Glycoprotein 130 Receptor Signaling Mediates A-Cell Dysfunction in a Rodent Model of Type 2 Diabetes

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2984 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

Diabetes 2014;63:2984–2995 | DOI: 10.2337/db13-1121

Dysregulated glucagon secretion accompanies islet in- receptor signaling may be useful for the treatment flammation in type 2 diabetes. We recently discovered of type 2 diabetes. that interleukin (IL)-6 stimulates glucagon secretion from human and rodent islets. IL-6 family cytokines Islet inflammation (1,2) and pancreatic a-cell dysfunction require the glycoprotein 130 (gp130) receptor to signal. (3–6) contribute to hyperglycemia in patients with type 2 In this study, we elucidated the effects of a-cell gp130 diabetes. Pancreatic islets from patients with type 2 diabe- receptor signaling on glycemic control in type 2 diabetes are infiltrated with macrophages (7,8), express elevated tes. IL-6 family cytokines were elevated in islets in ro- proinflammatory cytokines (9,10), and express features of dent models of this disease. gp130 receptor activation fibrosis (11), consistent with reports from animals and a increased STAT3 phosphorylation in primary -cells primates with this disease (7,12–18). The detrimental and stimulated glucagon secretion. Pancreatic a-cell fl ISLET STUDIES effects of in ammation on islet b-cell function were re- gp130 knockout (agp130KO)miceshowednodiffer- cently confirmed, when the interleukin (IL)-1 receptor an- ences in glycemic control, a-cell function, or a-cell tagonist reduced hyperglycemia and improved b-cell mass. However, when subjected to streptozotocin plus insulin secretion in patients with type 2 diabetes (1). high-fat diet to induce islet inflammation and pathophys- a iology modeling type 2 diabetes, agp130KO mice had Pancreatic -cell dysfunction is detectable in the early reduced fasting glycemia, improved glucose tolerance, stages of type 2 diabetes, where the inverse relationship reduced fasting insulin, and improved a-cell function. between pulsatile insulin and glucagon secretion is lost Hyperinsulinemic-euglycemic clamps revealed no dif- (19). This dysregulation is associated with relative hyper- ferences in insulin sensitivity. We conclude that in a set- glucagonemia, which contributes to the development of ting of islet inflammation and pathophysiology modeling fasting hyperglycemia associated with frank type 2 diabe- type 2 diabetes, activation of a-cell gp130 receptor sig- tes (6). Indeed, inhibition of glucagon action by various naling has deleterious effects on a-cell function, pro- means reduces hyperglycemia in rodent models of this moting hyperglycemia. Antagonism of a-cell gp130 disease (5,6).

1Department of Surgery, Faculty of Medicine, University of British Columbia, Child Corresponding author: Jan A. Ehses, [email protected]. and Family Research Institute, Vancouver, British Columbia, Canada Received 19 July 2013 and accepted 7 April 2014. 2Diabetes Research Unit, Novo Nordisk A/S, Måløv, Denmark This article contains Supplementary Data online at http://diabetes 3Institute of Biochemistry, Medical Faculty, Christian Albrechts University of Kiel, .diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1121/-/DC1. Kiel, Germany 4Institute of Metabolic Science, University of Cambridge Metabolic Research S.Z.C. and M.S. contributed equally to this work. Laboratories and National Institute for Health Research Biomedical Research © 2014 by the American Diabetes Association. Readers may use this article as Centre, Addenbrooke’s Hospital, Cambridge, U.K. long as the work is properly cited, the use is educational and not for proﬁt, and 5Department of Genetic Medicine and Development, Faculty of Medicine, Univer- the work is not altered. sity 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 diabetes.diabetesjournals.org Chow and Associates 2985

We recently identified IL-6 as a key cytokine elevated 8-chamber slides (Nunc) (29). Islet cells were serum during islet inflammation in rodents with type 2 diabetes starved followed by treatment with IL-6, hyper-IL (HIL)- (7,20), with effects on a-cell glucagon secretion, GLP-1 6 (30), leukemia inhibitory factor (LIF), or IL-27 (100 ng/mL secretion, and survival (21,22). IL-6 is part of a family was used for all cytokine treatments unless otherwise of IL-6–type cytokines that all share the same common stated). Cells were paraformaldehyde fixed, permeabilized signal transducer for signaling, the glycoprotein 130 with methanol, blocked with 5% goat serum (Invitrogen), (gp130) (23). In support of a role for IL-6 cytokines in and incubated with rabbit anti-pSTAT3 (1:100; Cell Signal- human disease, a recent study of global gene expression in ing Technology) and mouse anti-glucagon (1:1,000; Sigma- human islets identified a group of coexpressed genes as- Aldrich). Slides were incubated with Alexa 488 donkey sociated with type 2 diabetes. Within this group of genes, anti-rabbit (1:250; Jackson ImmunoResearch) and Alexa expression of two IL-6 family cytokines, IL-6 and IL-11, 594 donkey anti-mouse (1:450; Jackson ImmunoResearch) were both increased in islets from patients with type 2 and mounted using Vectashield with DAPI (Vector Labora- diabetes: IL-6 was increased 2.75-fold, while IL-11 mRNA tories). Imaging was acquired with a BX61 microscope and was increased 1.61-fold (24). Leica SP5 II confocal imaging system and quantified using Despite these advances, there remain significant gaps Image-Pro Analyzer. An average of 1,130 6 261 glucagon- in our knowledge regarding how the gp130 receptor positive a-cells were counted per experimental condition. regulates a-cell function. In our previous reports, we were Pancreatic a-cell apoptosis was assessed by TUNEL unable to distinguish systemic IL-6 effects from tissue- staining as described (21) and imaged using a BX61 mi- specific IL-6 cytokine-mediated effects on the a-cell. In croscope. An average of 3,164 6 722 glucagon-positive addition, we did not evaluate how IL-6 family cytokines a-cells were counted per experimental condition. regulate glycemia via actions on glucagon or GLP-1 secre- Flow Cytometry fl b tion in a setting of islet in ammation and decreased -cell Isolated islets were dispersed and incubated with Fc block mass modeling type 2 diabetes (21,22). Thus we generated (1:100; eBioscience). Islet cells were stained with CD45- a a -cell gp130 receptor knockout ( gp130KO) mice and in- eFluor 450 (1:250; clone 30-F11; eBioscience), CD11b-PE a vestigated the role of the -cell in a nongenetic rodent (1:1,200; clone M1/70; eBioscience), Ly-6c allophycocya- model of type 2 diabetes. nin (1:1,200; clone HK1.4; eBioscience), and the viability dye eFluor 506 (1:1,000; eBioscience). Unstained, single RESEARCH DESIGN AND METHODS stains, and fluorescence minus one controls were used for Animals setting gates and compensation with the help of the CFRI Male rodents were used for all experiments. Goto- FACS core facility using a BD LSR II instrument (BD Kakizaki (GK) rats (Taconic), Wistar rats (Taconic), + +/+ Biosciences). Cells were gated on live cells and CD45 cells BKS.Cg-m Leprdb/BomTac (db/db) mice (Taconic), C57BL/6J + + tm4(ACTB-tdTomato,-EGFP)Luo followed by CD11b Ly6c cells. Islets were pooled from mice (CDM), and Gt(ROSA)26Sor /J two mice per treatment per time point. (mT/mG) mice (The Jackson Laboratory) were from com- fl fl Secretion Assays mercial sources. Gp130 / and Gcg-Cre mice were provided Islets were plated on 804G extracellular matrix–coated by W. Müller (25) and P. Herrera (26), respectively. Gcg- plates for secretion assays. Aprotinin (250 kallikrein in- Cre mice were bred with mT/mG mice to produce Gcg-Cre hibitor units/mL; Sigma-Aldrich) and dipeptidyl peptidase- mT/mG mice. All rodents were housed at the Child and 4 inhibitor (50 mmol/L; Millipore) were added to each well, Family Research Institute (CFRI) animal facility, main- and islets were treated with IL-6 or HIL-6. Conditioned tained on a 12-h light/dark cycle, and fed a normal chow media were collected, and 70% acid ethanol was added diet (13 kcal% fat). All procedures were approved by the to islets for total hormone content. Glucagon and GLP-1 University of British Columbia Committee on Animal Care were measured by radioimmunoassay (Millipore). Glucose- or the Principles of Laboratory Care (Denmark). stimulated insulin secretion from islets was performed as Streptozotocin, High-Fat Diet, and Streptozotocin/ previously published (7). For total pancreatic insulin, glu- High-Fat Diet Mouse Models cagon, and GLP-1 content, hormones were extracted using Streptozotocin (STZ; Sigma-Aldrich) was prepared in ace- 70% acid ethanol and determined using Luminex technol- tate buffer and administered (25 mg/kg) via intraperitoneal ogy (Millipore). Total pancreatic protein concentration was injections for 5 consecutive days at 7–9 weeks of age. Con- determined using the Pierce BCA Protein Assay kit (Thermo trol mice were administered an intraperitoneal injection of Scientific). Ex vivo secretion of IL-6 and LIF was deter- acetate buffer. Some mice were put on high-fat diet (HFD; mined by Luminex, and soluble IL-6 receptor (sIL-6R) se- 58 kcal% fat with sucrose; Research Diets) 3 weeks follow- cretion was assayed by ELISA (31). ing intraperitoneal injection of acetate buffer or STZ. Islet Isolation and Immunocytochemistry Physiological Measurements Mouse and rat islets were isolated and cultured as For blood sampling, aprotinin (250 kallikrein inhibitor described (7,27,28). For pSTAT3 experiments, islets were units/mL plasma; Sigma-Aldrich) and dipeptidyl peptidase-4 dispersed and plated on 804G extracellular matrix–coated inhibitor (50 mmol/L; Millipore) were added to the 2986 a-Cell gp130 Receptor Signaling in Diabetes Diabetes Volume 63, September 2014 collection tubes. Mice were fasted overnight and injected X-100 with mouse anti-glucagon (1:2,000) followed by intraperitoneally with 1.5 g/kg body weight glucose for incubation with Alexa 647 goat anti-mouse (1:250; glucose tolerance tests (intraperitoneal glucose tolerance Abcam) and mounted with Vectashield. Images were test [IPGTT]). For insulin tolerance tests (ITTs), mice taken on a Leica SP5 II confocal imaging system. were fasted 2 h and injected intraperitoneally with For pancreatic b- and a-cell mass analysis, all islets in 1 unit/kg insulin (Novolin GE, Novo Nordisk). Plasma in- three sections (spaced 200 mm apart) were analyzed using sulin and glucagon were measured using ELISA (ALPCO Image-Pro Analyzer. Insulin and glucagon-positive area and Mercodia, respectively), total GLP-1 was measured was quantified using the area/count function. Endocrine using Meso Scale Discovery technology, and plasma IL-6 cell mass was calculated as previously described (21). was measured using Luminex technology (Millipore). Gene Expression Analysis Hyperinsulinemic-Euglycemic Clamps Total RNA was extracted from islets using the MN Hyperinsulinemic-euglycemic clamps were performed as NucleoSpin RNA II kit (Macherey-Nagel), RNA was re- previously described (32–34). Mice were fasted overnight versed transcribed and quantitative PCR was performed and anesthetized with acepromazine, midazolam, and fen- using PrimeTime primers/probes (IDT) in a ViiA 7 real- tanyl, with an initial dose by intraperitoneal injection and time PCR system (ABI) as previously described (35). Dif- 2 top-up doses by subcutaneous injection. Once fully immo- ferential expression was determined by the 2 DDCT bilized from anesthesia, the tail vein was cannulated, and method (36) with Rplp0 and 18S as reference genes. 3 D m a 1-h basal infusion of H- -glucose (1.2 Ci/h) was started Statistical Analysis to determine steady-state tracer. Duplicate blood samples Data are expressed as means 6 SEM/SD with the number of were taken from the saphenous vein at the end of the basal individual experiments presented in the figure legends. All period to measure fasting blood glucose levels, for deter- data were analyzed using the nonlinear regression analysis mination of endogenous glucose production, and for insu- program Prism (GraphPad), and significance was tested us- lin measurements. Hyperinsulinemia was induced with ing Student t test or ANOVA with post hoc tests for multiple a constant infusion of insulin (3.5 mU/kg/min). To main- comparison analysis. Significance was set at P , 0.05. tain euglycemia (;5.0 mmol/L), a variable infusion of 12.5% D-glucose was started simultaneously. Once euglyce- RESULTS mia had been achieved, steady state was maintained for 60 IL-6 Family Cytokines Are Elevated in Pancreatic Islets min, after which triplicate blood samples were taken for From Rodent Models of Type 2 Diabetes further analysis. Briefly, insulin concentrations were mea- We analyzed islet IL-6 family cytokine expression in two sured by ELISA, and plasma samples were counted using rodent models, the GK rat and db/db mouse. Both the GK a 1450 MicroBeta TriLux LSC and Luminescence Counter rat and the db/db mouse displayed elevated blood glucose (PerkinElmer) after extraction by trichloroacetic acid pre- and declining insulin levels at 12 and 15 weeks of age, 21 21 cipitation. Whole-body glucose use (mmol kg min )was respectively, suggestive of b-cell failure (Fig. 1A, B, D, and determined as the ratio of the specific activity of glucose to E). Isolated islets from 8–12-week-old GK rats had signif- 3 the rate of H-D-glucose appearance. Subsequently, endog- icantly increased levels of Il6, Lif, Clcf1, and Osm mRNA 21 21 enous glucose production (mmol kg min )couldbe relative to Wistar controls (Fig. 1C); similar effects were calculated as the difference between whole-body glucose observed in islets from 15-week-old db/db mice (compared uptake and exogenous glucose infusion. with Fig. 1C and F). Interestingly, both IL-6 family cyto- db/db Immunohistochemistry kines and glycemia were more elevated in mice Antigen retrieval was performed on sectioned tissues relative to GK rats. Thus elevated IL-6 family cytokine using 10 mmol/L citrate buffer (Fisher Scientific) followed expression accompanies hyperglycemia and declining in- by blocking with 1% goat serum and donkey/goat anti- sulin levels in rodent models of type 2 diabetes, and the mouse IgG (1:30; eBioscience). Sections were incubated magnitude of islet IL-6 cytokine expression may contrib- with guinea pig anti-insulin (1:1,000; Millipore) and ute to the prevailing hyperglycemia in these models. mouse anti-glucagon (1:2,000) followed by incubation gp130 Receptor Activation Stimulates STAT3 with Alexa 488 donkey anti–guinea pig (1:250; Jackson Phosphorylation and Glucagon Secretion From a-Cells ImmunoResearch) and Alexa 594 donkey anti-mouse To determine if gp130 receptor signaling is activated in (1:450). Sections were mounted using Vectashield with a-cells following IL-6 treatment, dispersed islets were DAPI and imaged on a BX61 microscope. stimulated with IL-6 or HIL-6 (IL-6 fused to the sIL-6R Pancreatic Gcg-Cre X mT/mG and mT/mG cryosections [37]; HIL-6 can stimulate all gp130 receptors in the ab- were paraformaldehyde fixed and equilibrated through sence of an IL-6R) and immunostained for pSTAT3. IL-6 a sucrose gradient. Tissue was frozen in optimum cutting induced phosphorylation of STAT3 in a-cells within 30 temperature compound (Tissue-Tek). Sections were per- min to a similar degree as HIL-6 (Fig. 2A). Next we meabilized with 0.1% Triton X-100 for 30 min and then assessed the effect of different IL-6 doses and time of ex- blocked in 5% goat serum and 1% BSA (Fisher Scientific). posure on glucagon secretion in islets. IL-6 increased glu- Sections were incubated in 1% BSA and 0.1% Triton cagon secretion in a concentration- and time-dependent diabetes.diabetesjournals.org Chow and Associates 2987

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)in4–15-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 con- sections revealed expression of EGFP in a proportion of tent (Fig. 2B and C). Consistent with our pSTAT3 data, a-cells stained with glucagon, demonstrating active Cre IL-6 stimulated glucagon secretion to a similar degree as recombinase in 44.1 6 1.5% of a-cells (Fig. 3A and B), HIL-6 (Fig. 2D and E), suggesting that a-cell gp130 re- similar to previous reports using Gcg-Cre mice (38). ceptor signaling is maximally activated by IL-6 and sup- Next we assessed pSTAT3 levels in response to gp130 porting our previous observation that pancreatic a-cells receptor activation in fl/fl controls and agp130KO islets. express the IL-6 transmembrane receptor (21). To ensure Stimulation of pSTAT3 with IL-6 and LIF was significantly that IL-6 did not induce a-cell death, causing glucagon decreased within a-cells of agp130KO islets (Fig. 3C). IL-27 release into the incubation medium, islets were treated failed to induce pSTAT3 within control islets, suggesting lack with IL-6 and apoptosis visualized by TUNEL staining. IL-6 of IL-27 receptors on a-cells. Finally, we assessed glucagon protected from cell death induced by a mixture of cyto- secretion in response to gp130 receptor activation in kines (IL-1b, TNFa, and IFNg) (Fig. 2F), consistent with agp130KO islets. IL-6 stimulated glucagon secretion was our previous observations under nutrient stress (21). Col- reduced by ;50% in agp130KO islets (Fig. 3D). There was lectively, these data indicate that gp130 signaling within no effect of IL-6 on GLP-1 secretion following 48-h exposure a-cells leads to phosphorylation of STAT3 and stimula- of mouse islets (Fig. 3E). Thus agp130KO islets have Cre- tion of glucagon secretion. mediated recombination occurring in a proportion of a-cells and reduced gp130 receptor signaling, coinciding with a 50% Generation and Validation of agp130KO Mice reduction in gp130 receptor–mediated glucagon secretion. To assess Cre recombinase activity within a-cells, Gcg-Cre To rule out any a-cell or intestinal L-cell developmental mice were crossed with mT/mG reporter mice. Pancreatic abnormalities in agp130KO mice, we assessed several 2988 a-Cell gp130 Receptor Signaling in Diabetes Diabetes Volume 63, September 2014

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 Pancreatic a-Cell Dysfunction and Islet Inﬂammation mice displayed no difference in body weight, glucose tol- in the STZ/HFD Mouse Model of Type 2 Diabetes erance, glucose-stimulated insulin secretion, or insulin To investigate the role of the a-cell gp130 receptor in sensitivity at 16–18 weeks of age (Fig. 3F–I). Fasting a mouse model of human type 2 diabetes, we generated GLP-1 levels were unchanged, and pancreatic a-cell func- a nongenetic model displaying features of type 2 diabetes. tion was normal, as assessed by glucose-mediated glucagon Mice were administered STZ, HFD, or STZ in combination suppression during a GTT and hypoglycemia-mediated glu- with HFD as depicted in Fig. 4A. STZ alone caused glucose cagon secretion during an ITT (Fig. 3J and K). Lastly, b-cell intolerance, tended to reduce insulinsecretioninresponse and a-cell mass were similar between genotypes (Fig. 3L to glucose in vivo, had minimal effects on glucose- and M). Thus agp130 receptor signaling does not appear to mediated glucagon suppression and hypoglycemia- affect a-cell development or function under normal chow- induced glucagon secretion in vivo, and tended to reduce fed conditions. b-cell mass and increase a-cell mass (Fig. 4B–L). HFD diabetes.diabetesjournals.org Chow and Associates 2989

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, STZ in combination with HFD caused increased body increased insulin secretion in response to glucose in vivo, weight, fed hyperglycemia, glucose intolerance, and marked impaired glucose-mediated glucagon suppression, had no a-cell dysfunction (Fig. 4B–H). In addition, b-cell mass was effect on hypoglycemia-induced glucagon secretion in vivo, reduced by ;50%, in parallel with impaired glucose- and had minimal effects on b-anda-cell mass (Fig. 4B–L). stimulated insulin secretion from islets ex vivo and reduced 2990 a-Cell gp130 Receptor Signaling in Diabetes Diabetes Volume 63, September 2014

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)(1.5g/kgglucose),ITT(D)(1unit/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)after9weeks.Pancreaticb-anda-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 diabetes.diabetesjournals.org Chow and Associates 2991 islet insulin content (Fig. 4J–N). a-Cell mass was also sig- observed between genotypes, along with no differences nificantly increased in STZ/HFD mice (Fig. 4K). Thus treat- in b- and a-cell mass (Fig. 5M–O). STZ/HFD-treated ment with STZ plus HFD resulted in a mouse with Gcg-Cre mice did not show any glucose intolerance com- impaired a-andb-cell function and hyperglycemia. pared with wild-type mice excluding any phenotypic effect To characterize islet inflammation post-STZ, islet of Cre recombinase expression in a-cells (Supplementary infiltrating monocytes (CD11b+Ly6c+ cells) were analyzed Fig. 1). Further, agp130KO mice on HFD alone showed by flow cytometry. The number of islet monocytes tended no phenotypic differences in glucose tolerance or a- and to increase at weeks 0.5 and 1 post-STZ and were signifi- b-cell function (Supplementary Fig. 2). cantly increased at week 2 post-STZ (Fig. 4O). To deter- Finally, to determine if changes in insulin sensitivity mine if islet IL-6 family cytokine expression was contributed to the protective phenotype in STZ/HFD- increased, we analyzed islet gene expression at multiple treated mice, we performed hyperinsulinemic-euglycemic times post-STZ and during subsequent HFD feeding (Fig. clamps. There were no differences in glucose infusion 4P). IL-27 mRNA was increased ;1 week post-STZ, Osm rate, whole-body insulin-stimulated glucose uptake, or mRNA was increased 2 weeks post-STZ, and IL-6 and Lif hepatic insulin sensitivity between genotypes (Fig. 6). In- mRNA were increased 3 weeks post-STZ. Other IL-6 fam- terestingly, hepatic glucose production under fasting con- ily cytokines were unchanged (Fig. 4P and not shown). ditions tended to be reduced in agp130KO mice (Fig. 6D), Consistent with these mRNA data, IL-6 and LIF protein consistent with reduced fasting glycemia in these mice. secretion were increased in islets isolated 3 weeks post- Taken together, these data show that gp130 receptor STZ, with no change in sIL-6R secretion (Fig. 4Q). Islet signaling contributes to a-cell dysfunction in a nonge- IL-6 secretion was not significantly increased at week 9 netic model of type 2 diabetes, and partial inhibition in our treatment groups (Fig. 4R), indicating that IL-6 of gp130 receptor signaling improves glucose tolerance cytokines are only transiently increased following STZ and protects from hyperglycemia through improved and not further increased by HFD. However, plasma IL-6 a-cell function. levels were significantly increased in our STZ/HFD group at the end of our study (Fig. 4S). Taken together, these DISCUSSION data show that STZ/HFD-treated mice have many hall- Normally, glucagon secretion is increased during fasting mark characteristics of type 2 diabetes, including pan- and suppressed as blood glucose rises following a meal in creatic a-cell dysfunction and increased expression of a counterregulatory fashion to insulin. However, during pancreatic islet IL-6 family cytokines. type 2 diabetes, glucagon secretion is inappropriately Partial Knockout of the a-Cell gp130 Receptor elevated, contributing to hyperglycemia (5,6). Pioneering Protects From Hyperglycemia Following STZ/HFD studies by Müller et al. (39) and elegant clamp studies by To determine the consequence of a-cell gp130 signaling Shah et al. (3) have demonstrated that lack of glucagon during islet inflammation and in a pathophysiological set- suppression contributes to postprandial hyperglycemia in ting modeling type 2 diabetes, we subjected agp130KO people with type 2 diabetes. The main mechanism under- mice to STZ alone or STZ followed by HFD feeding. lying this hyperglucagonemia is thought to be b-cell dys- agp130KO mice displayed no difference in body weight function and a decrease in b-cell–derived secretory following STZ or STZ/HFD (Fig. 5A). When subjected to products that are known to have inhibitory effects on STZ alone, agp130KO mice showed mildly improved glu- a-cell secretion, including insulin, g-aminobutyric acid, cose tolerance, with no differences in insulin sensitivity, and zinc ions (40). All of these factors inhibit glucagon insulin secretion, or a-cell function in vivo (Fig. 5C–I). secretion, and their secretion is likely decreased in frank However, following STZ/HFD, agp130KO mice had de- type 2 diabetes. Our data demonstrate that islet inflam- creased fasting glycemia, improved glucose tolerance, mation also contributes to a-cell dysfunction, with a central and decreased fasting insulin and showed improvements role for gp130 receptor signaling in this process and with in glucose modulated glucagon secretion in vivo (Fig. 5B– implications for type 2 diabetes. I). This was despite decreased fasting GLP-1 levels (Fig. agp130KO mice were protected from glucose intoler- 5E). ITTs indicated no difference in insulin sensitivity in ance following STZ alone, but not following HFD alone. agp130KO mice (Fig. 5F). No differences in pancreatic This was consistent with STZ treatment causing a tran- glucagon, insulin, or GLP-1 content (Fig. 5J–L) were sient 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 =5–6micepertimepoint(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. 2992 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)offl/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)from1–4 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 with macrophage inﬁltration following STZ. We recently expression. One caveat in our study is that isolating islets found that depletion of islet macrophages reduced islet induces IL-6 mRNA expression and secretion (41). It is amyloid polypeptide–induced islet IL-6 mRNA expression therefore likely that islet IL-6 is elevated for a more pro- (42). Whether islet macrophages are the cellular source of longed period of time than depicted in Fig. 4P. Interest- IL-6 family cytokines causing a-cell dysfunction should be ingly, islet IL-6 cytokine mRNA levels correlated very well addressed in future studies. diabetes.diabetesjournals.org 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 =7fl/fl and n =6agp130KO 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 sequence of events may also help explain how a transient not prolong or increase islet IL-6 family cytokine expres- increase in islet IL-6 cytokines can have long-lasting sion, but it did result in further impairments in a-cell effects on glycemic control. function compared with STZ or HFD treatments alone. Two independent groups recently investigated the One possible explanation for this synergism is that HFD effect of gp130 receptor cytokines on the a-cell. McGuinness 2 2 mediates a-cell dysfunction by causing a-cell insulin re- and colleagues found that IL-6 / micehadabluntedglu- sistance and that STZ exacerbates these effects by de- cagon response to endotoxin that was restored by replace- creasing insulin and inducing inflammation. This might ment of IL-6 (45). They went on to show that IL-6 amplifies explain the synergistic effect of STZ plus HFD on glucose- adrenergic-dependent glucagon secretion from mouse mediated glucagon suppression in vivo during an IPGTT. islets (46). In a separate study, Fernández-Millán et al. With respect to ITT responses, hypoglycemia-induced glu- showed that a-cell mass expansion and hyperglucagone- cagon secretion is strongly driven by the parasympathetic mia during suckling in rats is partly IL-6–dependent, nervous system (43,44). Thus STZ acts synergistically contributing to hyperglycemia postweaning (47). These with HFD to impair this response. Regardless of the studies, our previous work (21), and data presented here mechanism of action, these hypotheses are consistent support the notion that IL-6 cytokines are modulators of with effects of gp130 receptor signaling on a-cell func- a-cell glucagon secretion. Taking these findings into con- tion and glycemic control being more pronounced in the sideration, we propose that gp130 receptor actions are STZ/HFD model, compared with STZ or HFD treatments context-dependent and that IL-6 cytokines modulate alone. Thus gp130 receptor signaling acts as a modifier of a-cell glucagon secretion to allow for increased glucose a-cell function in a synergistic manner with HFD-induced output during normal physiology (e.g., during acute in- effects on a-cell function. fection or postexercise), while also driving excessive Improved a-cell function in agp130KO mice treated a-cell glucagon secretion and dysfunction during type 2 with STZ/HFD resulted in reduced fasting hyperglycemia, diabetes. likely due to reduced hepatic glucose output. Reduced Exposure of human islets to IL-6 for 4 days or enriched glycemia was likely responsible for the decreased fasting human pancreatic a-cells to IL-6 enhances GLP-1 secre- insulin observed in agp130KO mice. We ruled out any tion in addition to glucagon secretion, perhaps via upreg- contribution of increased insulin sensitivity in agp130KO ulation of pro(hormone)-convertase 1/3 (22). Mouse mice treated with STZ/HFD by performing hyperinsulinemic- islets exposed to IL-6 for 2 days did not have elevated euglycemic clamps. However, reduced fasting glycemia may GLP-1 secretion. Dedifferentiation of a-cells to a pre- have improved b-cell function in agp130KO mice, likely a-cell state has been shown to induce GLP-1 secretion by reducing the effects of glucotoxicity on the b-cell. This (48). In addition, prolonged culture of human islets in 2994 a-Cell gp130 Receptor Signaling in Diabetes Diabetes Volume 63, September 2014 vitro results in increased numbers of glucagon-positive 4. Baron AD, Schaeffer L, Shragg P, Kolterman OG. Role of hyperglucagonemia cells due to conversion of b-cells to a-cells (49). 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Saturated fatty acid and TLR signaling link b cell dysfunction and islet inflammation. Cell Metab 2012;15:518– 533 Acknowledgments. The authors thank G. Soukhatcheva, M. Komba, and 14. Jones HB, Nugent D, Jenkins R. Variation in characteristics of islets of L. Xu (CFRI) for technical assistance provided by the CFRI islet isolation core Langerhans in insulin-resistant, diabetic and non-diabetic-rat strains. Int J Exp facility and CFRI flow cytometry core and Bruce Verchere (University of British Pathol 2010;91:288–301 Columbia) for critical review of the manuscript. 15. Masters SL, Dunne A, Subramanian SL, et al. Activation of the NLRP3 in- Funding. This work was supported by funding from the CFRI (J.A.E.), the flammasome by islet amyloid polypeptide provides a mechanism for enhanced University of British Columbia (J.A.E.), the Canadian Institutes of Health Research IL-1 in type 2 diabetes. Nat Immunol 2010;11:897–904 (PCN-110793 and PNI-120292 to J.A.E.), the Canadian Diabetes Association b (OG-3-13-4097-JE), the Deutsche Forschungsgemeinschaft (Bonn, Germany; 16. Youm YH, Adijiang A, Vandanmagsar B, Burk D, Ravussin A, Dixit VD. fl SFB877; project A1 to S.R.-J.), and the Cluster of Excellence “Inflammation at Elimination of the NLRP3-ASC in ammasome protects against chronic obesity- – Interfaces” (to S.R.-J.). S.Z.C. is supported by a Canadian Institutes of Health induced pancreatic damage. Endocrinology 2011;152:4039 4045 Research Transplantation Training Program Scholarship. H.E. is supported by the 17. Chentouf M, Dubois G, Jahannaut C, et al. Excessive food intake, obesity fl Danish Council for Independent Research and the Novo Nordisk Foundation. D.N. and in ammation process in Zucker fa/fa rat pancreatic islets. PLoS ONE 2011;6: is supported by a University of British Columbia Transplantation Training Program e22954 fl and Vanier Canada Graduate Scholarship. 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