Week 26 James Banting

INSULIN
Secreted from the beta cells of the islet of Langerhans in the pancreas
The liver:
- The liver is the key organ that maintains blood glucose concentrations. It does this by
gluconeogenesis (de novo synthesis of glucose) or glycogenolysis (break down of glycogen)
- When insulin levels are low, and glucagon levels are high, there is increased gluconeogenesis and
increased glycogenolysis. “Makes more plasma glucose available by breaking everything down”
- When insulin levels are high after a meal there is glycogen synthesis and the conversion of glucose
to triglycerides for storage.
1. Insulin activates glycogen synthesis within the liver by stimulating the enzymes glucokinase and
glycogen synthase.
2. It diminishes the activity of glycogen phosphorylase (i.e. stopping break down of glycogen) and
also stops the activity of glucose-6-phosphotase (stopping the conversion of glycogen to glucose)
3. Insulin also promotes the storage of fats and inhibits the oxidation of fatty acids, promoting the
formation of triglycerides which are stored or exported in VLDL which goes to muscle and adipose
tissue
4. Insulin promotes the synthesis of protein and reduces its degradation within the liver

The muscle:
- Insulin has 4 major effects on muscle:
1. Stimulates GLUT4 (which the muscle uses to uptake glucose) Note: also stimulates this in adipose
tissue
2. Enhances the conversion of glucose to glycogen by activation of hexokinase and glycogen
synthase
3. Increases glucose breakdown and oxidation by stimulation of Phosphofructokinase and pyruvate
dehydrogenase
4. Stimulates the synthesis and slows the degradation of protein within the muscle
- Insulin on the muscle preserves body protein and fat, and increases the oxidation of carbohydrates.

The adipocytes:
- Insulin also has 4 major actions on the adipocytes
1. Stimulates the activation of GLUT4, allowing glucose uptake into the adipocytes
2. Promotes the break down of glucose to metabolites used for the synthesis of triglycerides
3. Promotes the formation of triglycerides
4. Induces the synthesis of LPL – acts on triglycerides and VLDL cleaving them to glycerol and fatty
acids, promoting storage in adipose tissue
 T1D v T2D
T1D T2D
Epidemiology 5-10% of all diabetes 90-95% of all diabetes
Childhood onset (<20 y.o) Adult onset (>40 y.o)
Aetiology Autoimmune β-cell destruction (90% HLA Hereditary + environment
association – HLA-DR3/4) Obesity, physical inactivity, metabolic syndrome!
Pathophysiology 1. Genetic susceptibility 1. Peripheral insulin resistance
2. Environmental trigger (often associated  Central obesity →↑inflammation, ↑FFA
with previous viral infection)  ER stress (UPR)
3. Production of auto-Ab e.g., anti-glutamic  → activation of PKC → serine +P of IRS-1 →
acid decarboxylase antibody (anti-GAD), ↓GLUT4 translocation to membrane
→ T cell mediated destruction of 80– 2. Pancreatic β cell dysfunction
90% of β cells  ↑demand on remaining → ER stress (UPR)
4. Absolute insulin deficiency → Cf. table (progression of T2D)
elevated blood glucose levels (and DKA)
Signs/symptoms Onset: sudden Onset: gradual
Acute presentation: diabetic ketoacidosis* Presentation: hyperosmolar hyperglycaemic state

Classic Classic + non-specific

 Polyuria (osmotic - urine)
 Polydipsia (secondary to polyuria) Complications:*** (AMBOSS + 25.7)
 Polyphagia Acute
 Hyperglycaemic crsis: DKA, HHS
Non-specific  Hypoglycaemia: inappropriate insulin dose
 Fatigue
 Blurred vision (osmotic - lens) Long term
 Calf cramps  Macrovascular
 Impaired wound healing  Microvascular
 Pruritis  Intracellular
 Weight loss
Investigations/DDx Random blood glucose (>200 mg/dL) Random blood glucose (>200 mg/dL)
Pathological FPG, OGTT, HbA1C Pathological FPG, OGTT, HbA1C

Specific auto-Ab for T1D Specific auto-Ab for T1D

C peptide: ↓(T1D – no insulin) v ↑(T2D – C peptide: ↓(T1D – no insulin) v ↑(T2D – insulin
insulin resistance) resistance)
Treatment Insulin therapy Lifestyle intervention → drugs → insulin therapy

Consequences of metabolic acidosis (diabetic ketoacidosis - DKA)

Physiology
T1D:
 No insulin → no suppression of lipolysis
 ↑ketogenesis → DKA
T2D:
 Small amounts of insulin → suppression of lipolysis
 Predominantly hyperglycaemic state (HHS)

Consequences
1. Osmotic diuresis and hypovolemia
 No insulin → hyperglycaemia
 → Osmotic diuresis + loss of electrolytes
 → hypovolemia
 RESULT: Dry mucous membranes, ↓tissue turgor, hypotension

2. Metabolic acidosis with ↑ anion gap

 No insulin → no suppression of lipolysis
 Lipolysis → hepatic ketone production (acetoacetic acid and beta-hydroxybutyric acid!)
 Serum bicarb consumed as buffer for acidic ketones (↓serum HCO3-)
 RESULT: ↑RR, nausea + vomiting!! (characteristic of DKA)

3. Intracellular potassium deficit

 HHS - osmotic diuresis + lack of insulin to ↑cellular K+ uptake
 → K+ excretion
 → Total body potassium deficit (even though serum K+ may be normal/↑)
 RESULT: during insulin replacement therapy, consider K+ replacement! (rapid uptake by
depleted cells)

NOTE: DKA characteristics (not in HHS)

 Rapid onset
 Abdominal pain (irritation of peritoneum due to ketoacidosis)
 Fruity breath (exhaled acetone)
 Hyperventilation (compensation for metabolic acidosis)

ENDOCRINE PANCREAS - STRUCTURE/FUNCTION

Anatomy
Gross anatomy

Microscopic anatomy
Islet of Langerhans embedded within exocrine pancreas
3 cell types:
 α cells: peripheral, produce glucagon
 β cells: central, produce insulin (INSIDE = INSULIN)
 δ cells: dispersed, produce somatostatin

Overview of function

Cell type Secretion Function Feedback

α cell Glucagon Catabolic Stimulated by:
 ↑Gluconeogenesis Hypoglycaemia
 ↑Glycogenolysis
 ↑Lipolysis Inhibited by:
 ↑Ketogenesis Hyperglycaemia, insulin,
somatostatin
β cell Insulin Anabolic Stimulated by:
Muscle: Hyperglycaemia, β2
 ↑Glucose uptake adrenoreceptor agonism
 ↑Glycogen synthesis
 ↑Lipogenesis Inhibited by:
 ↓Lipolysis α2 adrenoreceptor
 ↓Gluconeogenesis agonism
δ cell Somatostatin ↓Insulin and glucagon secretion Stimulated by:
↓ GH secretion ↓pH (gastric acid)

+ Regulate exocrine action* Inhibited by: Vagus

ϵ cell Ghrelin Stimulates appetite Stimulated by: fasting
↑GH secretion Inhibited by: food intake
PP/γ cell PPY ↑Gastric acid secretion Stimulated by: fasting
Promotes satiety Inhibited by: food intake

Normal insulin physiology
 Secretion: Insulin is synthesized in the β cells of the islets of Langerhans. The cleavage
of proinsulin (precursor molecule of insulin) produces the C-peptide (connecting peptide)
and insulin, which consists of two peptide chains (A and B chains).
  Action: Insulin has a variety of metabolic effects on the body, primarily contributing to the
generation of energy reserves and glycaemic control.
 Carbohydrate metabolism: Insulin is the only hormone in the body that lowers the
blood glucose level. 
 Protein metabolism: stimulates protein synthesis: stimulates amino acid uptake into
cells; inhibits proteolysis
 Lipid metabolism: maintains a fat depot and has an antiketogenic effect 
 Electrolyte regulation: stimulates intracellular potassium accumulation 

BIOCHEMISTRY OF GLUCOSE REGULATION AND

DIABETES (26.1, 26.2, 26.6, 26.8, 27.7) + cancer
NORMAL GLUCOSE METABOLISM
How glucose enters cells

1. Transporters
 Energy independent – glucose gradient required
 GLUT2 active post meal (to liver - first pass)
 GLUT3 constitutively active (to brain)
 GLUT4 governed by blood glucose concentration (to muscle)

2. Insulin action
 Translocation of GLUT4 from GSVs to membrane surface

Glucose utilisation and storage (function of glycolysis)

Muscle
 Glycogenolysis: glucose released from glycogen (G1P <=> G6P)
 A) Glycolysis: to generate ATP for muscle contraction
 B) Lacking glucose-6-phosphatase (does NOT convert into free glucose)

Liver/kidney cortex
 Glycogenolysis: glucose released from glycogen (G1P <=> G6P)
 A) Glycolysis to generate acetyl-CoA for FA synthesis
 B) Has glucose-6-phosphatase (converts G6P to glucose, maintain blood glucose)
 C) Gluconeogenesis (maintain blood glucose)

Note: tumour cells also metabolise glycogen for survival*

Glycolysis
1. Glycerol-3-phosphate shuffle (aerobic conditions)
 Transports NADH from glycolysis into mitochondria (membrane impermeable to NADH)
 A) Cytoplasmic G3P dehydrogenase
o DHAP to G3P (NADH back to NAD+)
 B) Mitochondrial G3P dehydrogenase
o G3P to DHAP (FAD to FADH2)
  Used by muscle cells to quickly regenerate NAD+ (for glycolysis) and synthesis ATP

NOTE: malate-aspartate shuffle also same function

2. Anaerobic conditions
 NAD+ sustained by lactate dehydrogenase
o Pyruvate → lactate (NADH → NAD+)
o Lactate transported to liver for gluconeogenesis

3. Key regulatory steps in glycolysis and glycogen breakdown

4. Regulating hormones
 Muscle
o Adrenaline (↑glycogen breakdown + glycolysis – sustain muscle activity)
o Insulin (↑glycogen storage in muscle)
 Liver/kidney cortex
o Glucagon (↑glycogen breakdown + gluconeogenesis - ↑blood glucose)
o Insulin (↑glycogen storage in liver + glycolysis – store glucose as glycogen and FA)

Gluconeogenesis (Cori cycle, Alanine cycle)

1. Overview - ↑BLOOD GLUCOSE

 Cori cycle – lactate from tissues to glucose
 Alanine cycle – alanine from muscle breakdown to glucose
 Glycerol – from TAGs to glucose (storage: pyruvate → acetyl-CoA → FA + glycerol = TAGs)
 Note: rate limiting steps in glycolysis – 4 different enzymes in gluconeogenesis
 o

2. Gluconeogenesis key steps and regulation

Key points:
 Lactate or alanine → pyruvate
 Transported into mitochondria (malate/aspartate shuttle involved)
o PEPCK enzyme: PEP → gluconeogenesis
 G3P shuttle helps to replenish NAD+ needed for gluconeogenesis (if not enough DHAP,
gluconeogenesis won’t occur)

3. Metformin MOA (inhibits gluconeogenesis)

Key points:
 Metformin inhibits MITOCHONDRIAL G3P dehydrogenase
o 1)↓DHAP
o 2)↑NADH/NAD+ ratio
 Suppresses lactate to pyruvate = ↓pyruvate
Regulation of carbohydrate metabolism
1. Muscle
 DANGER: Adrenaline (↑glucose for glycolysis/ATP)
o → activation of glycogen phosphorylase
 FOOD: Insulin (↑glycogen synthesis)
o → activation of glycogen synthase
o ↑GLUT4 to membrane

2. Liver
 FASTING: Glucagon (↓glycogen synthesis, ↓TAG synthesis, ↑blood glucose)
o → inhibition of glycogen synthase
o ↓glycolysis (inhibits rate limiting enzymes)
o → activation of glycogen phosphorylase
o → activation of gluconeogenesis
 FOOD: Insulin (↑glycogen synthesis, ↑TAG synthesis) (also kidney cortex)
o → activation of glycogen synthase
o ↑glycolysis (↑acetyl-CoA → TAGs)

Mitochondria and reactive oxygen species

1. Pathways for ATP production
 Glycolysis → acetyl-CoA for TCA
 FA degradation (β-oxidation) → acetyl-CoA for TCA and
NADH for ox phos
 TCA cycle → NADH for ox phos

2. Generation of ROS
 Hypoxia → formation of superoxide
 CoQ usually reduced in two steps
o Under hypoxia → unstable partially reduced CoQ
transfers unpaired e- to O2, generating superoxide
 Mitochondrial DNA susceptible to oxidative damage
o No histones
o Poor DNA repair
o Major source of ROS (CoQ)
o Can’t encode for new mitochondrial complexes

Insulin signal transduction

Insulin production/release

1. Structure
 Active form is 2 chains (A and B)
 Inter and intra-chain disulphide bonds
 Preproinsulin → proinsulin → mature insulin
2. Pancreatic β cells as sole source
 Euchromatin
 Transcription factors
 Processing (biochemical factors)
o Note: ER dependent – under high load → unfolded protein response (UPR/ER stress)

3. Glucose stimulated insulin release

 Glucose entry via insulin INDEPENDENT GLUT2 (β-cell)
 ↑ATP → ↓*K+ (ATP gated channel)
 Depolarisation and Ca2+ influx
 Insulin exocytosis
 NOTE:
o Also GLP-1 stimulated insulin release (↑cAMP, Ca2+
independent)
o Circadian rhythm also involved
o *MEDICATION: ↑ insulin release by blocking K+ channel
(sulphonyl ureas)

Insulin action at target cell (molecular level)

1. Insulin receptor
 Transmembrane protein
 α2β2 structure (2 extracellular α and 2 transmembrane β domains)
 Tyrosine kinase domain

2a. Signal transduction pathway (nuclear, slow)

 Insulin → insulin receptor (auto +P tyrosine residues)
 → phosphorylated IRS-1 (scaffold protein)
 Multiple signals (nuclear, metabolic)
 MAPK

2b. Signal transduction pathway (metabolic, fast)

 → phosphorylated IRS-1 →→→ PKB activation
o ↑Glucose uptake via GLUT4 translocation to membrane
o ↑Glycogen synthesis through glycogen synthase
activation (inhibition of GSK3)

3.Signal transduction pathway (other)

 Target transcription factors for degradation
 E.g. ↓glycogen phosphorylase
Biochemistry of diabetes
Predisposing factors to T2DM
1. Obesity!
 ↑BMI → ↑relative risk of diabetes

2. Insulin resistance (earliest detectable abnormality in T2DM) - characteristics

 ↑Starting levels
 ↓peak levels
 Slower time to reach peak
 Slower return kinetics
3. Molecular mechanisms of insulin resistance
 A) Intracellular lipids → PKC activation
 B) Obesity → ↑cytokines/inflammation → PKC activation
 C) ER stress → UPR → PKC activation
 →→PKC activation → ↑serine +P on IRS-1 (inhibits activation by tyrosine +P and leads to
degradation) = ↓GLUT4 to membrane + other signalling events

Prediabetes transition to overt diabetes

1. Phases
 Hyperinsulinema in response to insulin resistance
 Impaired glucose tolerance
 Hyperglycemia in fasting state (and low insulin)

2. β cell dysfunction
 A) ER stress due to ↑insulin production → UPR
o Puts ↑ stress on remaining β cells → vicious cycle
o NOTE: give insulin injections to T2D to protect remaining β cells
 B) Obesity (exposure to FFA) → loss of colocalisation
o Ca2+ channels open, but occurs in the “wrong” place
o Fails to evoke secretion of insulin

Complications of diabetes
1. Hyperglycemia Current and potential treatments
 Microvascular  Lifestyle, drugs
 Macrovascular  Insulin alternatives
 Surgical (bariatric)
2. Changes in protein metabolism  Future
 Protein breakdown o Target insulin
 Sent to liver for gluconeogenesis signalling
 → ↑hyperglycemia o Induce new β-cells

3. Liver metabolism
 Becomes PRODUCER of glucose (gluconeogenesis and glycogenolysis)
 FA as source of fuel → accumulation of ketone bodies in liver → fuel for brain (but DKA)
 Changes in diabetes resemble PROLONGED FASTING but with HIGH blood glucose

4. DKA
5. Hypoglycaemia (as a result of anti-diabetic therapy)
Metabolic adaptation of cancer cells (how cancer cells use glucose)

Warburg effect (reliance on glycolysis for ENERGY)

1. ATP generation shift from TCA to glycolysis (aerobic!)
 Overexpression of fetal GLUT1 (↑glucose intake)
 Rate limiting glycolytic enzymes overexpressed e.g. hexokinase, PFK1, LDH (↑glycolytic rate)
 Pyruvate kinase M2 (PKM2) overexpressed, with LOWER activity than normal PK (→ PPP*)
 Pyruvate dehydrogenase (PDH) activity is suppressed (↓TCA cycle)

*↑ PEP due to ↑glycolysis, so lower activity level is fine – also allows for PEP back to G6P to provide
precursors for PPP!
2. Detection of cancer cells (18F-FDG)
 18F-FDG is a glucose derivative, used as tracer
 Can’t be further metabolised, accumulates in cancer cells

Oxidative stress (combating ROS from hypoxia)

1. Glutathione
 Cancer cells use glutathione as an anti-oxidant
 Mops up ROS, but then needs to be reduced again to continue
 Need NADPH to regenerate

2. Pentose phosphate pathway (NADPH and biosynthetic precursor generation)

 Oxidative phase
o Generation of NADPH (for ROS)
o Generation of NADPH (for reductive biosynthesis)
 Non-oxidative phase
o Generation of precursors for biosynthesis

3. Hypoxia induced HIF-1

 → Glycogen synthase (early stages, store glycogen)
 → Glycogen phosphorylase (late hypoxic stages, release G6P for PPP)
o Generate MORE NADPH!!

PRECURSORS for high cell proliferation rate and growth

1. PPP (Oxidative and non-oxidative)

2. Glutamine metabolism (for TCA cycle intermediates)

 ↑expression of enzymes for glutamine metabolism
 → generates intermediates of TCA cycle