Download Glycoprotein 130 Receptor Signaling Mediates a-Cell

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Diabetes Volume 63, September 2014
Samuel Z. Chow,1 Madeleine Speck,1 Piriya Yoganathan,1 Dominika Nackiewicz,1 Ann Maria Hansen,2
Mette Ladefoged,2 Björn Rabe,3 Stefan Rose-John,3 Peter J. Voshol,4 Francis C. Lynn,1
Pedro L. Herrera,5 Werner Müller,6 Helga Ellingsgaard,7 and Jan A. Ehses1
Glycoprotein 130 Receptor
Signaling Mediates a-Cell
Dysfunction in a Rodent Model
of Type 2 Diabetes
ISLET STUDIES
Diabetes 2014;63:2984–2995 | DOI: 10.2337/db13-1121
Dysregulated glucagon secretion accompanies islet inflammation in type 2 diabetes. We recently discovered
that interleukin (IL)-6 stimulates glucagon secretion
from human and rodent islets. IL-6 family cytokines
require the glycoprotein 130 (gp130) receptor to signal.
In this study, we elucidated the effects of a-cell gp130
receptor signaling on glycemic control in type 2 diabetes. IL-6 family cytokines were elevated in islets in rodent models of this disease. gp130 receptor activation
increased STAT3 phosphorylation in primary a-cells
and stimulated glucagon secretion. Pancreatic a-cell
gp130 knockout (agp130KO) mice showed no differences in glycemic control, a-cell function, or a-cell
mass. However, when subjected to streptozotocin plus
high-fat diet to induce islet inflammation and pathophysiology modeling type 2 diabetes, agp130KO mice had
reduced fasting glycemia, improved glucose tolerance,
reduced fasting insulin, and improved a-cell function.
Hyperinsulinemic-euglycemic clamps revealed no differences in insulin sensitivity. We conclude that in a setting of islet inflammation and pathophysiology modeling
type 2 diabetes, activation of a-cell gp130 receptor signaling has deleterious effects on a-cell function, promoting hyperglycemia. Antagonism of a-cell gp130
1Department of Surgery, Faculty of Medicine, University of British Columbia, Child
and Family Research Institute, Vancouver, British Columbia, Canada
2Diabetes Research Unit, Novo Nordisk A/S, Måløv, Denmark
3Institute of Biochemistry, Medical Faculty, Christian Albrechts University of Kiel,
Kiel, Germany
4Institute of Metabolic Science, University of Cambridge Metabolic Research
Laboratories and National Institute for Health Research Biomedical Research
Centre, Addenbrooke’s Hospital, Cambridge, U.K.
5Department of Genetic Medicine and Development, Faculty of Medicine, University of Geneva, Geneva, Switzerland
6Faculty of Life Sciences, University of Manchester, Manchester, U.K.
7The Centre of Inflammation and Metabolism and the Centre for Physical Activity
Research, Department of Infectious Diseases, Rigshospitalet, University of
Copenhagen, Copenhagen, Denmark
receptor signaling may be useful for the treatment
of type 2 diabetes.
Islet inflammation (1,2) and pancreatic a-cell dysfunction
(3–6) contribute to hyperglycemia in patients with type 2
diabetes. Pancreatic islets from patients with type 2 diabetes are infiltrated with macrophages (7,8), express elevated
proinflammatory cytokines (9,10), and express features of
fibrosis (11), consistent with reports from animals and
primates with this disease (7,12–18). The detrimental
effects of inflammation on islet b-cell function were recently confirmed, when the interleukin (IL)-1 receptor antagonist reduced hyperglycemia and improved b-cell
insulin secretion in patients with type 2 diabetes (1).
Pancreatic a-cell dysfunction is detectable in the early
stages of type 2 diabetes, where the inverse relationship
between pulsatile insulin and glucagon secretion is lost
(19). This dysregulation is associated with relative hyperglucagonemia, which contributes to the development of
fasting hyperglycemia associated with frank type 2 diabetes (6). Indeed, inhibition of glucagon action by various
means reduces hyperglycemia in rodent models of this
disease (5,6).
Corresponding author: Jan A. Ehses, [email protected].
Received 19 July 2013 and accepted 7 April 2014.
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1121/-/DC1.
S.Z.C. and M.S. contributed equally to this work.
© 2014 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and
the work is not altered.
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We recently identified IL-6 as a key cytokine elevated
during islet inflammation in rodents with type 2 diabetes
(7,20), with effects on a-cell glucagon secretion, GLP-1
secretion, and survival (21,22). IL-6 is part of a family
of IL-6–type cytokines that all share the same common
signal transducer for signaling, the glycoprotein 130
(gp130) (23). In support of a role for IL-6 cytokines in
human disease, a recent study of global gene expression in
human islets identified a group of coexpressed genes associated with type 2 diabetes. Within this group of genes,
expression of two IL-6 family cytokines, IL-6 and IL-11,
were both increased in islets from patients with type 2
diabetes: IL-6 was increased 2.75-fold, while IL-11 mRNA
was increased 1.61-fold (24).
Despite these advances, there remain significant gaps
in our knowledge regarding how the gp130 receptor
regulates a-cell function. In our previous reports, we were
unable to distinguish systemic IL-6 effects from tissuespecific IL-6 cytokine-mediated effects on the a-cell. In
addition, we did not evaluate how IL-6 family cytokines
regulate glycemia via actions on glucagon or GLP-1 secretion in a setting of islet inflammation and decreased b-cell
mass modeling type 2 diabetes (21,22). Thus we generated
a-cell gp130 receptor knockout (agp130KO) mice and investigated the role of the a-cell in a nongenetic rodent
model of type 2 diabetes.
RESEARCH DESIGN AND METHODS
Animals
Male rodents were used for all experiments. GotoKakizaki (GK) rats (Taconic), Wistar rats (Taconic),
BKS.Cg-m+/+Leprdb/BomTac (db/db) mice (Taconic), C57BL/6J
mice (CDM), and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J
(mT/mG) mice (The Jackson Laboratory) were from commercial sources. Gp130fl/fl and Gcg-Cre mice were provided
by W. Müller (25) and P. Herrera (26), respectively. GcgCre mice were bred with mT/mG mice to produce Gcg-Cre
mT/mG mice. All rodents were housed at the Child and
Family Research Institute (CFRI) animal facility, maintained on a 12-h light/dark cycle, and fed a normal chow
diet (13 kcal% fat). All procedures were approved by the
University of British Columbia Committee on Animal Care
or the Principles of Laboratory Care (Denmark).
Streptozotocin, High-Fat Diet, and Streptozotocin/
High-Fat Diet Mouse Models
Streptozotocin (STZ; Sigma-Aldrich) was prepared in acetate buffer and administered (25 mg/kg) via intraperitoneal
injections for 5 consecutive days at 7–9 weeks of age. Control mice were administered an intraperitoneal injection of
acetate buffer. Some mice were put on high-fat diet (HFD;
58 kcal% fat with sucrose; Research Diets) 3 weeks following intraperitoneal injection of acetate buffer or STZ.
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8-chamber slides (Nunc) (29). Islet cells were serum
starved followed by treatment with IL-6, hyper-IL (HIL)6 (30), leukemia inhibitory factor (LIF), or IL-27 (100 ng/mL
was used for all cytokine treatments unless otherwise
stated). Cells were paraformaldehyde fixed, permeabilized
with methanol, blocked with 5% goat serum (Invitrogen),
and incubated with rabbit anti-pSTAT3 (1:100; Cell Signaling Technology) and mouse anti-glucagon (1:1,000; SigmaAldrich). Slides were incubated with Alexa 488 donkey
anti-rabbit (1:250; Jackson ImmunoResearch) and Alexa
594 donkey anti-mouse (1:450; Jackson ImmunoResearch)
and mounted using Vectashield with DAPI (Vector Laboratories). Imaging was acquired with a BX61 microscope and
Leica SP5 II confocal imaging system and quantified using
Image-Pro Analyzer. An average of 1,130 6 261 glucagonpositive a-cells were counted per experimental condition.
Pancreatic a-cell apoptosis was assessed by TUNEL
staining as described (21) and imaged using a BX61 microscope. An average of 3,164 6 722 glucagon-positive
a-cells were counted per experimental condition.
Flow Cytometry
Isolated islets were dispersed and incubated with Fc block
(1:100; eBioscience). Islet cells were stained with CD45eFluor 450 (1:250; clone 30-F11; eBioscience), CD11b-PE
(1:1,200; clone M1/70; eBioscience), Ly-6c allophycocyanin (1:1,200; clone HK1.4; eBioscience), and the viability
dye eFluor 506 (1:1,000; eBioscience). Unstained, single
stains, and fluorescence minus one controls were used for
setting gates and compensation with the help of the CFRI
FACS core facility using a BD LSR II instrument (BD
Biosciences). Cells were gated on live cells and CD45+ cells
followed by CD11b+Ly6c+ cells. Islets were pooled from
two mice per treatment per time point.
Secretion Assays
Islets were plated on 804G extracellular matrix–coated
plates for secretion assays. Aprotinin (250 kallikrein inhibitor units/mL; Sigma-Aldrich) and dipeptidyl peptidase4 inhibitor (50 mmol/L; Millipore) were added to each well,
and islets were treated with IL-6 or HIL-6. Conditioned
media were collected, and 70% acid ethanol was added
to islets for total hormone content. Glucagon and GLP-1
were measured by radioimmunoassay (Millipore). Glucosestimulated insulin secretion from islets was performed as
previously published (7). For total pancreatic insulin, glucagon, and GLP-1 content, hormones were extracted using
70% acid ethanol and determined using Luminex technology (Millipore). Total pancreatic protein concentration was
determined using the Pierce BCA Protein Assay kit (Thermo
Scientific). Ex vivo secretion of IL-6 and LIF was determined by Luminex, and soluble IL-6 receptor (sIL-6R) secretion was assayed by ELISA (31).
Islet Isolation and Immunocytochemistry
Physiological Measurements
Mouse and rat islets were isolated and cultured as
described (7,27,28). For pSTAT3 experiments, islets were
dispersed and plated on 804G extracellular matrix–coated
For blood sampling, aprotinin (250 kallikrein inhibitor
units/mL plasma; Sigma-Aldrich) and dipeptidyl peptidase-4
inhibitor (50 mmol/L; Millipore) were added to the
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collection tubes. Mice were fasted overnight and injected
intraperitoneally with 1.5 g/kg body weight glucose for
glucose tolerance tests (intraperitoneal glucose tolerance
test [IPGTT]). For insulin tolerance tests (ITTs), mice
were fasted 2 h and injected intraperitoneally with
1 unit/kg insulin (Novolin GE, Novo Nordisk). Plasma insulin and glucagon were measured using ELISA (ALPCO
and Mercodia, respectively), total GLP-1 was measured
using Meso Scale Discovery technology, and plasma IL-6
was measured using Luminex technology (Millipore).
X-100 with mouse anti-glucagon (1:2,000) followed by
incubation with Alexa 647 goat anti-mouse (1:250;
Abcam) and mounted with Vectashield. Images were
taken on a Leica SP5 II confocal imaging system.
For pancreatic b- and a-cell mass analysis, all islets in
three sections (spaced 200 mm apart) were analyzed using
Image-Pro Analyzer. Insulin and glucagon-positive area
was quantified using the area/count function. Endocrine
cell mass was calculated as previously described (21).
Hyperinsulinemic-Euglycemic Clamps
Total RNA was extracted from islets using the MN
NucleoSpin RNA II kit (Macherey-Nagel), RNA was reversed transcribed and quantitative PCR was performed
using PrimeTime primers/probes (IDT) in a ViiA 7 realtime PCR system (ABI) as previously described (35). Differential expression was determined by the 22DDCT
method (36) with Rplp0 and 18S as reference genes.
Hyperinsulinemic-euglycemic clamps were performed as
previously described (32–34). Mice were fasted overnight
and anesthetized with acepromazine, midazolam, and fentanyl, with an initial dose by intraperitoneal injection and
top-up doses by subcutaneous injection. Once fully immobilized from anesthesia, the tail vein was cannulated, and
a 1-h basal infusion of 3H-D-glucose (1.2 mCi/h) was started
to determine steady-state tracer. Duplicate blood samples
were taken from the saphenous vein at the end of the basal
period to measure fasting blood glucose levels, for determination of endogenous glucose production, and for insulin measurements. Hyperinsulinemia was induced with
a constant infusion of insulin (3.5 mU/kg/min). To maintain euglycemia (;5.0 mmol/L), a variable infusion of
12.5% D-glucose was started simultaneously. Once euglycemia had been achieved, steady state was maintained for 60
min, after which triplicate blood samples were taken for
further analysis. Briefly, insulin concentrations were measured by ELISA, and plasma samples were counted using
a 1450 MicroBeta TriLux LSC and Luminescence Counter
(PerkinElmer) after extraction by trichloroacetic acid precipitation. Whole-body glucose use (mmol kg21 min21) was
determined as the ratio of the specific activity of glucose to
the rate of 3H-D-glucose appearance. Subsequently, endogenous glucose production (mmol kg21 min21) could be
calculated as the difference between whole-body glucose
uptake and exogenous glucose infusion.
Immunohistochemistry
Antigen retrieval was performed on sectioned tissues
using 10 mmol/L citrate buffer (Fisher Scientific) followed
by blocking with 1% goat serum and donkey/goat antimouse IgG (1:30; eBioscience). Sections were incubated
with guinea pig anti-insulin (1:1,000; Millipore) and
mouse anti-glucagon (1:2,000) followed by incubation
with Alexa 488 donkey anti–guinea pig (1:250; Jackson
ImmunoResearch) and Alexa 594 donkey anti-mouse
(1:450). Sections were mounted using Vectashield with
DAPI and imaged on a BX61 microscope.
Pancreatic Gcg-Cre X mT/mG and mT/mG cryosections
were paraformaldehyde fixed and equilibrated through
a sucrose gradient. Tissue was frozen in optimum cutting
temperature compound (Tissue-Tek). Sections were permeabilized with 0.1% Triton X-100 for 30 min and then
blocked in 5% goat serum and 1% BSA (Fisher Scientific).
Sections were incubated in 1% BSA and 0.1% Triton
Gene Expression Analysis
Statistical Analysis
Data are expressed as means 6 SEM/SD with the number of
individual experiments presented in the figure legends. All
data were analyzed using the nonlinear regression analysis
program Prism (GraphPad), and significance was tested using Student t test or ANOVA with post hoc tests for multiple
comparison analysis. Significance was set at P , 0.05.
RESULTS
IL-6 Family Cytokines Are Elevated in Pancreatic Islets
From Rodent Models of Type 2 Diabetes
We analyzed islet IL-6 family cytokine expression in two
rodent models, the GK rat and db/db mouse. Both the GK
rat and the db/db mouse displayed elevated blood glucose
and declining insulin levels at 12 and 15 weeks of age,
respectively, suggestive of b-cell failure (Fig. 1A, B, D, and
E). Isolated islets from 8–12-week-old GK rats had significantly increased levels of Il6, Lif, Clcf1, and Osm mRNA
relative to Wistar controls (Fig. 1C); similar effects were
observed in islets from 15-week-old db/db mice (compared
with Fig. 1C and F). Interestingly, both IL-6 family cytokines and glycemia were more elevated in db/db mice
relative to GK rats. Thus elevated IL-6 family cytokine
expression accompanies hyperglycemia and declining insulin levels in rodent models of type 2 diabetes, and the
magnitude of islet IL-6 cytokine expression may contribute to the prevailing hyperglycemia in these models.
gp130 Receptor Activation Stimulates STAT3
Phosphorylation and Glucagon Secretion From a-Cells
To determine if gp130 receptor signaling is activated in
a-cells following IL-6 treatment, dispersed islets were
stimulated with IL-6 or HIL-6 (IL-6 fused to the sIL-6R
[37]; HIL-6 can stimulate all gp130 receptors in the absence of an IL-6R) and immunostained for pSTAT3. IL-6
induced phosphorylation of STAT3 in a-cells within 30
min to a similar degree as HIL-6 (Fig. 2A). Next we
assessed the effect of different IL-6 doses and time of exposure on glucagon secretion in islets. IL-6 increased glucagon secretion in a concentration- and time-dependent
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Figure 1—IL-6 family cytokine expression is increased in rodents with type 2 diabetes. Nonfasted glycemia (A) and insulin levels (B) in
Wistar and GK rats. IL-6 family cytokine expression levels in islets from 8–12-week-old Wistar and GK rats (C). Expression levels were
assessed by quantitative real-time PCR, normalized to the housekeeping gene Rplp0, and expressed as fold control (Wistar). Nonfasted
glycemia (D) and insulin levels (E) in 4–15-week-old db/db mice. IL-6 family cytokine expression levels in islets from 4-, 8-, and 15-week-old
db/db mice (F). Expression levels were assessed by quantitative real-time PCR, normalized to the housekeeping gene Rplp0, and
expressed as fold control (4 weeks db/db). Data represent mean 6 SEM from 3–4 Wistar and 3–4 GK rats (A–C). Data represent
mean 6 SEM from 5–20 db/db mice (D–E) and mean 6 SD from islets pooled from $3 mice/age (F). White bars represent Wistar/
4-week db/db, gray bars represent 8-week db/db, and black bars represent GK or 15-week db/db. ★P < 0.05, ★★P < 0.01,
★★★P < 0.001 as tested by Student t test. nd, not detected.
manner, with no significant differences in glucagon content (Fig. 2B and C). Consistent with our pSTAT3 data,
IL-6 stimulated glucagon secretion to a similar degree as
HIL-6 (Fig. 2D and E), suggesting that a-cell gp130 receptor signaling is maximally activated by IL-6 and supporting our previous observation that pancreatic a-cells
express the IL-6 transmembrane receptor (21). To ensure
that IL-6 did not induce a-cell death, causing glucagon
release into the incubation medium, islets were treated
with IL-6 and apoptosis visualized by TUNEL staining. IL-6
protected from cell death induced by a mixture of cytokines (IL-1b, TNFa, and IFNg) (Fig. 2F), consistent with
our previous observations under nutrient stress (21). Collectively, these data indicate that gp130 signaling within
a-cells leads to phosphorylation of STAT3 and stimulation of glucagon secretion.
Generation and Validation of agp130KO Mice
To assess Cre recombinase activity within a-cells, Gcg-Cre
mice were crossed with mT/mG reporter mice. Pancreatic
sections revealed expression of EGFP in a proportion of
a-cells stained with glucagon, demonstrating active Cre
recombinase in 44.1 6 1.5% of a-cells (Fig. 3A and B),
similar to previous reports using Gcg-Cre mice (38).
Next we assessed pSTAT3 levels in response to gp130
receptor activation in fl/fl controls and agp130KO islets.
Stimulation of pSTAT3 with IL-6 and LIF was significantly
decreased within a-cells of agp130KO islets (Fig. 3C). IL-27
failed to induce pSTAT3 within control islets, suggesting lack
of IL-27 receptors on a-cells. Finally, we assessed glucagon
secretion in response to gp130 receptor activation in
agp130KO islets. IL-6 stimulated glucagon secretion was
reduced by ;50% in agp130KO islets (Fig. 3D). There was
no effect of IL-6 on GLP-1 secretion following 48-h exposure
of mouse islets (Fig. 3E). Thus agp130KO islets have Cremediated recombination occurring in a proportion of a-cells
and reduced gp130 receptor signaling, coinciding with a 50%
reduction in gp130 receptor–mediated glucagon secretion.
To rule out any a-cell or intestinal L-cell developmental
abnormalities in agp130KO mice, we assessed several
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Figure 2—IL-6 and HIL-6 activate a-cell STAT3 signaling and stimulate glucagon secretion from islets. STAT3 activation in primary mouse
a-cells following 30-min cytokine treatment (A). Representative image indicates staining for glucagon (red), pSTAT3 (green), and DAPI
(blue). Glucagon secretion (B) and glucagon content (C) following IL-6 treatment of mouse islets at the indicated doses and times.
Glucagon secretion (D) and glucagon content (E) from IL-6– and HIL-6–treated mouse islets following 48 h. TUNEL-positive a-cells
were analyzed following 24-h exposure to IL-6, cytokine mix (200 pg/mL IL-1b, 1 ng/mL TNFa, 5 ng/mL IFNg), or cytokine mix plus
IL-6 (F). Data represent mean 6 SEM from n = 3 mice performed in three independent experiments (A), n = 3–6 mice performed in two
independent experiments (B and C), n = 6 mice performed in three independent experiments (D and E), and n = 4 mice in two independent
experiments (F). ★P < 0.05; ★★P < 0.01; ★★★P < 0.001 vs. untreated control; #P < 0.05 vs. cytokine mix as tested by ANOVA with
Dunnett (A–E) or Newman-Kuels posttest (F). Ctrl, control; cyt, cytokine; CM, cytokine mix.
physiological parameters in agp130KO mice. agp130KO
mice displayed no difference in body weight, glucose tolerance, glucose-stimulated insulin secretion, or insulin
sensitivity at 16–18 weeks of age (Fig. 3F–I). Fasting
GLP-1 levels were unchanged, and pancreatic a-cell function was normal, as assessed by glucose-mediated glucagon
suppression during a GTT and hypoglycemia-mediated glucagon secretion during an ITT (Fig. 3J and K). Lastly, b-cell
and a-cell mass were similar between genotypes (Fig. 3L
and M). Thus agp130 receptor signaling does not appear to
affect a-cell development or function under normal chowfed conditions.
Pancreatic a-Cell Dysfunction and Islet Inflammation
in the STZ/HFD Mouse Model of Type 2 Diabetes
To investigate the role of the a-cell gp130 receptor in
a mouse model of human type 2 diabetes, we generated
a nongenetic model displaying features of type 2 diabetes.
Mice were administered STZ, HFD, or STZ in combination
with HFD as depicted in Fig. 4A. STZ alone caused glucose
intolerance, tended to reduce insulin secretion in response
to glucose in vivo, had minimal effects on glucosemediated glucagon suppression and hypoglycemiainduced glucagon secretion in vivo, and tended to reduce
b-cell mass and increase a-cell mass (Fig. 4B–L). HFD
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Figure 3—agp130KO mice exhibit partial loss of a-cell gp130 receptor function and normal glucose homeostasis and a-cell function under
chow-fed conditions. Gcg-Cre mice were bred with mT/mG mice to assess activity of a-cell Cre recombinase (A and B). Images indicate
nonrecombinant cells (tdTomato), recombinant cells (EGFP), and glucagon-positive cells (purple). EGFP was expressed in 44.1 6 1.5% of
a-cells (B). STAT3 activation in primary mouse a-cells from fl/fl and agp130KO mice following 30-min cytokine treatment (C). Glucagon (D)
and GLP-1 (E) secretion from fl/fl and agp130KO mouse islets following IL-6 treatment. Basal glucagon secretion was 2.26 6 0.54 for fl/fl
and 2.35 6 0.53 for agp130KO as percentage glucagon content. Basal GLP-1 secretion was 1.97 6 0.39 for fl/fl and 1.31 6 0.17 for
agp130KO as percentage GLP-1 content. Body weight of fl/fl and agp130KO mice at 16–18 weeks of age (F). IPGTT (G) (1.5 g/kg), insulin
levels during IPGTT ( H), and ITT ( I) (1 unit/kg). Fasting GLP-1 levels ( J) and glucagon levels during IPGTT and ITT (K). Pancreatic b- and
a-cell mass (L and M). Gray bars represent fl/fl control, and light red bars represent agp130KO. Scale bars represent 50 mm. Data represent
n = 3–5 mice per genotype ( A and B), n = 3–4 mice per genotype performed in three independent experiments (C), n = 6 mice per genotype
performed in two independent experiments (D and E), and 3–12 mice per genotype (F–M). ★P < 0.05; ★★P < 0.01 as tested by Student t
test. Ctrl, control; fl/fl, gp130fl/fl; KO, agp130KO.
alone increased body weight, caused glucose intolerance,
increased insulin secretion in response to glucose in vivo,
impaired glucose-mediated glucagon suppression, had no
effect on hypoglycemia-induced glucagon secretion in vivo,
and had minimal effects on b- and a-cell mass (Fig. 4B–L).
STZ in combination with HFD caused increased body
weight, fed hyperglycemia, glucose intolerance, and marked
a-cell dysfunction (Fig. 4B–H). In addition, b-cell mass was
reduced by ;50%, in parallel with impaired glucosestimulated insulin secretion from islets ex vivo and reduced
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Figure 4—Pancreatic a-cell dysfunction and islet inflammation in STZ-, HFD-, and STZ/HFD-treated mice. Schematic of chow, STZ (25
mg/kg), HFD, and STZ/HFD treatment groups (A) with weekly fed blood glucose monitoring (B). IPGTT (C) (1.5 g/kg glucose), ITT (D) (1 unit/kg
insulin), and body weight (E) of chow, STZ, HFD, and STZ/HFD mice. Plasma insulin (F) and glucagon (G and H) during IPGTT and ITT,
and fed insulin ( I) after 9 weeks. Pancreatic b- and a-cell mass (J and K) in treated mice with representative images ( L). Glucose-stimulated
insulin secretion (M) and insulin content (N) of islets ex vivo after 9 weeks. Pancreatic islet infiltrating monocytes (CD11b+Ly6c+ cells) in
chow and STZ-treated mice (O). Expression levels of IL-6 family cytokines post-STZ injection and during HFD relative to chow mice (P). IL-6,
sIL-6R, and LIF protein secretion from 100 islets/well isolated 3 weeks post-STZ (Q). Islet-derived IL-6 (R) from 100 islets/well and
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islet insulin content (Fig. 4J–N). a-Cell mass was also significantly increased in STZ/HFD mice (Fig. 4K). Thus treatment with STZ plus HFD resulted in a mouse with
impaired a- and b-cell function and hyperglycemia.
To characterize islet inflammation post-STZ, islet
infiltrating monocytes (CD11b+Ly6c+ cells) were analyzed
by flow cytometry. The number of islet monocytes tended
to increase at weeks 0.5 and 1 post-STZ and were significantly increased at week 2 post-STZ (Fig. 4O). To determine if islet IL-6 family cytokine expression was
increased, we analyzed islet gene expression at multiple
times post-STZ and during subsequent HFD feeding (Fig.
4P). IL-27 mRNA was increased ;1 week post-STZ, Osm
mRNA was increased 2 weeks post-STZ, and IL-6 and Lif
mRNA were increased 3 weeks post-STZ. Other IL-6 family cytokines were unchanged (Fig. 4P and not shown).
Consistent with these mRNA data, IL-6 and LIF protein
secretion were increased in islets isolated 3 weeks postSTZ, with no change in sIL-6R secretion (Fig. 4Q). Islet
IL-6 secretion was not significantly increased at week 9
in our treatment groups (Fig. 4R), indicating that IL-6
cytokines are only transiently increased following STZ
and not further increased by HFD. However, plasma IL-6
levels were significantly increased in our STZ/HFD group
at the end of our study (Fig. 4S). Taken together, these
data show that STZ/HFD-treated mice have many hallmark characteristics of type 2 diabetes, including pancreatic a-cell dysfunction and increased expression of
pancreatic islet IL-6 family cytokines.
Partial Knockout of the a-Cell gp130 Receptor
Protects From Hyperglycemia Following STZ/HFD
To determine the consequence of a-cell gp130 signaling
during islet inflammation and in a pathophysiological setting modeling type 2 diabetes, we subjected agp130KO
mice to STZ alone or STZ followed by HFD feeding.
agp130KO mice displayed no difference in body weight
following STZ or STZ/HFD (Fig. 5A). When subjected to
STZ alone, agp130KO mice showed mildly improved glucose tolerance, with no differences in insulin sensitivity,
insulin secretion, or a-cell function in vivo (Fig. 5C–I).
However, following STZ/HFD, agp130KO mice had decreased fasting glycemia, improved glucose tolerance,
and decreased fasting insulin and showed improvements
in glucose modulated glucagon secretion in vivo (Fig. 5B–
I). This was despite decreased fasting GLP-1 levels (Fig.
5E). ITTs indicated no difference in insulin sensitivity in
agp130KO mice (Fig. 5F). No differences in pancreatic
glucagon, insulin, or GLP-1 content (Fig. 5J–L) were
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observed between genotypes, along with no differences
in b- and a-cell mass (Fig. 5M–O). STZ/HFD-treated
Gcg-Cre mice did not show any glucose intolerance compared with wild-type mice excluding any phenotypic effect
of Cre recombinase expression in a-cells (Supplementary
Fig. 1). Further, agp130KO mice on HFD alone showed
no phenotypic differences in glucose tolerance or a- and
b-cell function (Supplementary Fig. 2).
Finally, to determine if changes in insulin sensitivity
contributed to the protective phenotype in STZ/HFDtreated mice, we performed hyperinsulinemic-euglycemic
clamps. There were no differences in glucose infusion
rate, whole-body insulin-stimulated glucose uptake, or
hepatic insulin sensitivity between genotypes (Fig. 6). Interestingly, hepatic glucose production under fasting conditions tended to be reduced in agp130KO mice (Fig. 6D),
consistent with reduced fasting glycemia in these mice.
Taken together, these data show that gp130 receptor
signaling contributes to a-cell dysfunction in a nongenetic model of type 2 diabetes, and partial inhibition
of gp130 receptor signaling improves glucose tolerance
and protects from hyperglycemia through improved
a-cell function.
DISCUSSION
Normally, glucagon secretion is increased during fasting
and suppressed as blood glucose rises following a meal in
a counterregulatory fashion to insulin. However, during
type 2 diabetes, glucagon secretion is inappropriately
elevated, contributing to hyperglycemia (5,6). Pioneering
studies by Müller et al. (39) and elegant clamp studies by
Shah et al. (3) have demonstrated that lack of glucagon
suppression contributes to postprandial hyperglycemia in
people with type 2 diabetes. The main mechanism underlying this hyperglucagonemia is thought to be b-cell dysfunction and a decrease in b-cell–derived secretory
products that are known to have inhibitory effects on
a-cell secretion, including insulin, g-aminobutyric acid,
and zinc ions (40). All of these factors inhibit glucagon
secretion, and their secretion is likely decreased in frank
type 2 diabetes. Our data demonstrate that islet inflammation also contributes to a-cell dysfunction, with a central
role for gp130 receptor signaling in this process and with
implications for type 2 diabetes.
agp130KO mice were protected from glucose intolerance following STZ alone, but not following HFD alone.
This was consistent with STZ treatment causing a transient increase in IL-6 and LIF secretion from islets, while
systemic IL-6 (S) during fed after 9 weeks. White bars/circles represent chow control, horizontally striped bars/white squares represent STZ,
vertically striped bars/white triangles represent HFD, and black bars/triangles represent STZ/HFD. Scale bars represent 50 mm. Data represent mean 6 SEM from n = 5 mice (B–I), n = 3–11 mice (J–L) per experimental group, n = 3–5 mice per experimental group (M and N), n =
4–5 mice per time point (O), n = 5–6 mice per time point (P), n = 4–5 mice for each experimental group (Q–S). ★P < 0.05; ★★P < 0.01;
★★★P < 0.001; ★★★★P < 0.0001 as tested by two-way ANOVA with Bonferroni post test (B–D), ANOVA with Dunnett post test (E–H),
or Student t test (M–Q) compared with chow controls. Ctrl, control; ns, not significant.
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a-Cell gp130 Receptor Signaling in Diabetes
Diabetes Volume 63, September 2014
Figure 5—agp130KO mice display improved glucose homeostasis and a-cell function following STZ/HFD. Body weight (A) and fasting
glycemia (B) of fl/fl and agp130KO mice at 16–18 weeks of age following STZ or STZ/HFD. IPGTT (C) (1.5 g/kg) with quantification of area
under curve above basal (D), fasting GLP-1 (E), and ITT (F) (1 unit/kg) with corresponding plasma insulin (G) and glucagon levels (H–I ).
Insulin (J ), glucagon (K), and GLP-1 (L) pancreatic content of fl/fl and agp130KO mice following STZ/HFD. Pancreatic b- and a-cell mass
(M–N), and representative islet images (O) of STZ/HFD fl/fl and agp130KO mice. Representative image indicates glucagon (red), insulin
(green), and nuclei (blue; DAPI). Scale bars represent 50 mm. Data represent mean 6 SEM from n = 4–6 mice per genotype treated with STZ
(A–I ), n = 22–23 mice per genotype treated with STZ/HFD (A–D), n = 4 mice per genotype (E), n = 4–8 mice per genotype treated with
STZ/HFD (F), n = 11–14 mice per genotype treated with STZ/HFD (G–I ), n = 4–8 mice per genotype (J–N) from 1–4 independent cohorts
of mice. Black border white bars/triangles represent STZ fl/fl, red border white bars/triangles represent STZ agp130KO, black border
gray bars/circles represent STZ/HFD fl/fl, and red border red bars/squares represent STZ/HFD agp130KO. ★P < 0.05; ★★P < 0.01 as
tested by Student t test. In C, ★P < 0.05 and #P < 0.05 as tested by Student t test compared with fl/fl controls. AUC, area under the
curve; KO, agp130KO; ns, not significant.
HFD alone did not increase islet IL-6 family cytokine
expression. One caveat in our study is that isolating islets
induces IL-6 mRNA expression and secretion (41). It is
therefore likely that islet IL-6 is elevated for a more prolonged period of time than depicted in Fig. 4P. Interestingly, islet IL-6 cytokine mRNA levels correlated very well
with macrophage infiltration following STZ. We recently
found that depletion of islet macrophages reduced islet
amyloid polypeptide–induced islet IL-6 mRNA expression
(42). Whether islet macrophages are the cellular source of
IL-6 family cytokines causing a-cell dysfunction should be
addressed in future studies.
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Chow and Associates
2993
Figure 6—No difference in insulin sensitivity in agp130KO mice following STZ/HFD. Hyperinsulinemic-euglycemic clamps were performed
after STZ plus 6 weeks of HFD feeding in fl/fl and agp130KO littermates. Blood glucose during clamping (A) and glucose infusion rate (B).
Glucose turnover (C), suppression of hepatic glucose production (D), and insulin levels (E) during clamp. Data represent mean 6 SEM from
n = 7 fl/fl and n = 6 agp130KO mice from two independent cohorts. P value was determined using Student t test. GIR, glucose infusion rate;
KO, agp130KO.
Interestingly, the combination of STZ plus HFD did
not prolong or increase islet IL-6 family cytokine expression, but it did result in further impairments in a-cell
function compared with STZ or HFD treatments alone.
One possible explanation for this synergism is that HFD
mediates a-cell dysfunction by causing a-cell insulin resistance and that STZ exacerbates these effects by decreasing insulin and inducing inflammation. This might
explain the synergistic effect of STZ plus HFD on glucosemediated glucagon suppression in vivo during an IPGTT.
With respect to ITT responses, hypoglycemia-induced glucagon secretion is strongly driven by the parasympathetic
nervous system (43,44). Thus STZ acts synergistically
with HFD to impair this response. Regardless of the
mechanism of action, these hypotheses are consistent
with effects of gp130 receptor signaling on a-cell function and glycemic control being more pronounced in the
STZ/HFD model, compared with STZ or HFD treatments
alone. Thus gp130 receptor signaling acts as a modifier of
a-cell function in a synergistic manner with HFD-induced
effects on a-cell function.
Improved a-cell function in agp130KO mice treated
with STZ/HFD resulted in reduced fasting hyperglycemia,
likely due to reduced hepatic glucose output. Reduced
glycemia was likely responsible for the decreased fasting
insulin observed in agp130KO mice. We ruled out any
contribution of increased insulin sensitivity in agp130KO
mice treated with STZ/HFD by performing hyperinsulinemiceuglycemic clamps. However, reduced fasting glycemia may
have improved b-cell function in agp130KO mice, likely
by reducing the effects of glucotoxicity on the b-cell. This
sequence of events may also help explain how a transient
increase in islet IL-6 cytokines can have long-lasting
effects on glycemic control.
Two independent groups recently investigated the
effect of gp130 receptor cytokines on the a-cell. McGuinness
and colleagues found that IL-62/2 mice had a blunted glucagon response to endotoxin that was restored by replacement of IL-6 (45). They went on to show that IL-6 amplifies
adrenergic-dependent glucagon secretion from mouse
islets (46). In a separate study, Fernández-Millán et al.
showed that a-cell mass expansion and hyperglucagonemia during suckling in rats is partly IL-6–dependent,
contributing to hyperglycemia postweaning (47). These
studies, our previous work (21), and data presented here
support the notion that IL-6 cytokines are modulators of
a-cell glucagon secretion. Taking these findings into consideration, we propose that gp130 receptor actions are
context-dependent and that IL-6 cytokines modulate
a-cell glucagon secretion to allow for increased glucose
output during normal physiology (e.g., during acute infection or postexercise), while also driving excessive
a-cell glucagon secretion and dysfunction during type 2
diabetes.
Exposure of human islets to IL-6 for 4 days or enriched
human pancreatic a-cells to IL-6 enhances GLP-1 secretion in addition to glucagon secretion, perhaps via upregulation of pro(hormone)-convertase 1/3 (22). Mouse
islets exposed to IL-6 for 2 days did not have elevated
GLP-1 secretion. Dedifferentiation of a-cells to a prea-cell state has been shown to induce GLP-1 secretion
(48). In addition, prolonged culture of human islets in
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a-Cell gp130 Receptor Signaling in Diabetes
vitro results in increased numbers of glucagon-positive
cells due to conversion of b-cells to a-cells (49). We surmise that signals causing a-cell dedifferentiation or conversion of b-cells to a-cells act together with IL-6
cytokines to stimulate GLP-1 secretion from a-cells. In
the future, the mechanisms underlying IL-6–stimulated
a-cell GLP-1 secretion will need to be determined and
their role tested in vivo.
Despite decreased systemic GLP-1 levels in agp130KO
mice (likely due to receptor deletion in L cells), activation
of a-cell gp130 receptor signaling in a setting of reduced
b-cell mass and HFD-induced insulin resistance had detrimental effects on normal a-cell function and glycemic
control. This may suggest that effects of gp130 receptor
cytokines on a-cell function are more important for glycemic control than effects on L-cell GLP-1 secretion in
type 2 diabetes. While it remains to be determined what
the mechanisms are that facilitate IL-6 cytokine actions
on glucagon secretion, insight into how IL-6/gp130 influences a-cell secretory processes and/or a-cell differentiation may allow us to identify druggable targets that
modify the actions of the a-cell and could thereby be
used for the treatment of type 2 diabetes.
Acknowledgments. The authors thank G. Soukhatcheva, M. Komba, and
L. Xu (CFRI) for technical assistance provided by the CFRI islet isolation core
facility and CFRI flow cytometry core and Bruce Verchere (University of British
Columbia) for critical review of the manuscript.
Funding. This work was supported by funding from the CFRI (J.A.E.), the
University of British Columbia (J.A.E.), the Canadian Institutes of Health Research
(PCN-110793 and PNI-120292 to J.A.E.), the Canadian Diabetes Association
(OG-3-13-4097-JE), the Deutsche Forschungsgemeinschaft (Bonn, Germany;
SFB877; project A1 to S.R.-J.), and the Cluster of Excellence “Inflammation at
Interfaces” (to S.R.-J.). S.Z.C. is supported by a Canadian Institutes of Health
Research Transplantation Training Program Scholarship. H.E. is supported by the
Danish Council for Independent Research and the Novo Nordisk Foundation. D.N.
is supported by a University of British Columbia Transplantation Training Program
and Vanier Canada Graduate Scholarship. J.A.E. has salary support from a CFRI
scientist award and a Canadian Diabetes Association scholar award.
Duality of Interest. No potential conflicts of interest relevant to this article
were reported.
Author Contributions. S.Z.C. designed and performed experiments, analyzed data, and wrote the manuscript. M.S. designed and performed experiments
and analyzed data. P.Y., D.N., A.M.H., and M.L. designed and performed experiments. B.R. and S.R.-J. performed experiments and provided reagents. P.J.V. and
H.E. designed experiments. F.C.L., P.L.H., and W.M. provided reagents and mice.
J.A.E. supervised the study, designed and performed experiments, analyzed data,
and wrote the manuscript. All authors edited the manuscript. J.A.E. is the guarantor
of this work and, as such, had full access to all the data in the study and takes
responsibility for the integrity of the data and the accuracy of the data analysis.
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