptidyl peptidase-4 (DPP-4) inhibitors, which prolong incretin halflife; amylin agonists, which act as a neuroendocrine hormone to enhance satiety and
reduce food intake; and sodium-glucose transporter 2 (SGLT2) inhibitors, which
reduce glucose reabsorption in the proximal tubule of the kidney.
11.3
Markers of Oxidative Stress
While it appears clear that hyperglycemia leads to oxidative stress damage in tissues, the actual clinical value of measurements of ROS is less clear (Table 11.3).
ROS reacts with DNA as the primary target to produce various byproducts such as
8-hydroxydeoxyguanosine (8-OHdG) (Hwang and Kim 2007). Oxidative stress can
also be measured by oxidative modification of proteins, peptides and amino acids,
lipids, cell membranes, and small molecules. For example, markers such as nitrotyrosine, modified albumin, oxidized low density lipoprotein (ox-LDL), malondialdehyde (MDA), and arachidonic acid derived oxidation products (plasma
F(2)-isoprostanes) have been used to measure ROS.
Many enzymes, such as superoxide dismutase (SOD), nitric oxide synthase
(NOS), myeloperoxidase (MPO), and glutathione peroxidase, process nitric oxide,
a key ROS. In addition, small molecule REDOX acceptors also process ROS. These
include vitamin C, vitamin E, and uric acid (Pitocco 2013; Sung et al. 2013).
Glutathione (GSH) levels are a direct measurement of ROS generation (Motawi
et al. 2013). For example, GSH levels decrease in diabetic patients due to increased
mitochondrial oxidative activity and the formation of byproducts of NADH dehydrogenase. Oxidative damage of DNA, amino acids, lipids, and small molecules
also has unique repair systems such as DNA ligase and methionine sulfoxide reductase (Hwang and Kim 2007). Lipoprotein-associated phospholipase A2 (Lp-PLA2)
hydrolyzes oxidized phospholipids to generate lysophosphatidylcholine and oxidized fatty acids, and Lp-PLA2 activates the innate immune system.
Oxidative repair and formation is in flux, and clinical correlations often use
several biomarker measurements to account for overall oxidative stress (Sung et al.
2013). Oxidative stress is further complicated by the amplification caused by pro-

Table 11.3 Markers of oxidative
stress
Measurement
DNA damage
Markers
8-OHdG
Lipid
oxidation
ox LDL, MDA
197
Key cellular pathways
NOS
DNA ligase
SOD
NOX
NADH/GSH
Anti-oxidants (diet)
Innate immune cells
Lipase
Innate immune cells
inflammatory cytokines released by immune cells and stimulated by bacterial lipopolysaccharides (LPS) during infections. Therefore, many biomarkers, including
those for immune cell activation, are accounted for in clinical assessments. Urinary
biomarkers of oxidative damage has been an attractive topic of research due to the
noninvasive sampling process involved (Ilyasova et al. 2012). Thus, the range and
depth of biomarkers tested is broad and wide, and correlations vary greatly between
the actual species measured. Of the biomarkers tested to date, 8-OHdG, ox LDL,
and MDA are among the most clinically verified (See Table 11.3).
In obesity and metabolic syndrome, rapid weight loss correlates with reduced
oxidative stress as measured by 8-OHdG, oxLDL, and MDA. Oxidative stress measured by these three biomarkers significantly increases (P < 0.05) with fast blood
glucose and HbA1c in type 1 diabetics (Goodarzi et al. 2010; Holvoet et al. 1998).
Statistically significant higher values of ROS markers were observed in diabetic
patients along with slight reduction in the synthesis of nitric oxide and a decrease in
the antioxidant levels (Ramakrishna and Jailkhani 2007; Al-Rawi 2011). Of the
oxidative stress marker measured, markers of DNA damage, 8-OHdG and MDA,
appear to be the most clinically sensitive to diabetes. Dieting and improved antioxidant intakes significantly improves the levels of these markers in diabetic subjects,
and the improvements correlate with improved fasting plasma glucose value and
HbA1c (Armstrong et al. 1996).
Alternative approaches to measure oxidative stress by NO, SOD, GSH, or
REDOX acceptors poorly differentiate normal patients from diabetics with hyperglycemia (Motawi et al. 2013). Small decreases in MPO (15 %) and phospholipase
activities (Lp-PLA2) (4 %), after adjustment for age and sex, were observed in diabetic patients (Tumova et al. 2013). The levels of plasma F(2)-isoprostanes, oxLDL, and Lp-PLA2 as markers of oxidative damage were unchanged across the risk
categories of metabolic syndrome (Seet et al. 2010). A small decrease of 12 % in
oxidized low density lipoprotein (ox-LDL) levels was reported in diabetics with
hyperglycemia. Additionally, ox-LDL levels increase in diabetic patients with coronary artery disease (CAD) but do not increase in diabetic patients without CAD, and
these levels are impacted by sulfonylureas therapy (Motawi et al. 2013).
Hypoxia and uremic toxins increase oxidative stress during kidney and heart
disease. Antioxidant therapies show significant reduction in event risk but have not
198
M. Pugia
been shown to improved survival (Sung et al. 2013). Innate immune cell involvement shows a significant increase in oxidative stress and event risk. However, the
predictive value of ROS markers is weakly correlated (R ~ 0.4) with diabetes given
the complexity of oxidative stress status of the body. Many factors, such as age,
diet, and other diseases, contribute to oxidative stress levels. Therefore, biomarker
use in a clinical setting is impractical for general diabetic patient care, as all factors
must be accounted for (Motawi et al. 2013). Although recent trials on anti-oxidants
treatments (like alpha-lipoic acid) in type 2 diabetes have not shown decreases in
ROS or AGE levels (Porasuphatana et al. 2012), ROS markers remain a valuable
research tool.
11.4
Impact of Obesity
Adipose tissue is now recognized as having an endocrine function that serves as a

source of adipokines and as having an innate immune function that serves as a
source of cytokine and chemokine signals (Preedy and Hunter 2011). Release of
adipokines allows insulin independent glucose uptake and fatty acid oxidation in
muscle and liver. Obesity impairs the secretion of adipokines and causes insulinemia, hyperglycemina, lipidemia, and reduced appetite suppression (See
Table 11.1). Impaired adipokine secretion helps explain the impact of excess fat
storage during glycemia where an impaired AMP-activated protein kinase (AMBK)
pathway results in decreased glycolysis and reduced fatty acid oxidation. Obesity
and insulin resistance increase the release of free fatty acids from the liver. Lipid
loading causes the adipose tissue to become inflamed and hypoxic, and it induces
cytokine and chemokine release and stimulation of innate immune cells. However,
these new understandings of how adipokines relate to insulin resistance and the
resultant hyperglycemia has had limited benefits for todays diagnosis and
treatment.
Lifestyle changes such as exercise and diet for weight loss and glycemic control
have not stemmed the tide of the epidemic of obese adults with diabetes. Bariatric
surgery has become a common treatment for weight loss and glycemic control in
obese adults with diabetes (Maggard-Gibbons 2013), and studies have shown that at
the 2-year follow up after bariatric surgery, adiponectin, leptin, insulin resistance,
and body mass indexes (BMI) continue to show improvement (Trakhtenbroit et al.
2009; Ballantyne 2005). Glucose metabolism and insulin resistance are improved
short term by caloric restriction, and decreased fat mass leads to long-term improvements in insulin resistance and returns adipokines secretion closer to normal levels
(Cumb 2005; Sasaki et al. 2014). It is common for bariatric surgery patients to also
have non-alcoholic fatty liver disease (NAPLD), and NAPLD progresses to nonalcoholic steatohepatitis (NASH) in morbidly obese subjects. Hepatic insulin resistance and steatosis precede the development of type 2 diabetes. Thus, bariatric
surgery improves steatosis, fibrosis, lipidemia, and inflammation markers in NASH
and NAPLD patients (Sasaki et al. 2014).
199
Adipokines have led to several new therapeutic approaches for treatment of obesity and diabetes. Adiponectin administration in rodents is insulin-sensitizing and
decreases body weight, but this result is not applied to humans (Haluzik 2005).
Thiazolidinedione (TZD) activation of peroxisome proliferator-activated receptors
(PPAR) increases adiponectin and transcription of important regulators in transport, synthesis, storage, and oxidation of fatty acids. Leptin therapy normalizes
gonadotrophin secretion and menstrual function but is only used in rare human
disorders that cause congenital leptin deficiency and lipodystrophy. Leptin treatment causes reductions in hyperglycemia, hypertriglyceridemia, and hepatic steatosis (Gorden 2003)
Gherlin and peptide tyrosine tyrosine (aka peptide YY, PYY, pancreatic peptide,
and YY3-36) are also important factors that impact obesity through appetite. Gherlin
and PYY are released by cells in the ileum and colon in response to feeding (Soares
et al. 2008; Knudsen et al. 2014). Inhibition of ghrelin impacts growth hormone
secretagogue receptor (GHSR) and neuropeptide Y (NPY) pathways and reduce
appetite, food intake, body weight, and adiposity. Thus, the gherlin pathway is an
attractive therapeutic target. When PYY is injected and enters the central nervous
system, it acts as an orexigenic and increases appetite. It also has functions related
to digestion and absorption and is associated with both increased and decreased
appetite. Obese people secrete less PYY than non-obese people, and studies have
shown that it inhibits appetite in rodents and primates. Leptin treatments have also
been shown to reduce appetite, but obese people develop a resistance to leptin, so it
is not an effective treatment for obesity.
11.5
Adipokine Markers
While adipokine biomarkers are closely associated with obesity and diabetes, the
predictive value of these markers has not made the grade for routine diagnostic use
(See Table 11.1). Adiponectin, leptin, and gherlin have the strongest correlations
and are useful for pathway research (See Chap. 1 and Table 11.4), and levels of these
three markers improve after bariatric surgery (Ballantyne 2005; Gumbs et al. 2005).
Table 11.4 Markers of adipose stress
Measurement
Impaired glycolysis and fatty
acid oxidation
Markers
Adiponectin
Appetite suppression
Gherlin
Fat mass and energy balance

(intake/exercise)
Leptin
Key cellular pathway

AMPK
PPAR
GHSR
Neuropeptide Y (NPY)
JAK STAT
GHSR
Neuropeptide Y (NPY)
JAK STAT
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M. Pugia
Leptin is directly correlated to patient fat mass and diet/exercise balance (Meiera
and Gressner 2004). In addition, leptin and adiponectin correlate with hypertrophic
adipose tissue in obesity and reduced glycolysis, reduced fatty acid oxidation, insulin resistance, and type 2 diabetes. An increase in the ratio of adiponectin to leptin
improves the correlations (Satoh 2004). Ghrelin levels also generally correlate with
obesity. Leptin and ghrelin exert important roles on body weight regulation, eating
behavior, and reproduction by acting on the central nervous system and target reproductive organs. The relevance of other adipokines has not been resolved or strongly
correlated and are still of question (See Chap. 1).
11.6
Fatty Acid Markers
Obesity increases adipose release of free fatty acids (FFA) and triglycerides (TG),
and adipose tissues can produce FFA and reduce TG by the acylation stimulating
protein (ASP). Interaction of the ASP precursor (compliment C3), factor B, and
adipsin produce ASP. Elevated FFA concentrations are predictive of pre-diabetic
progression to diabetes but are not diagnostic of diabetes, and a decrease in fasting
FFA adversely impacts glucose-induced insulin release by pancreatic -cells. FFA
acid uptake and metabolism are required for normal islet function (Lam et al. 2002).
Insulin increases the production of ASP. Fatty acid oxidation, PPAR gamma activation, and mitochondrial activity in the muscle and liver are also upregulated by
insulin (Turner et al. 2007). Fasting plasma ASP concentration correlates with fasting serum triglyceride during the oral fat tolerance test (OFTT) and negatively correlates with the glucose disposal rate and high-density lipoprotein (HDL)
concentrations (Koistinen 2001). However, there is no correlation between fasting
plasma ASP concentration and TG in lean healthy men, and fasting plasma ASP
concentrations are only mildly elevated in obese and diabetic subjects (Koistinen
2001; Peake et al. 2005). Given these correlations, fatty acid stress is currently best
measured by typical and proven diagnostic markers of lipidemia (See Table 11.5).
ASP is highly correlated to ketosis-prone diabetes (Liu et al. 2014). Other adipose
markers such as lipase, FABP, and adipose factor are involved in fatty acid stress
during obesity but have poor diagnostic predictive value for diabetic onset (See
Chap. 1).
Nuclear receptors such as sterol regulatory binding protein (SREBP), farnesiod
X receptor (FXR), and liver X receptor (LXR) are pharmacological targets for
improving lipidemia, bile acid production, and glucose metabolism in type 2 diabeTable 11.5 Markers of fatty acid stress
Category
Impair fatty acid tolerance
Markers
Fatty acids (FFA)
Triglycerides (TG)
Lipids (HDL/LDL)

ASP
DCAT
SREBP, FXR and LXR
201
tes (Ding 2014). Synthetic derivatives of triiodothyronine, retinoic acids, fatty

acids, eicosanoids, oxysterols, bile acids, steroids, xenobiotics, mineralocorticoids,
glucocorticoids, androgens, estrogens, progesterone, retinoic acids, synthetic estrogens, riccardin, and podocarpic acid amides have all been identified as potent agonists of SREBP, FXR, and LXR (Lui 2005; Makishima 2005). Acyl CoA
diacylglycerol transferase (DGAT) is an important enzyme in TG synthesis and
another important pharmacological target (Subauste and Burant 2003). Treatment
with n-3 fatty acids as inhibitors of DGAT decreases the availability of fatty acids
for triglyceride synthesis (Subauste and Burant 2003; DeVita and Pinto 2013).
Statins are inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (HMG) and
lower cholesterol. Reduced cholesterol directly reduces DGAT expression by
increasing the transcription factor SREBP and upregulates fatty acid oxidizing
enzymes and inhibitors (Chen and Farese 2005).
11.7
Inflammation Markers in Diabetes
Adipocytes produce tumor necrosis factor alpha (TNF), IL-1, and IL-6 during
obesity and are shown to cause insulin resistance (Kern et al. 2001; Lorenzo et al.
2008; Cawthorn and Sethi 2008; Coppack 2001). Adipose tissue also contains a
significant amount of stromovascular fraction that is made up of preadipocytes,
endothelial cells, smooth muscle cells, fibroblasts, leukocytes, and macrophages
which can produce substantially more TNF than adipocytes (Cawthorn and Sethi
2008). Production of IL-1 and IL-6 is regulated differently from TNF, and interactions between macrophages and adipocytes control adipocyte differentiation and
cytokine expression (Xie et al. 2010; Cawthorn and Sethi 2008). TNF induces
cytokine secretion by inflamed cells and is cytotoxic. IL-6 promotes differentiation
of B cells and stimulates antibody secretion by plasma cells. IL-1 stimulates T cells,
attracts macrophages and neutrophils, and activates NK cells (Gao et al. 2014). Cell
signaling by TNF, IL-1, and IL-6 decreases GLUT4 in adipocytes. Both IL-6 and
TNF- inhibit insulin action through the Akt-PIK3 pathway. They also both initiate
a host of other cytokines (e.g. IL-8, IL-18) and chemokines (e.g. MIC-1, MIP-2,
MCP-1, CINC-1) to recruit macrophages and other innate immune cells to inflamed
tissue. Chemokines direct chemotaxis to distressed cells and cause neutrophils,
macrophages, T-lymphocytes, eosinophils, natural killer cells, and other innate
immune cells to build-up in the adipose. These pathways can be characterized by
several markers (See Table 11.6).
During insulin resistance, cytokines and chemokine secretion is elevated in adipose tissue and muscle (Beavers and Nicklas 2011; Cawthorn and Sethi 2008).
Adipose tissue produces a five-fold increase in TNF or IL-1 and a 40-fold increase
in IL-6 secretion which correlates with obesity measured by BMI (Kern et al. 2001;
Zhao et al. 2014). Plasma values of IL-8, IL-18, and Monocyte chemotactic protein
(MCP-1) are elevate to a lesser extent during insulin resistance, and plasma levels
of IL-1, TNF, and IL-6 also correlate with insulin resistance and adipose dys-
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Table 11.6 Inflammatory markers adipose stress

Category
Cytotoxic adipose response in obesity
Prolonged inflammation exposure due to
obesity and diabetes
Macrophage response
Markers
TNF-, IL-6, and
IL-1
CRP
TNFRs
YKL-40, IL-6 ,
MCP-1

NFB activation
Akt-PIK3 inhibition
TNF-
IL-1
IL-6
M2 pro-inflammatory
polarization
function (Beavers and Nicklas 2011; Kern et al. 2001). However, clinical utility is
limited, as values of IL-1, TNF, and IL-6 show high intra and inter-individual
variability with overlap between healthy and diabetic individuals (Kern et al. 2001;
Breslin et al. 2012; Beavers and Nicklas 2011). Nonspecificity is also an issue, as
cytokines are also markers of endothelial dysfunction and vascular inflammation
(Beavers and Nicklas 2011).
Tumor necrosis factor receptor (TNFR) is released as a soluble form (sTNFR)
into plasma during diabetic inflammation (Cawthorn and Sethi 2008), and in animal
studies, neutralization of TNF by sTNFR leads to a significant improvement in
insulin resistance (Hotamisligil et al. 1993). However, in both obese and nonobese
adults with damaging lipidemia, plasma sTNFR levels increase and are not more
correlated to obese adipose than TNF (Cawthorn and Sethi 2008; Good et al.
2006). Plasma sTNFR has been correlated to progression of chronic kidney disease
(CKD) (Krolewski et al. 2012). This chronic response likely reflects prolong TNF
activation and not acute innate immune activity (See Table 11.6). In contrast, lipocalin (LCN-2), aka neutrophil gelatinase-associated lipocalin (NGAL), is secreted
by neutrophils during renal ischemia reperfusion injury and is a marker of acute
kidney injury (AKI) (Soni et al. 2009). However NGAL is only weakly associated
with obesity and diabetes through levels of fasting triglycerides and LPS-binding
protein and FFA intake (Moreno-Navarrete et al. 2010)
C-Reactive protein (CRP) synthesis is upregulated by cytokines, predominantly
IL-6, and is a precise measure of systemic mild inflammation that returns to baseline 1224 months after weight loss from bariatric surgery (Gebhart et al. 2014).
Mild inflammation as measured by CRP is consistently shown to be an independent
predictor of metabolic syndrome, obesity, and diabetes (Seet et al. 2010) (See
Table 11.6). CRP activates the complement pathway, binds phosphocholine, and
promotes clearance of apoptotic cells (Szalai 2004). CRP levels are significantly
increased in obese patients during hyperglycemic and diabetic ketoacidosis crises as
compared to obese controls (Stentz et al. 2004), and these CRP values return to
normal after insulin treatment and resolution (Stentz et al. 2004; Peuchant 1997).
Increased fat mass correlates with CRP (Tsuriya et al. 2011). However, these CRP
changes are small in magnitude and cannot predict type 2 diabetes or the prediabetic
state but are clearly indicative of prolonged chronic inflammation.
Mild elevations of peripheral white blood cell (WBC), neutrophil, granulocyte,
lymphocyte, and monocyte counts are associated with the risk of diabetes (Gkrania-
203
Klotsas et al. 2010; Vuong et al. 2014). However, these innate immune cell levels
are not able to differentiate progression to diabetes. In diseased adipose tissue, macrophages are polarized into pro-inflammatory M1 forms by IL-10, CD8 positive T
cells, interferon (INF-), and immunoglobulins from B cells. Alternatively, macrophages are polarized in healthy adipose tissue into anti-inflammatory M2 forms by
regulatory T cells (Tregs), eosinophils, and IL-4. As polarization occurs in tissue,
few blood based markers for macrophage responsivity exist today. However, general macrophage markers such as chitinase-3-like protein 1 (YKL-40), which is
secreted by macrophages in inflamed tissues and causes activation of the Akt prosurvival signaling (anti-apoptotic pathway), can be used. YKL-40 levels are elevated in patients with vascular disease, hypertension, diabetes, and obesity (Ridker
2014). However, YKL-40 is also elevated in neutrophils. MCP-1 is another example
of a marker secreted primarily by macrophages, endothelial cells, and adipocytes
(Patel et al. 2013).
11.8
Endothelial System Markers
Vascular dysfunction occurs in diabetic and obesity as the endothelium loses it

selectivity as a permeable barrier (Versari et al. 2009). The eNOS, RAAS and
kallikrein-kinin response systems all fail to maintain normal vascular homeostasis
with hyperglycemia, reactive oxidative species (ROS), free fatty acid (FFA) stress
and pro- inflammatory signaling (Roberts and Porter 2013; Brady et al. 2012).
Blood pressure increases in diabetic obesity. Circulating pro-inflammatory cytokine
signaling (TNF-, IL-1) activates MAPK p38 and causes releases of vascular
adhesion molecules (ICAM, VCAM, E-selectin) by endothelial cell (Szmitko et al.
2003b; Collins et al. 1995) (See Chap. 1). Vascular adhesion molecules attract
innate immune cells to endothelium. Tissue remodeling with hypertrophy increases
in the heart, kidney, liver, peripheral vascular and artery systems (Versari et al.
2009).
When innate immune cells pass the endothelium barrier and infiltrate the surrounding tissue, they also initiate repair and wound healing mechanisms (Szmitko
et al. 2003a). Serine proteases that are released from innate immune cells activate
protease-activated receptors (PARs). This group of receptors activates platelet
aggregation, vascular smooth muscle proliferation, promotes thrombosis, prolongs
inflammation, stimulates endothelial cell proliferation, increases collagen synthesis,
stimulates connective tissue synthesis, and activates platelet-derived growth factor
production (Barry et al. 2006; Szmitko et al. 2003b). The activation of PAR increases
endothelial cell release of plasmin, thrombin, and trypsin and allows urokinase
(uPA) activation of tissue remodeling (See Fig. 1.2 of Chap. 1). Plasminogen
activator inhibitor 1 (PAI-1) is a modulator of fibrinolysis that slows cell proliferation and restores normal cell differentiation (Ghosh and Vaughan 2012). PAI-1 correlates to fibrosis (decreasing elasticity) and membrane thickening throughout the
body.
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M. Pugia
Table 11.7 Markers of endothelial

stress in diabetes
Category
Vascular fibrinolysis
Markers
PAI-1
Anti-proliferative
factor
Bik/Uri

PAR 1-4
VCAM-1/ICAM-1
uPA/tPA
PAR inhibition
uPA inhibition
Akt-PIK3 activation
There are several markers of endothelial dysfunction in diabetes and obesity that
have value for patient assessments (See Table 11.7). Plasminogen activator inhibitor-1 (PAI-1) levels significantly increase during hyperglycemic and diabetic ketoacidosis in obese patients as compared to obese controls (Stentz et al. 2004;
Lopes-Virella et al. 2008). PAI-1 activity assays correlate with metabolic syndrome
better than PAI-1 antigen assays do (Al-Hamodi et al. 2011), and tissue plasminogen activator (tPA) activity is not elevated in these patients. Uristatin (Uri) and
Bikunin (Bik) respond to tissue regeneration by inhibiting PAR and uPA (Pugia
et al. 2007), and Bik and Uri are urinary markers that correlate with glomerular
nephritis and suppress cellular proliferation while activating Akt-PIK3.
There are several additional markers of endothelial dysfunction that are involved
in diabetic conditions but have not yet proven clinical value. Endogenous tissue
inhibitor of metalloproteinases (TIMP), angiotensin I converting enzyme, and angiotensinogen correlate with atherosclerosis but do not correlate directly with diabetes
(Szmitko et al. 2003a, b; Goldfine et al. 2011). Endothelin-1 (ET-1) is a potent vasoconstrictor that is released by the endothelium in obese subjects. However, circulating levels of ET-1 reflect overall clearance and not vascular production (Weil et al.
2011). Vascular adhesion molecules (ICAM, VCAM, E-selectin) are increased in
poorly controlled diabetes (Motawi et al. 2013; Ridker 2014; Lopes-Virella et al.
2008). However, vascular adhesion molecules and fibrinogen are less predictive of
hypertension, diabetes, and obesity than inflammation markers (Ridker 2014; Motawi
et al. 2013). Matrix metalloproteinase (MMP) levels decrease in patients with diabetes and hyperglycaemia and correlates with reduced C-peptide (Lewandowski et al.
2011; Shiau et al. 2006). However, MMP levels are increased in patients with atherosclerosis (Szmitko et al. 2003a; Wang 2003). Both sCD40 and adrenomedullin levels
are increased in patients with atherosclerosis and diabetes but play an unspecific role
in diabetes (Szmitko et al. 2003a; Wong et al. 2014; Gottster et al. 2013).
11.9
Markers of Dysfunctional Cell Growth
Obesity mediated diabetes is characterized by inflamed, hypertorphic, and dysfunctional tissues. This dysfunctional tissue is observed in the adipose, liver, pancreas,
vascular system, and kidney (Halberg et al. 2009; Suzuki et al. 2014; Li et al. 2014;
Rosenberger et al. 2008; Haligur et al. 2012), and these tissues all share the characteristic of being under oxygenated. Hypoxia typically induces an angiogenic
205
response with endothelial cell migration and organization into capillary-like structures. However, dysfunctional tissue growth occurs in obesity and diabetes as characterized by fibrosis and innate immune cell infiltration. For example, the control of
pre-adipocytes, specialized fibroblast-like progenitor cells that normally differentiate into mature adipocytes under control of growth factors, is over-ridden in diabetic
and obese patients (Sorisky et al. 2000).
Hypoxic tissues release TGF and IL-6, which chemotactically attract macrophages and promote fibroblast, endothelial, and progenitor cell growth (Bourlier
et al. 2012; Halberg et al. 2009). Matrix metalloproteinase (MMP) is responsible for
the proteolytic activation of TGF, and TGF causes endothelial cell expression of
fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF)
which block apoptosis via MAPK p38 and allow cell growth during tissue remodeling (Ferrari et al. 2006). Additionally, endothelial hyaluronan receptor 1 (LYVE-1)
and hypoxia-inducible factor (HIF) are upregulated in hypoxic tissue and contribute
to dysfunctional tissue growth and fibrosis (Sun et al. 2013). Leptin deficiency contributes to transcription of hypoxia related genes (Yingzhong et al. 2006). Other
growth factors such as growth hormone (GH), insulin-like growth factors (IGF),
epidermal growth factor (EGF), placental growth factor (PDF), hepatocyte growth
factor (HGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF),
and growth differentiation factors (GDF) are also important players in diabetes, and
they work together to control differentiation of various cell types and connective
tissue (Chiarella et al. 2004; Doronzo et al. 2006; Lu et al. 1999; Cook et al. 2002;
Seedorf 1995, Cirri et al. 2005). Cell signaling to generate normal tissue growth
versus dysfunctional tissue growth under stress conditions is a complex process
(Seedorf 1995).
New markers are needed to identify and monitor the repair processes during
diabetic hypoxia (See Table 11.8). Hypoxic conditions increase HIF and TGF values
which induce changes in the expression of >1000 genes (Jiang et al. 2011;
Ranganathan et al. 2007). Serum TGF-1 levels increase in diabetic nephropathy
(El Mesallamy 2012). Plasma TGF-2 decreases, and urinary TGF-1 increases in
type-1 Diabetes Mellitus (Azar et al. 1999; Flores et al. 2004). TGF-1 directly correlates to insulin administration and is an effective measure of therapeutic response
to pro-fibrotic stimulus (Flores et al. 2004; Pscherer et al. 2013). Reduction of
hypoxia signaling also improves insulin sensitivity and decreases adiposity (Jiang
et al. 2011). Plasma HIF-1 levels are closely correlated and have predictive value
to coronary calcification in diabetes (Li et al. 2014). Furthermore, VEGF levels
significantly increase in diabetes and atherosclerosis, whereas VEGF receptor (soluble Flt-1) does not significantly increase in diabetic patients but has value as a
marker of angiogenesis (Blann et al. 2002). Many other growth factors such as
FGF-19, GDF-15, HGF, NGF, and IGFBP are insulin dependent and undergo
changes in diabetes (Vila et al. 2011; Goldfine et al. 2011; Cirri et al. 2005;
Matsumoto et al. 2007; Gerhard et al. 2013; Azar et al. 1999). Since these growth
factors correlate with insulin, measuring insulin sensitivity is more diagnostically
addressable. Urinary EGF levels are insulin independent and are lower in diabetic
patients, but these changes are thought to be related to declining kidney function
and age (Lev-Ran et al. 1990; Kawaguchi et al. 1993).
206
M. Pugia
Table 11.8 Markers of

dysfunctional cell growth in
diabetes
Category
Pro-fibrotic
stimulus
Markers
TGF-
Hypoxia
HIF
Insulin desensitizing
Ig-CTF
Angiogenesis
VEGF

SMAD
MAPK p38
Akt-PI3K
NOS
VEGF
EPO
TACE
IDE
PKC
MAPK ERK
Paxillin
Akt-PI3K
Insulin and growth hormone have a strong impact on cell growth and differentiation (Chiarella et al. 2004). Tissue and cell growth and survival are activated through
the Akt-PI3K pathway (Siddle 2011; Chiarella et al. 2004; Sorisky et al. 2000).
Insulin activation of Akt-PI3K increases expression of HIP-1, VEGF, GDF, PPAR,
IGF, and IGFBP (Doronzo et al. 2006; Lu et al. 1999; Cook et al. 2002). Insulin
inhibits PDGF (Cirri et al. 2005). FGF and EGF are insulin independent growth
factors that activate MAPK ERK (Kir et al. 2011). EGF also activates HGF
(Scheving et al. 2002). However, MAPK ERK is also stimulated by insulin receptors and IGFR and causes cross talk with EGF (Seedorf 1995; Kir et al. 2011; Cross
et al. 1997). The Akt-PI3K pathway is therefore of great regulatory interest for tissue growth in diabetes.
As shown in this volume, inhibition of insulin degradation through adiponectin
receptor-1 C-terminal fragments (AdipoR1 CTF) is providing new understanding of
tissue insulin resistance and intracellular insulin control (See Table 11.8). Oxygen
and glucose deprivation increase expression of TNF converting enzyme (TACE)
(Hurtado et al. 2001), and TACE is key in AdipoR CTF-Ig formation. TACE is also
the mechanism of release for soluble forms of TNF, TGF, and IL-6 from their
membrane-bound precursors in hypoxia and is essential in all steps of immune cell
recruitment and inflammatory response (Black 2002; Garton et al. 2006; Ichikawa
et al. 2004). Overactive mitochondria activity in the muscle could increase AdipoR
CTF as a means for desensitizing insulin action that is directed by immunoglobulin
attachment. Patients undergoing anti-TNF therapies have lower plasma Ig-CTF levels
that are similar to levels observed in long term diabetes patients (See Chaps. 5 and 6).
11.10
Innate Immunity Markers
Inflammation, vascular dysfunction, and hypoxia attract innate immune cells such
as neutrophils, macrophages, natural killer cells, and eosinophils into the diabetic
tissue (Lee and Lee 2014; Olefsky and Glass 2010; Johnson et al. 2012).
207
Immunoglobulin and complement proteins are also attracted. Dysfunctional tissue

is cleaned-up through immune mediated apoptosis (Fink and Cookson 2005;
Olefsky and Glass 2010) (See Chap. 1, Fig. 1.3). Regulated cell death (apoptosis)
causes cell loss in the adipose, liver, pancreas, and kidney during diabetes (Sorisky
et al. 2000; Younossi et al. 2008; Chandra et al. 2001). Apoptosis is initiated after
the innate immune cells infiltrate the endothelium and release serine proteases,
granzyme B, cathepsin G and proteinase 3 (PR 3) (Wiedow and Meyer-Hoffert
2005; Johnson et al. 2003). Pro-inflammatory signaling (e.g. TNF-, IL-1) and
activation of NFB remove protection from granzyme B apoptosis (See Chap. 1)
and trigger apoptosis through caspase 8 activation (Cawthorn and Sethi 2008).
Apoptosis prevents the maturation of pre-adipocytes into normal healthy tissue
(Sorisky et al. 2000). Neutrophils, macrophages, T-lymphocytes, eosinophils, natural killer cells and other innate immune cells also mediated damage by secreting
TNF- (Olefsky and Glass 2010). Immune cell activation of alternative complement
pathway is through C3b deposition followed by factor B activation and ending with
opsonized and lysed tissue cells (Carroll 2004a, b; Mamane et al. 2009; Phieler
et al. 2013; Schulze et al. 1993)
T- Cell and B- cell lymphocytes and other adaptive immune cells are also clearly
observed in diabetic tissues (Travers et al. 2015; Johnson et al. 2012; Lalor 2002;
Descamps-Latscha 1993). Classical complement pathway deposits immune complexes (IgG & IgM) followed by factor B activation and ending with opsonized and
lysis of tissue cells (Carroll 2004a, b, 4; Alsaad and Herzenberg 2007; Callard
1975). Apoptotic cell antigens are detected by IgM on B cell which are activated by
T cells in the lymphoid compartment to produce IgG. This is followed by plasma
and memory cell production by the spleen and bone marrow. Apoptotic antigens are
then detected in dysfunction tissue by free IgG and activate the complement complex. The complex attracts neutrophils and macrophages to induce cell death and
clean up, and the diabetic autoimmune response destroys the ability of the pancreas
to produce insulin (Yoon and Jun 2005). Work in this volume showed CTF adsorption causes insulin resistance. Antibodies attached to CTF are polyclonal and are
directed at multiple antigens but could play a role in the adaptive response. Insulin
sensitivity is required for B-cell and T-cell activation. Whether Ig-CTF protects
cells from adaptive immune response or further activates the response, remains to
be seen.
11.11
Conclusion
New markers of adipose, glycation, endothelial, and oxidative stress can be combined with markers of dysfunctional cell growth, inflammation, and fatty acid stress
to give a systematic view of the diabetic inflammatory process. However, there is
still a need to identify and monitor injury due to immune cell activation during diabetes, and markers are needed to sort out the complex interaction of innate and
adaptive immune cells within tissues. Anti-inflammatory drugs to date have had
limited effectiveness to reduce diabetic complications (Agrawal and Kant 2014).
208
M. Pugia
The goal of new markers should be to improve therapeutic outcomes and reduce the
cost of diabetic complications. Therapies directed toward reducing the inflammatory response and protecting specific tissues such as the kidney, vascular systems,
liver, pancreas, and eyes are needed.
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Index
A
Acute kidney injury (AKI)
biomarker analysis, 147148
in cardiopulmonary bypass surgery
patients, 144
patient population, 145147
post-operative prediction, 152
pre-operative predictors, 151154
pro-inflammatory biomarkers, 144
risk assessment tools, 144
risk score, 144
sample collection, 147
statistical analysis, 148151
uTi, 144, 145
Adipokine markers, 194, 199200
Adiponectin receptor C-terminal fragment
(AdipoR/AipoR CTF)
animal models, 9798
antibodies, 6566
bioassays, 95
biomarker measurements, 98
blood sample preparation, 80
brain morphologic observations, 87
cell and tissue staining procedure, 86
cell culture methods, 95, 99
cell culture treatment, 9596, 99
cell lines, 95
cell proliferation counts, 97
cellular insulin, 97, 100102
conjugates and standards, 6667
correlation to inflammation, 118, 119
diabetic collection, 116
disease progression, 98, 102,
104106
ELISA, 96
blood levels, 74
calibrator reproducibility and stability, 72
cross reactivity, 72
immunoglobulin bound form
measurement, 6768
immunoglublin chain
identification, 71
interference testing, 73
patient recovery, 73
fat morphologic observations, 86
fatty-acid oxidation, 62, 63
FBG measurement, 112
gestational diabetes
collection, 114115
Ig-CTF correlations, 119, 121
Ig-CTF distribution, 118, 120
predictive values, Ig-CTF, 120, 122
gluconeogenesis, 62, 63
glucose clamp, 113
G protein-coupled receptor, 6364
HbA1c measurement, 112
IDE, 7879
Ig-CTF and CTF measurements, 116
Ig-CTF R1 values, sample measurement,
116118
iMALDI approach, 6869
immunoglobulin binding mechanism, 7071
immunuo fluorescent staining, 8789
inhibition studies
ADAM17/TACE and IDE activity, 8184
InnoZyme Insulysin-IDE
Immunocapture Activity Assay Kit,
80
InnoZyme TACE Activity Kit, 80
insulin

DOI 10.1007/978-3-319-21927-1
215
216
Adiponectin receptor C-terminal fragment
(AdipoR/AipoR CTF) (cont.)
receptor response, 88, 90
sensitivity/resistance, 112, 113
sensitizing agent, 62
intracellular calcium, 9697, 99100
in-vivo insulin, 101103, 105106
leptin resistance, 94
liver morphologic observations, 87
mass spectroscopy peptide enrichment
assays, 7172
mechanism of action, 104, 107
MEROPS search, 78
muscle
glucose uptake, 62, 63
morphologic observations, 86
OGTT, 112
oligomeric isoforms, 62
pancreas morphologic observations, 87
patient reproducibility and stability, 73
peptide preparation, 6465
plasma forms, 85
precision and accuracy testing, 73
pre-diabetic
collection, 115, 116
correlation, 121123
protease identification, 7980
reference range testing, 114
SELDI-MALDI method, 69
SISCAPA method, 68
TACE, 79
T-cadherin, 63
tissue
and blood analysis, 81
forms and distribution, 85
sample preparation, 80
TNF-, 62
TZD treatment, 62
western blot method, 67, 70, 96, 9899
western blot techniques, 81
X-ray crystallography analysis, 62
Adipose stress markers, 201203
Advanced glycation end products (AGEs),
195196
vs. CD59 markers, 6
glycated hemoglobin, 5
human serum albumin, 5
molecular damages, 5
nontraditional markers, 5
receptors activation, 56
AGEs See Advanced glycation end products
(AGEs)
Aprotinin, 172, 183185
Index
B
secretase (BACE), 7879
Biacore testing, 65
Bikunin (Bik)
anti-inflammatory cellular signaling
aprotinin, 183185
Ca2+ in-flux, 179, 186
cellular signal response, 177179
DPP-4, 178181
membrane disruption, 179, 180,
185186
necrosis, 185
peptide analogs, 181183
PI3K-Akt pathway, 181, 182,
186187
proliferation, 186
proteinuria, 184
trypsin inhibition, 182, 184
urinary trypsin inhibitor, 172
Bioassays, 95
C
Cardiomyocyte (C2C12) models
cell culture treatment, 99
intracellular calcium, 99100
western blot analysis, 9899
Cardiovascular disease (CVD), 18, 3839
adiponectin response, 168
atherosclerosis, 157
demographic characteristics, 161, 162, 164
diagnostic risk factor, 161, 163, 164
epidemiological evidence, 158
indicators, 157158
PAR activation, 158159
statistics methods, 161
CD59 glycoprotein
animal experimental model, 41
glycated (see Glycated CD59 (GCD59))
glycation-inactivation, 3940, 4243
MAC inhibition mechanism, 3536
Chronic kidney disease (CKD), 131, 137, 139
Complement system
diabetic complications
cardiovascular disease, 3839
nephropathy, 3637
neuropathy, 38
retinopathy, 37
MAC
alternative pathway, 31, 33
classical pathway, 31, 33
inhibitory protein (see CD59
glycoprotein)
217
Index
MBL/lectin pathway, 31, 33
non-lytic effects, 3435
therapeutic application, 44
C-reactive protein (CRP)
acute phase reactants, 158
pro-inflammatory response, 166, 168
vascular stenosis, inflammation markers,
164166
C-terminal fragment (CTF) See Adiponectin
receptor C-terminal fragment
(AdipoR/AipoR CTF)
CVD See Cardiovascular disease (CVD)
D
Dipeptidyl peptidase 4 (DPP-4), 174, 178181
Dysfunctional cell growth markers
AdipoR1 CTF, 206
Akt-PI3K pathway, 206
hypoxia, 204206
E
ELISA See Enzyme linked immunosorbent
assays (ELISA)
Endothelial dysfunction
adipose angiotensin, 12
Bikunin, 13
chronic activation, 12
kinin formation, 1112
nitric oxide, 11
TFPI action, 13
Endothelial stress markers, 203204
Enzyme linked immunosorbent assays
(ELISA), 96, 174
blood levels, 74
calibrator reproducibility and stability, 72
cross reactivity, 72
immunoglobulin bound form measurement,
6768
immunoglublin chain identification, 71
interference testing, 73
patient recovery, 73
F
Fasting blood glucose (FBG), 112
Fatty acid markers, 200201
Fibrosis
Bikunin action, 17
coagulation, 16
mechanism, 16, 17
Free fatty acids (FFAs), 910
G
GCD59 See Glycated CD59 (GCD59)
Gestational diabetes
collection, 114115
Ig-CTF correlations, 119, 121
Ig-CTF distribution, 118, 120
predictive values, Ig-CTF, 120, 122
Glomerular filtration rate (GFR), 129, 131,
132
Glycated CD59 (GCD59)
ELISA, 46
HbA1c assay, 45
OGTT test, 47
plasma level, 4647
H
Hyperglycemia
complications with, 3
glycation stress (see Advanced glycation
end products (AGEs))
insulin resistance, 34
oxidative stress, 67
stress markers
AGE, 195196
glycation stress markers, 195
impaired insulin metabolism, 194195
insulin resistance, 193195
stress factor, 193, 194
therapeutic approaches, 196
I
Inflammation
endothelial dysfunction
adipose angiotensin, 12
Bikunin, 13
chronic activation, 12
kinin formation, 1112
nitric oxide, 11
TFPI action, 13
fibrosis
Bikunin action, 17
coagulation, 16
mechanism, 16, 17
innate immune apoptosis
complement system, 15
cytokines, 1415
lymphocytes, 1415
mechanism, 1314
serine proteases, 15
T-cells, 15
218
Infliximab, 114
Innate immune mediated apoptosis
complement system, 15
cytokines, 1415
lymphocytes, 1415
mechanism, 1314
serine proteases, 15
T-cells, 15
Innate immunity markers, 206207
Insulin-degrading enzyme (IDE), 7879
Insulin resistance, 34
M
Membrane attack complex (MAC)
alternative pathway, 31, 33
classical pathway, 31, 33
inhibitory protein (see CD59 glycoprotein)
MBL/lectin pathway, 31, 33
non-lytic effects, 3435
MEROPS search, 78
Metabolic syndrome (MetSyn), 166, 167
Modified Eagle Medium (MEM), 9596
N
Nephropathy, 1819, 3637
Neuropathy, 20, 38
O
Obesity, 194, 198199
adipose signaling
adiponectin, 8
ghrelin level, 89
hypoxia, 10
leptin signals, 7
free fatty acids, 910
inflammatory response (see Inflammation)
prevalence, U.S., 34
Oral glucose tolerance test (OGTT), 112
Oxidative stress markers, 197
biomarker measurements, 196197
ROS, 196, 198
P
Protease activate receptors (PAR) signalling,
1516
R
Reactive oxidative species, 196198
Reactive oxygen species (ROS), 67
Index
Retinopathy, 19, 37
ROS See Reactive oxidative species; Reactive
oxygen species (ROS)
T
Thiazolidinediones (TZD) treatment, 62
TNF-converting enzyme (TACE), 79
U
Urinary trypsin inhibitor (uTi), 144, 145
anti-inflammatory cellular signaling
Akt and PIK3 measurements, 175
apoptosis assays, 176
cell culturing procedure, 173174
DPP-4 peptidase activity assay, 174
intra-cellular analysis, 176
proliferation, 181, 186
protease and inhibitor balance, 177,
178
serine protease activity, 174
trypsin-specific chromogenic substrate,
176177
western-blot analysis, 175
CRP
acute phase reactants, 158
pro-inflammatory response, 166, 168
vascular stenosis, inflammation
markers, 164166
CVD
adiponectin response, 168
atherosclerosis, 157
demographic characteristics, 161, 162,
164
diagnostic risk factor, 161, 163, 164
epidemiological evidence, 158
indicators, 157158
statistics methods, 161
Kunitz type inhibitor, 172
marker analysis, 160161
metabolic syndrome (MetSyn), 166, 167
patient assessment, 159160
sample collection, 160161
uristatin immunoassay
anti-inflammatory response, 127131,
139
clinical analysis, 133134
cross-reactivity, 132
dipstick method, 132
follow-up care, 137138
health screen collection, 134135
hospital infection collection, 136
219
Index
hospital patient collection, 136
kidney model, 128131
proteinuria/hematuria, 138139
sample and data bank, 134135
urinary tract infection collection, 136
urine samples, 137
viral infections, 138
WBC, 129132
Western blot, 136137
uristatin response, 168
Uristatin (Uri) immunoassay
anti-inflammatory response, 127131,
139
clinical analysis, 133134
cross-reactivity, 132
dipstick method, 132
follow-up, 137138
health screen collection, 134135
hospital infection collection, 136

hospital patient collection, 136
kidney model, 128131
proteinuria/hematuria, 138139
sample and data bank, 134135
urinary tract infection collection, 136
urine samples, 137
viral infections, 138
WBC (see White blood cells (WBCs))
western blot, 136137
uTi See Urinary trypsin inhibitor (uTi)
W
White blood cells (WBCs)
anti-inflammatory response, 129130
cell death, 131132
neutrophilic elastase, 129

Inflammatory Pathways in Diabetes

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Inflammatory Pathways in Diabetes

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Progress in Inflammation Research

Series Editor: Michael J. Parnham

Michael Pugia Editor

Progress in Inflammation Research

Additional material to this book can be downloaded from http://extras.springer.com

Library of Congress Control Number: 2015952543

Adenosine deaminase binding protein 26

Oxidized low density lipoprotein

Impact of Inflammation and Innate Immunity Response

Impact of Complement on Diabetic Disease

Complement and Complement Regulatory Proteins

Role of C Terminal Fragment of Adiponectin Receptor

Development of Adiponectin Receptor C-Terminal

Protease Inhibition and Biological Distribution

Cell and Biological Models for the C Terminal

C-Terminal Fragment of Adiponectin Receptor

Uristatin Assay for Prediction of Renal

Uristatin Immunoassay Usage in Glomerular

Acute Response of Uristatin in Surgery. . . . . . . . . . . . . . . . . . . . . . . .

Cardiovascular and Metabolic Syndrome Impact

Uristatin Anti-inflammatory Cellular Signaling . . . . . . . . . . . . . . . . .

Progress in New Markers for Diabetes Inflammation . . . . . . . . . . . .

ELISA method for IgG-CTF assay

Manju Basu Department of Chemistry and Biochemistry,

Mitchell H. Rosner, MD, FACP Division of Nephrology,

Impact of Inflammation and Innate Immunity

Healthcare Impact of Diabetes

Diabetes has a significant impact on overall healthcare costs. The population of

Diabetes in US causes 173 billion in costs

Fat cell signaling

Leptin ,Adiponectin, etc

Beta cell loss

Impact of Inflammation and Innate Immunity Response in Obesity Mediated Diabetes

Hyperglycemia is shown to increase oxidative stress and the production of

Impact of Inflammation and Innate Immunity Response in Obesity Mediated Diabetes

nephropathy, retinopathy, obesity, hypertension, cardiovascular diseases, and heart

Adiponectin has structural similarity to the C1q/TNF family of proteins (Preedy

Impact of Inflammation and Innate Immunity Response in Obesity Mediated Diabetes

Free Fatty Acid Storage

Inflammation in Obesity and Diabetes

With the development of obesity, there is an increase in adipocyte mass, greater

Impact of Inflammation and Innate Immunity Response in Obesity Mediated Diabetes

Dysfunctional adipose tissue produces cytokines such as TNF, TGF-, and

Impact of Inflammation and Innate Immunity Response in Obesity Mediated Diabetes

Chapter 9 reviews the innate anti-inflammatory response which is activated in

Immune Mediated Apoptosis

Chronic low-grade inflammation in adipose tissue induces an innate immune cell

Impact of Inflammation and Innate Immunity Response in Obesity Mediated Diabetes

Replication and transcription

phospholipase C (PLC), adenylcyclase (AC), phosphatidylinositol 3-kinase (PLA2),

Impact of Inflammation and Innate Immunity Response in Obesity Mediated Diabetes

aggregation and endothelial cell proliferation. Thrombospondin 1 from platelets

Diabetes increases the risk of myocardial infarction, stroke, premature peripheral

Impact of Inflammation and Innate Immunity Response in Obesity Mediated Diabetes

typically starts with inflammation as glomerulonephritis and ends with wide

Impact of Inflammation and Innate Immunity Response in Obesity Mediated Diabetes

Espinoza-Jimnez A, Pen AN, Terrazas LI (2012) Alternatively activated macrophages in types 1

Impact of Inflammation and Innate Immunity Response in Obesity Mediated Diabetes

Pacurari M et al (2014) The Renin-angiotensin-aldosterone system in vascular inflammation and

Impact of Inflammation and Innate Immunity Response in Obesity Mediated Diabetes