1052 Diabetes Volume 66, April 2017

Jacqueline M. Ratter,1,2 Hanne M.M. Rooijackers,1 Cees J. Tack,1

Anneke G.M. Hijmans,1 Mihai G. Netea,1 Bastiaan E. de Galan,1 and
Rinke Stienstra1,2

Proinflammatory Effects of
Hypoglycemia in Humans With
or Without Diabetes
Diabetes 2017;66:1052–1061 | DOI: 10.2337/db16-1091

Severe hypoglycemic events have been associated with Hypoglycemia is the most common complication of insulin
increased cardiovascular mortality in patients with therapy in people with type 1 diabetes (1). Patients with
diabetes, which may be explained by hypoglycemia- type 1 diabetes experience, on average, two hypoglycemic
induced inflammation. We used ex vivo stimulations of events per week and one severe event per year (2). The
peripheral blood mononuclear cells (PBMCs) and mono- extent to which hypoglycemia contributes to cardiovascular
cytes obtained during hyperinsulinemic-euglycemic (5.0
disease risks in diabetes is debated: An association between
mmol/L)-hypoglycemic (2.6 mmol/L) clamps in 11 healthy
severe hypoglycemia and increased mortality from cardio-
participants, 10 patients with type 1 diabetes and normal
COMPLICATIONS

vascular events has been established in patients with type 2

awareness of hypoglycemia (NAH), and 10 patients with
type 1 diabetes and impaired awareness (IAH) to test diabetes (3–6) but is less consistent in type 1 diabetes
whether the composition and inflammatory function of (6–10), even though hypoglycemia occurs much more fre-
immune cells adapt to a more proinflammatory state quently in patients with type 1 than in those with type 2
after hypoglycemia. Hypoglycemia increased leukocyte diabetes (11).
numbers in healthy control participants and patients with An increase in circulating proatherothrombotic factors
NAH but not in patients with IAH. Leukocytosis strongly in response to acute insulin-induced hypoglycemia can link
correlated with the adrenaline response to hypoglycemia. hypoglycemia to cardiovascular complications (12–14). In
Ex vivo, PBMCs and monocytes displayed a more robust addition, hypoglycemia has been reported to increase leu-
cytokine response to microbial stimulation after hypogly- kocyte counts and circulating proinflammatory cytokines in
cemia compared with euglycemia, although it was less both healthy individuals (15–18) and patients with type 1
pronounced in patients with IAH. Of note, hypoglycemia diabetes (14,19), supporting the concept that hypoglyce-
increased the expression of markers of demargination
mia-induced systemic inflammation contributes to cardio-
and inflammation in PBMCs. We conclude that hypogly-
vascular complications (12,13,15).
cemia promotes mobilization of specific leukocyte sub-
Adrenaline, the main counterregulatory hormone response
sets from the marginal pool and induces proinflammatory
functional changes in immune cells. Inflammatory re-
to hypoglycemia in patients with type 1 diabetes, may play
sponses were less pronounced in IAH, indicating that a role in the hypoglycemia-induced proinflammatory re-
counterregulatory hormone responses are key modula- sponse. When adrenaline is administered to healthy indi-
tors of hypoglycemia-induced proinflammatory effects. viduals (normoglycemic conditions), it specifically mobilizes
Hypoglycemia-induced proinflammatory changes may leukocytes equipped with cytotoxic effector potential from
promote a sustained inflammatory state. the marginal pool (vascular epithelium) (20). However,

1Department of Internal Medicine, Radboud University Medical Center, Nijmegen, J.M.R. and H.M.M.R. share first authorship.
the Netherlands B.E.d.G. and R.S. share senior authorship.
2Division of Human Nutrition, Wageningen University, Wageningen, the Netherlands
© 2017 by the American Diabetes Association. Readers may use this article as
Corresponding author: Jacqueline M. Ratter, [email protected]. long as the work is properly cited, the use is educational and not for profit, and the
Received 6 September 2016 and 17 January 2017 work is not altered. More information is available at http://www.diabetesjournals
Clinical trial reg. no. NCT02308293, clinicaltrials.gov. .org/content/license.

This article contains Supplementary Data online at http://diabetes

.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1091/-/DC1.
diabetes.diabetesjournals.org Ratter and Associates 1053

knowledge about the role of adrenaline in hypoglycemia- having abstained from caffeine, alcohol, and smoking for
induced changes related to inflammation is lacking. 24 h. Patients with diabetes received specific instructions
Patients with impaired awareness of hypoglycemia (IAH) to avoid (nocturnal) hypoglycemia the day before the clamp.
are at particularly high risk of hypoglycemia (21) because Experiments were rescheduled in case of hypoglycemia in
they lack hypoglycemia warning symptoms and have atten- the 24 h before the clamp. Upon arrival, two intravenous
uated adrenaline responses (1,22). If adrenaline contributes cannulae were inserted, one into the antecubital vein of each
to hypoglycemia-induced proinflammatory responses, such forearm. One forearm was placed in a heated box (55°C) so
effects may be altered in patients with type 1 diabetes and that arterialized venous blood could be obtained for frequent
IAH. Under euglycemic conditions, patients with IAH were blood sampling. The cannula in the contralateral arm was
found to have higher leukocyte counts and a higher rate of used for infusion of glucose 20% (Baxter, Deerfield, IL) and
endothelial dysfunction and preclinical atherosclerosis than insulin (insulin aspart; Novo Nordisk, Bagsværd, Denmark).
sex- and age-matched patients without IAH (23). In accor- Baseline plasma glucose levels were determined (Biosen
dance, Joy et al. (16) reported that antecedent hypoglyce- C-Line; EKF Diagnostics, Cardiff, U.K.), and a two-step
mia, which underlies the emergence of IAH, results in hyperinsulinemic (60 mU/m2/min)-euglycemic (5.0 6 0.2
greater endothelial dysfunction, but inflammatory responses mmol/L)-hypoglycemic (2.6 6 0.1 mmol/L) glucose clamp
to hypoglycemia are not enhanced after prior hypoglycemia. was initiated. Plasma glucose levels were determined every
Thus, hypoglycemia has been shown to increase circu- 5 min, and after a short euglycemic phase (;20 min),
lating proinflammatory cytokines, but the underlying plasma glucose levels were gradually decreased to
mechanisms, the role of repeated hypoglycemia, and the 2.6 mmol/L and maintained there for 60 min. Blood sam-
relationship with counterregulatory hormone responses, ples for measurement of adrenaline were taken at eugly-
particularly adrenaline, are incompletely understood. cemia and every 20 min during hypoglycemia. Insulin and
Because an enhanced proinflammatory state is not glucagon were determined at euglycemia and at 60 min of
exclusively reflected in the levels of circulating cytokines, hypoglycemia.
we studied the effects of acute hypoglycemia on the
composition and inflammatory output of immune cells. Analytical Methods
We investigated these aspects by using ex vivo stimula- Plasma insulin was assessed by an in-house radioimmu-
tions of peripheral blood mononuclear cells (PBMCs) noassay (26). After extraction (27), plasma glucagon was
obtained at various time points during hyperinsuline- measured with a commercially available radioimmunoas-
mic-euglycemic-hypoglycemic clamps in healthy partici- say kit (Eurodiagnostica, Malmö, Sweden). Plasma growth
pants and patients with type 1 diabetes. To assess the role hormone and cortisol were determined by routine analy-
of sympathoadrenal responses to hypoglycemia in inducing sis with an electrochemiluminescent immunoassay on a
potential proinflammatory effects, we also included patients Modular Analytics E170 (Roche, Manheim, Germany).
with type 1 diabetes and IAH characterized by impaired Plasma adrenaline and noradrenaline were analyzed by
counterregulatory hormone responses to hypoglycemia. high-performance liquid chromatography combined with
fluorometric detection (28). Peripheral total and differen-
RESEARCH DESIGN AND METHODS tial white blood cell counts were determined by routine
patient sample analysis (flow cytometric analysis on a
Participants
Sysmex XE-5000).
We recruited 11 participants without diabetes, 10 patients
with type 1 diabetes and normal awareness of hypoglyce- Isolation of PBMCs and CD14+ Monocytes
mia (NAH), and 10 patients with type 1 diabetes and IAH. Blood samples were processed for isolation of cells immedi-
Patients were otherwise healthy and did not use drugs that ately after being drawn to ensure equal quality of the samples
interfered with glucose metabolism other than insulin. because previous experiments showed that cytokine responses
Hypoglycemia awareness state, initially assessed by a Dutch are altered when blood samples are processed for isolation at
version of the Cox questionnaire in which a score of 0–1 of different time points after being drawn (data not shown).
5 indicates normal awareness and a score $ 3 indicates Isolation of PBMCs was performed by differential centrifuga-
impaired awareness (24,25), was determined on the basis tion over Ficoll-Paque PLUS (GE Healthcare). PBMCs were
of adrenaline and symptomatic responses to hypoglycemic washed three times with PBS and counted with a Coulter
clamp. Eighteen of the 20 patients were correctly charac- counter (Coulter Electronics). CD14+ monocytes were purified
terized as having either IAH or NAH through the Cox from freshly isolated PBMCs by using MACS MicroBeads
questionnaire. The institutional review board of the Rad- (Miltenyi Biotec, Teterow, Germany) for positive selection
boud University Medical Center (Nijmegen, the Nether- according to the manufacturer’s instructions.
lands) approved the study, and all participants gave
written informed consent before participation. Stimulation Experiments
For analysis of cytokine release, glucose consumption, and
Experimental Design lactate production, 5 3 105 PBMCs or 1 3 105 monocytes
All participants presented between 8:00 and 8:30 A.M. at were used per well in a 96-well plate. Cells were cultured
the clinical research facility after an overnight fast and in RPMI medium (no glucose; Gibco) supplemented with
1054 Proinflammatory Effects of Hypoglycemia Diabetes Volume 66, April 2017

10 mg/mL gentamicin (Gibco), 10 mmol/L pyruvate (Gibco), reverse transcription using the iScript cDNA Synthesis
10 mmol/L HEPES (Sigma-Aldrich), and 5.5 mmol/L glu- Kit (Bio-Rad). Primer sequences used for quantitative
cose (Sigma-Aldrich) and stimulated with either RPMI real-time PCR (qRT-PCR) are listed in Supplementary
medium, 10 ng/mL of the TLR4 agonist lipopolysaccha- Table 2. Power SYBR Green PCR Master Mix (Applied
ride (LPS) from Escherichia coli (Sigma-Aldrich), 10 Biosystems) was used for qRT-PCR in the CFX384 Touch
mg/mL of the TLR2 agonist Pam3CysSK4 (Pam3Cys) Real-Time PCR Detection System (Bio-Rad). Expression
(EMC Microcollections, Tübingen, Germany), 1 mg/mL data were normalized to the housekeeping gene human
Mycobacterium tuberculosis (H37Rv) lysate, or 1 3 106 b2M.
heat-killed organisms/mL Candida albicans conidia for Statistical Analysis
24 h. Cell culture supernatants were collected and stored
Data were tested for normality by using the Shapiro-Wilk
at 220°C.
test and Q-Q plots. Within-group differences were com-
Cytokine Measurements pared with paired Student t or Wilcoxon signed rank tests
The production of interleukin-1b (IL-1b), tumor necrosis when data were not normally distributed. Between-group
factor-a (TNF-a) (R&D Systems), IL-10, IL-6 (Sanquin), differences were analyzed by ANOVA followed by pairwise
and MCP-1 (eBioscience) was measured by ELISA. In the Bonferroni post hoc tests to delineate statistical signifi-
analysis of cytokine production by CD14+ monocytes, par- cance and for nonparametric data, with the Kruskal-Wallis
ticipants were excluded when cytokine production upon and post hoc Mann-Whitney U tests. For correlation anal-
stimulation of cells after both euglycemia and hypoglycemia ysis, Pearson correlation coefficient was used for normally
was below detection limits. The number of nonresponders distributed variables and Spearman rank sum test for
was comparable between groups. nonnormally distributed data. All data are expressed as
mean 6 SEM unless otherwise specified. P , 0.05 was
Glucose Consumption and Lactate Measurements
considered statistically significant. Statistical analyses
Glucose and lactate concentrations were measured in cell
were performed with SPSS version 20 software (IBM
culture supernatants. Measurements were based on an
enzymatic reaction in which glucose or lactate is oxidized Corporation).
and the resulting H2O2 is coupled to the conversion of RESULTS
Amplex Red reagent to fluorescent resorufin by horserad-
Study participants were well matched for age, sex, and
ish peroxidize. The fluorescence of resorufin (excitation/
BMI (Table 1). Duration of diabetes and HbA1c did not
emission maxima 570/585 nm) was measured on a 96-well
differ significantly between patient groups. Plasma glu-
plate reader (BioTek). Glucose consumption was calcu-
cose levels (Fig. 1A) and plasma insulin levels (data not
lated by subtracting the glucose concentration measured shown) were similar in all groups during both the
in cell culture supernatants from that in culture medium euglycemic and the hypoglycemic phase, whereas glucagon
incubated for 24 h without cells.
levels increased in response to hypoglycemia in healthy
RNA Isolation and Quantitative Real-Time PCR control participants but did not change in either patient
For mRNA expression analyses, PBMCs (1.5 3 106 group (Supplementary Table 1). Adrenaline levels during
PBMCs/condition) were lysed in TRIzol reagent (Invitro- hypoglycemia were significantly lower in patients with
gen) directly after isolation and stored at 280°C until IAH than in healthy control participants and patients
RNA isolation was performed according to the manufac- with NAH (0.39 6 0.07, 1.90 6 0.46, and 1.94 6
turer’s instructions. RNA was transcribed into cDNA by 0.29 nmol/L, respectively).

Table 1—Participant characteristics

Patients with type 1 Patients with type 1
Healthy control diabetes and NAH diabetes and IAH
participants (n = 11) (n = 10) (n = 10)
Age (years) 24.5 6 5.3 24.2 6 5.1 25.3 6 6.0
Sex
Male 6 4 5
Female 5 6 5
BMI (kg/m2) 22.9 6 1.8 22.7 6 2.2 23.4 6 1.4
HbA1c
% — 7.5 6 0.6 6.9 6 0.7
mmol/mol — 58.5 6 6.9 52.1 6 7.8
Duration of diabetes (years) — 10.9 6 4.7 13.9 6 8.1
Total daily insulin dose (IU) — 48.2 6 12.6 48.2 6 13.2
Data are mean 6 SD or n.
diabetes.diabetesjournals.org Ratter and Associates 1055

Figure 1—Hypoglycemia induces leukocytosis in healthy control (HC) participants and patients with type 1 diabetes mellitus (T1DM) and NAH
but not in T1DM and IAH who have attenuated adrenaline responses to hypoglycemia. A: Time course of plasma glucose levels during the
clamp. Dashed lines represent the end of the euglycemic phase and the beginning and end of the hypoglycemic phase. B–E and G: Number of
circulating leukocytes (B), neutrophils (C), lymphocytes (D), and monocytes (E) and composition of leukocytes (G) measured with a routine
patient sample analysis at euglycemia and after 1 h of hypoglycemia. F: Correlation between the difference in leukocyte numbers during
hypoglycemia vs. euglycemia and the difference in adrenaline levels between hypoglycemia and euglycemia. *P < 0.05; **P < 0.01.
1056 Proinflammatory Effects of Hypoglycemia Diabetes Volume 66, April 2017

Hypoglycemia Increases Total Leukocyte Count Hypoglycemia Generally Increases Expression of

The total leukocyte count increased in response to hy- Markers for Demargination and Cells With Cytotoxic
poglycemia in healthy control participants and patients Effector Potential
with NAH but not in patients with IAH (Fig. 1B). This Because adrenaline levels increase markedly in response to
increase was mainly due to an increase in the number of hypoglycemia and because adrenaline drives demargination
lymphocytes and, to a lesser extent, an increase in the of leukocytes (20), we investigated whether hypoglycemia
number of monocytes (Fig. 1C–E). Consequently, neutro- altered gene expression levels of demargination markers in
phil-to-lymphocyte ratios decreased in response to hypo- isolated PBMCs. Hypoglycemia increased the expression of
glycemia (Fig. 1G). The change in total leukocyte count the integrin CD11a in PBMCs of healthy control partici-
correlated positively with the adrenaline response to hy- pants and patients with NAH but not in patients with IAH
poglycemia (R2 = 0.70; P , 0.001) (Fig. 1F). The positive (Fig. 4A). Hypoglycemia also increased the expression of
correlation was strongest in lymphocytes (R2 = 0.75; P , the chemokine receptor CX3CR1 in PBMCs of healthy
0.001) but was also seen in monocytes (R2 = 0.33; P = 0.003) control participants (Fig. 4B).
and neutrophils (R2 = 0.29; P = 0.007) (Supplementary We next assessed the expression of marker genes of
Fig. 1). By looking at the separate groups, the positive various immune cell types in PBMCs exposed to hypogly-
correlation between the change in total leukocyte count cemia or euglycemia (Fig. 4C–G). Hypoglycemia increased
and adrenaline response was significant in healthy control the expression of CD8 but not of CD4 or CD56 in PBMCs in
participants and patients with NAH, but not in patients all groups. Moreover, although hypoglycemia did not alter
with IAH. expression of CD14, it increased the expression of CD16, a
marker for the nonclassic monocyte subset that produces
Hypoglycemia Increases Ex Vivo Proinflammatory more cytokines than the classic monocytes in response to
Cytokine Production certain stimulations (29).
PBMCs from healthy control participants and patients
with NAH isolated after 1 h of hypoglycemia and stim- Hypoglycemia Increases Ex Vivo Cytokine
ulated with the TLR4 agonist LPS produced more proin- Production of CD14+ Cells
flammatory cytokines (IL-6, IL-1b, and TNF-a) than Because monocytes are the major producers of proinflamma-
PBMCs isolated after euglycemia. Hypoglycemia had no tory cytokines within the heterogeneous PBMC cell popula-
effect on LPS-stimulated cytokine production of PBMCs tion, we specifically investigated the effect of hypoglycemia
isolated from patients with IAH (Fig. 2A–C). Hypoglyce- on the inflammatory function of CD14+ monocytes. Hypo-
mia increased the production of the chemokine MCP-1 in glycemia did not affect the percentage of isolated CD14+
healthy control participants (Fig. 2D) but did not affect monocytes within the PBMC fraction in any of the three
levels of the anti-inflammatory cytokine IL-10 in any groups (Fig. 5A). When stimulated ex vivo, CD14+ cells pro-
group (Fig. 2E). duced more proinflammatory cytokines, particularly TNF-a,
Hypoglycemia enhanced the TNF-a response of PBMCs if isolated after hypoglycemia compared with euglycemia (Fig.
stimulated with Pam3Cys, M. tuberculosis, and C. albicans 5B). Nevertheless, CD14+ cells did not have increased gene
in all three groups. Hypoglycemia also increased the IL-6 expression levels of surface markers characterizing proin-
response to M. tuberculosis in healthy control participants flammatory monocytes (CD11a, CXRCR1, CCR5, CCR2)
and patients with NAH but had virtually no effect on the (Supplementary Fig. 3).
IL-6 and IL-1b responses to Pam3Cys or C. albicans in any DISCUSSION
of the three groups (Supplementary Fig. 2). Altogether,
This study investigated the effect of acute hypoglycemia on
hypoglycemia enhanced cytokine responses of PBMCs,
the composition and inflammatory function of circulating
with the most prominent increase in TNF-a responses
immune cells. We demonstrate that exposure to hypoglycemia
(Fig. 2F). Of note, cytokine release of stimulated PBMCs
leads to demargination of specific immune cell subtypes and
from patients with IAH isolated during euglycemia tended
enhances the inflammatory response of PBMCs and CD14+
to be higher than the cells from healthy control partici-
monocytes. Of note, the hypoglycemic response of PBMCs
pants and patients with NAH, although the differences
was partly blunted in patients with type 1 diabetes and IAH,
were not statistically significant.
highlighting the role of adrenaline in immune cell recruit-
Hypoglycemia Does Not Affect Glycolytic Metabolism ment and in the acute inflammatory response to hypoglyce-
of PBMCs mia. The data support the concept that hypoglycemia shifts
We then investigated whether hypoglycemia affected circulating immune cells toward a more proinflammatory
glycolytic metabolism of PBMCs. As expected, stimulation state. When sustained, such an enhanced inflammatory state
with LPS significantly increased glucose consumption and could contribute to atherogenesis in people with diabetes.
lactate production of PBMCs in all groups (Fig. 3A and B). In line with previous findings (15), the results demon-
However, no difference was found in either glucose con- strate that hypoglycemia induces leukocytosis. The strong
sumption or lactate production between cells exposed to correlation with adrenaline responses to hypoglycemia
hypoglycemic versus euglycemic conditions, regardless of suggests a role for adrenaline, which is supported by
whether stimulated with LPS. our observations in patients with type 1 diabetes and
diabetes.diabetesjournals.org Ratter and Associates 1057

Figure 2—PBMCs isolated after hypoglycemia produce more proinflammatory cytokines than PBMCs isolated after euglycemia. A–E: IL-6
(A), IL-1b (B), TNF-a (C), MCP-1 (D), and IL-10 (E) production of PBMCs isolated from euglycemic or hypoglycemic conditions and
stimulated for 24 h with LPS. F: Fold change in cytokine production (IL-6, IL-1b, TNF-a) by PBMCs upon hypoglycemia vs. euglycemia.
The gray dashed line represents euglycemic values. PBMCs were stimulated with the TLR4 agonist LPS, the TLR2 agonist Pam3Cys, C.
albicans, or lysate of M. tuberculosis. Data are mean (continuous lines) 6 SEM (dotted lines). *P < 0.05; **P < 0.01. HC, healthy control;
T1DM, type 1 diabetes mellitus.
1058 Proinflammatory Effects of Hypoglycemia Diabetes Volume 66, April 2017

PBMCs are a heterogeneous mix of cell populations, and

changes in composition could explain the increased
cytokine production observed in response to hypoglyce-
mia. However, measurements of the cellular composition
of several PBMC samples did not reveal major changes
(percentage of lymphocytes, monocytes, and granulocytes)
after hypoglycemia. Of note, CD11a and CX3CR1 gene
expression levels were increased in PBMCs exposed to
hypoglycemia, suggesting an increase in the number of
demarginated cells (20). Recruitment of a distinct cell pop-
ulation with a different phenotype and function likely con-
tributes to the observed change in inflammatory responses
after exposure to hypoglycemia. Similar to leukocytosis
experimentally induced by adrenaline (20), hypoglycemia
increased the number of cells with cytotoxic effector po-
tential, such as lymphocytes expressing CD8. Additionally,
hypoglycemia increased gene expression levels of CD16, sug-
gesting an increase in circulating CD16+ monocytes and
natural killer cells, also secondary to adrenaline-mediated
leukocytosis (20). Future studies applying flow cytometric
analysis of circulating immune cells to provide additional in-
formation on the specific surface expression of selected pro-
teins on certain cell populations would be of particular
interest.
Figure 3—No differences in glycolytic metabolism of PBMCs iso- Cytokine production was similarly altered in CD14+
lated from hypoglycemia vs. euglycemia. Glucose consumption (A)
cells and PBMCs exposed to hypoglycemia. Although we
and lactate secretion (B) measured in the supernatants of PBMCs
isolated from euglycemic or hypoglycemic conditions and cultured cannot distinguish between the different monocyte subsets
for 24 h with or without stimulation with LPS. *P < 0.05; **P < 0.01. (classic, intermediate, nonclassic) within the population
HC, healthy control; T1DM, type 1 diabetes mellitus. of isolated CD14+ cells, the increased cytokine response
of PBMCs is likely based on the enhanced cytokine pro-
duction capacity of CD14+ monocytes because the percentage
of monocytes was similar at hypoglycemia and euglycemia.
IAH who have blunted counterregulatory hormone responses Another factor that may contribute to an altered
to hypoglycemia. These studies now extend previous findings inflammatory output of immune cells is a shift in cellular
by investigating the inflammatory function of isolated im- metabolism induced by changes in the metabolic environ-
mune cells ex vivo. We observed that TNF-a production ment. For instance, a highly active glycolytic metabolism
significantly increases in PBMCs exposed to hypoglycemia, has been shown to drive proinflammatory cytokine pro-
independent of the pathogenic stimulus (LPS, Pam3Cys, duction in M1 macrophages and is important for activated
C. albicans, or M. tuberculosis), which strongly implies that the effector T cells (31). However, similar glucose consump-
hypoglycemic event causes a universal potentiation of inflam- tion and lactate production of cells isolated from hypogly-
matory function of the cells. Because equal numbers of cemic versus euglycemic conditions make it unlikely that
PBMCs were used in stimulations to compare the two glyce- changes in inflammatory responses are due to changes in
mic conditions, the increased levels of circulating proinflam- glycolytic metabolism of immune cells. We cannot fully
matory cytokines found in previous studies (15,17–19,30) exclude an involvement of glycolytic metabolism because
are not only due to the increase in the number of circulating acute changes can occur in vivo but might be masked at
immune cells in response to hypoglycemia but also likely the time point that we measured lactate levels in vitro.
reflect changes in the functional status of immune cells. The results revealed abrogation of hypoglycemia-induced
In contrast to TNF-a responses, the effect of hypogly- leukocytosis and an attenuated inflammatory response
cemia on IL-6 and IL-1b production was less pronounced to hypoglycemia in patients with IAH, potentially as a
and more variable between the various stimuli, suggesting consequence of extensive prior exposure to hypoglycemia.
that changes in pathogen-specific signaling pathways are The attenuated inflammatory response in patients with
involved. Such changes could affect either the expression of IAH underscores the contribution of counterregulatory
pattern recognition receptors on the cell surface or expres- hormones, especially adrenaline, in hypoglycemia-induced
sion of their downstream effectors. If intracellular signaling proinflammatory effects. Of note, hypoglycemia increased
pathways are indeed affected by hypoglycemia, this could CD8 and CD16 expression in PBMCs of patients with IAH,
also prime the immune cells to respond differently to other indicating an increase in circulating cells with cytotoxic
stimuli, such as proatherogenic factors. effector potential in these patients even while total leukocyte
diabetes.diabetesjournals.org Ratter and Associates 1059

Figure 4—Increased expression of markers for demargination and cells with cytotoxic effector potential after hypoglycemia as assessed by
qRT-PCR in PBMCs exposed to euglycemia or hypoglycemia. Relative expression of CD11a (A), CX3CR1 (B), CD4 (C), CD8 (D), CD56 (E),
CD14 (F), and CD16 (G). *P < 0.05; **P < 0.01. HC, healthy control; T1DM, type 1 diabetes mellitus.
1060 Proinflammatory Effects of Hypoglycemia Diabetes Volume 66, April 2017

cardiovascular mortality in patients with type 1 diabetes

and IAH compared with those with NAH (10) but contrasts
with studies focusing on vascular effects that reported
higher rates of preclinical atherosclerosis in patients with
repeated hypoglycemia (23) and greater proatherothrom-
botic responses and endothelial dysfunction after recurrent
hypoglycemia (16). Studies are needed to determine the
long-term consequences of repeated hypoglycemia and
IAH on inflammation and immune cells, their inflamma-
tory function, and their involvement in atherogenesis.
The strengths of this study include the use of glucose
clamps in three matched groups of participants under similar
glycemic conditions, which enabled us to differentiate be-
tween the impact of diabetes and IAH. A larger sample size
would have allowed us to differentiate better between
patients with IAH and NAH, especially with regard to baseline
values. Although we analyzed gene expression of demargi-
nation markers and of specific cell types, flow cytometric
analysis would have provided a more detailed characteriza-
tion of changes in composition and inflammatory status of
leukocytes. Future research should focus on mechanistic
studies in lymphocytes and look into the role of neutrophils
to extend the current gene expression data. Another
limitation of this study is that participants with and without
diabetes were healthy and relatively young. Inflammatory
responses to hypoglycemia might differ in older patients, in
those with a history of cardiovascular disease, or in those
with poor glycemic control.
We conclude that hypoglycemia leads to demargination,
an increase in circulating immune cells with cytotoxic effector
Figure 5—CD14+ monocytes isolated after hypoglycemia produce
more proinflammatory cytokines than CD14+ cells isolated after
potential, and an induction of proinflammatory functional
euglycemia. A: Percentage of CD14+ cells isolated by MACS. B: changes in PBMCs and CD14+ monocytes. Acute inflamma-
Fold change in cytokine production (IL-6, IL-1b, TNF-a) by CD14+ tory responses to hypoglycemia were partly blunted in pa-
cells isolated from hypoglycemic vs. euglycemic conditions. CD14+ tients with type 1 diabetes and IAH, highlighting that
cells were stimulated with the TLR4 agonist LPS, the TLR2 agonist
Pam3Cys, C. albicans, or lysate of M. tuberculosis. Data are mean counterregulatory hormone responses are key modulators
(continuous lines) 6 SEM (dotted lines). HC, healthy control; T1DM, of proinflammatory responses to hypoglycemia. These data
type 1 diabetes mellitus. indicate that hypoglycemia induces a shift in inflammatory
function of immune cells, which could promote a sustained
proinflammatory state in patients with diabetes.
count did not increase upon hypoglycemia. This finding
might be explained by the minimal, albeit still significant,
increase in adrenaline levels in response to hypoglycemia. Acknowledgments. The authors thank all the volunteers for participating in
Leukocyte numbers and cytokine responses during euglyce- this work. The authors also thank Evita Wiegers (Department of Radiology and Nuclear
mia appeared to be higher but were not significantly elevated Medicine, Radboud University Medical Center) for assistance during the glucose clamps.
in patients with IAH compared with healthy control Funding. Research support from the Dutch Diabetes Research Foundation
participants or patients with NAH. Although attributing (DFN 2012.00.1542) and the European Foundation for the Study of Diabetes is
this trend to prior exposure to hypoglycemia is tempting, a gratefully acknowledged. M.G.N. is supported by a European Research Council
larger sample size would be required to address this question. Consolidator grant (310372). R.S. is supported by a VIDI grant from the Nether-
One could speculate that an attenuated proinflamma- lands Organisation for Scientific Research.
tory response to acute hypoglycemia as observed in the Duality of Interest. No potential conflicts of interest relevant to this article
were reported.
patients with IAH might provide some protection against
Author Contributions. J.M.R., H.M.M.R., B.E.d.G., and R.S. designed the
harmful effects of subsequent hypoglycemia. Frequent study with input from C.J.T. and M.G.N. J.M.R., H.M.M.R., A.G.M.H., and R.S.
hypoglycemic events, typical for patients with type 1 di- performed the experiments. J.M.R. and H.M.M.R. analyzed the data. All authors
abetes, have been reported to protect against hypoglycemia- discussed the results and implications and commented on the manuscript at all
induced mortality (32) or neuronal damage (33) in rats. stages. R.S. is the guarantor of this work and, as such, had full access to all the
These adaptive effects of recurrent hypoglycemia appear data in the study and takes responsibility for the integrity of the data and the
to be in line with the reported absence of increased accuracy of the data analysis.
diabetes.diabetesjournals.org Ratter and Associates 1061

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