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Proc Natl Acad Sci U S A. Sep 2, 2008; 105(35): 13163–13168.
Published online Aug 21, 2008. doi:  10.1073/pnas.0801059105
PMCID: PMC2529061
Physiology

Interleukin-6 regulates pancreatic α-cell mass expansion

Abstract

Interleukin-6 (IL-6) is systemically elevated in obesity and is a predictive factor to develop type 2 diabetes. Pancreatic islet pathology in type 2 diabetes is characterized by reduced β-cell function and mass, an increased proportion of α-cells relative to β-cells, and α-cell dysfunction. Here we show that the α cell is a primary target of IL-6 actions. Beginning with investigating the tissue-specific expression pattern of the IL-6 receptor (IL-6R) in both mice and rats, we find the highest expression of the IL-6R in the endocrine pancreas, with highest expression on the α-cell. The islet IL-6R is functional, and IL-6 acutely regulates both pro-glucagon mRNA and glucagon secretion in mouse and human islets, with no acute effect on insulin secretion. Furthermore, IL-6 stimulates α-cell proliferation, prevents apoptosis due to metabolic stress, and regulates α-cell mass in vivo. Using IL-6 KO mice fed a high-fat diet, we find that IL-6 is necessary for high-fat diet-induced increased α-cell mass, an effect that occurs early in response to diet change. Further, after high-fat diet feeding, IL-6 KO mice without expansion of α-cell mass display decreased fasting glucagon levels. However, despite these α-cell effects, high-fat feeding of IL-6 KO mice results in increased fed glycemia due to impaired insulin secretion, with unchanged insulin sensitivity and similar body weights. Thus, we conclude that IL-6 is necessary for the expansion of pancreatic α-cell mass in response to high-fat diet feeding, and we suggest that this expansion may be needed for functional β-cell compensation to increased metabolic demand.

Keywords: alpha-cell mass, beta-cell function, high-fat diet, pancreatic islet

Type 2 diabetes is a metabolic disorder characterized by hyperglycemia, due to insulin resistance and pancreatic islet dysfunction. Most research on the pathology of the islet in type 2 diabetes has focused on the failure of the β-cell to secrete sufficient amounts of insulin in response to increased demand. Yet, the dysregulation of glucagon secretion, as proposed 30 years ago to contribute to hyperglycemia, and the disproportionately increased number of α-cells relative to β-cells in these individuals has been neglected (16).

Interleukin-6 is a pleiotropic cytokine that influences metabolic regulation during both normal physiology and disease (7). Plasma IL-6 levels are acutely elevated following muscle contraction and chronically during obesity (8, 9). With respect to its actions in regulating whole body metabolism, IL-6 is involved in the central control of obesity, in the regulation of insulin action, and in the mobilization of energy stores during exercise (10, 11). In obesity, systemically elevated IL-6 levels are a predictive factor for the development of type 2 diabetes (9, 12). Finally, IL-6 is also a potent regulator of cellular proliferation and survival, as most clearly demonstrated in liver and immune cells (7).

Upon IL-6 binding to its receptor, the IL-6R forms a complex with the signal transducing transmembrane glycoprotein, gp130, and signals are transduced via activation of STAT3 and ERK signaling, regulating downstream targets such as c-myc and bcl-2 (7, 13, 14). There exist soluble and transmembrane forms of both the IL-6R and gp130. The soluble IL-6R acts agonistically by binding transmembrane gp130, whereas the soluble gp130 receptor acts as an antagonist. Since gp130 is ubiquitously expressed, specificity of IL-6R signaling is mainly mediated by tissue specific IL-6R expression, and/or the local action of soluble IL-6Rs (7).

The role of IL-6 in the pancreatic islet is unclear. We have recently shown in a clinical study that blockade of IL-1 improved glycemia by improving pancreatic β-cell insulin secretion, while concomitantly strongly reducing circulating IL-6 levels (15). Given that elevated IL-6 levels are an independent predictor of type 2 diabetes (9, 12), we evaluated the role of IL-6 in the regulation of the pancreatic endocrine islet. Herein, we identify the pancreatic α-cell as a primary target of IL-6 actions. The pancreatic α-cell expresses a high level of IL-6R compared with other rodent tissues. Further, IL-6 promotes α-cell specific effects, including increasing glucagon expression and secretion, increasing α-cell proliferation, and preventing against metabolic stress induced α-cell apoptosis, in vitro. In vivo, high-fat (HF) diet feeding increases systemic IL-6 levels, which are necessary for expansion of α-cell mass and maintenance of fasting circulating glucagon. In the absence of IL-6, and without expansion of α-cell mass, IL-6 KO mice display glucose intolerance after long-term HF feeding, due to incomplete functional β-cell compensation.

Results

The Pancreatic Islet, Specifically the α-Cell, Expresses High Levels of the IL-6 Receptor.

We initially conducted a tissue expression profile of the IL-6R and its signal transducing protein, gp130 in rodents: both mouse and rat. In mice, IL-6R mRNA was highly expressed in the pancreatic islet compared with other tissues (Fig. 1A). The ubiquitously expressed glycoprotein, gp130 (which is not specific for IL-6R signaling), was also detected in the pancreatic islet [supporting information (SI) Fig. S1]. PCR on isolated mouse and human islets confirmed IL-6R and gp130 expression (Fig. S1). This high islet cell expression of the IL-6R was confirmed by comparison of various rat tissues (Fig. 1B). Furthermore, rat α-cells showed a higher level of IL-6R transcript compared with β-cells, or the rat INS-1 β-cell line (Fig. 1B). Comparison of FACS-sorted rat α- and β-cells (purity both ≈90%, based on insulin and glucagon staining) supported the gene array data insomuch as a higher level of IL-6R transcript was found in the α-cell enriched fraction (Fig. 1C). Comparison of IL-6R mRNA to another α- and β-cell housekeeping gene, ACADM (medium-chain acyl-coA dehydrogease) (16), reveals that its mRNA expression is 80% of that of ACADM mRNA in α-cells and 60% in β-cells, supporting the above. IL-6R mRNA levels in α-cells were 21%, 157%, 41%, and 69% and compared with mRNA for the GIP (glucose-dependent insulinotropic polypeptide) receptor (17), prolactin receptor (18), neuroserpin, and cholecystokinin A receptor (19) respectively (n = 2), confirming the relative abundant expression of the IL-6R in islet α cells. In β-cells, IL-6R mRNA levels were 6%, 7%, 12%, and 3% compared with these same receptors respectively (n = 2). Gp130 mRNA expression was not specifically elevated in rat α-cells (Fig. 1D). On the protein level, we detected the IL-6R in whole mouse islets, purified α-cells, and purified β-cells (Fig. 1E). Immunostaining of mouse pancreatic tissue sections and isolated islets localized the IL-6R within the pancreatic islet to the α-cell (Fig. S1). Specificity of the antibody was confirmed by isotype controls, and absorption tests for both immunohistochemistry (Fig. S1) and Western blot analysis (data not shown). Cytoplasmic staining of the IL-6R is a common finding in various tissues as the IL-6R exists in a membrane and soluble form (2022). Finally, activation of the IL-6R was coupled to increased STAT3 phosphorylation confirming functional expression of this receptor on mouse and human islets (Fig. 1F). Thus, based on gene array data, PCR of FACS-sorted α-cells, protein expression, and immunohistochemistry, we have localized IL-6R expression in the pancreatic islet to the pancreatic α-cell, however we cannot exclude the fact that the β-cell might also express the IL-6R.

Fig. 1.
IL-6R is expressed in the pancreatic α-cell and is functionally coupled to STAT3 phosphorylation. (A and B) Tissue expression profile of mouse and rat IL-6R mRNA expression determined by Affymetrix gene array (n = 3–5). (C and D) Quantitative ...

Interleukin-6 Regulates Pro-Glucagon mRNA and α-Cell Function.

We went on to investigate the effects of IL-6R stimulation on the pancreatic α-cell. Given that islets secrete IL-6 in the ng/ml range (23), we treated islets with 1–200 ng/ml exogenous IL-6. Already after 4 h, IL-6 stimulation of human islets increased pro-glucagon mRNA expression (Fig. 2A) with no effect on insulin mRNA (Fig. 2C). Both isolated mouse (Fig. S2) and human islets incubated with IL-6 displayed increased glucagon release in a time-dependent manner (Fig. 2B and normalized data in Fig. S2). The dose dependence of these IL-6 effects on human pro-glucagon mRNA and glucagon release are shown in Fig. S2. No effect of IL-6 was observed on either acute (Fig. S3) or chronic insulin release (Fig. 2D), indicating an α-cell specific effect. The specificity of these IL-6 effects were demonstrated by blocking IL-6-induced glucagon release from human islets with the IL-6R antagonist, Sant7 (Control: 15.9 ± 3.6, IL-6: 36.93 ± 5.9, IL-6 + Sant 7: 15.8 ± 1.2 glucagon secretion as percentage of content (n = 1 in quintuplicate)). Furthermore an additional IL-6 family member, oncostatin-M (OSM), did not stimulate glucagon release from human islets (Control: 14.4 ± 1.5, 200 ng/ml OSM: 10.8 ± 4.4 glucagon secretion as percentage of content (n = 2).

Fig. 2.
Interleukin-6 regulates pro-glucagon mRNA and glucagon secretion with no effect on insulin mRNA and release. (A and C) Pro-glucagon and insulin mRNA in human islets after exposure to 200 ng/ml IL-6 (n = 3–4). (B and D) Glucagon and insulin release ...

The effect of IL-6 on α-cell secretory function was further tested by preincubation of human islets with IL-6 followed by a 1-h static incubation in either high glucose (20 mM; unstimulated) or low glucose (2 mM; stimulated), or in the presence of 10 mM arginine (Fig. 2E). IL-6 preincubation for 4 and 24 h resulted in increased glucagon secretion under both high and low glucose conditions, however, there was no significant effect on arginine-stimulated glucagon secretion. Furthermore, there was no effect of IL-6 on human islet glucagon content (Fig. S2).

In contrast to these α-cell effects, IL-6 does not directly stimulate insulin secretion over 2–96 h in human islets (Fig. 2D), or acutely in human and mouse islets in the presence of 7.5 mM glucose (Fig. S3). Further, no effects on β-cell function, as assessed by glucose-stimulated insulin secretion, were induced by IL-6 treatment up to 48 h (Fig. S3). However, 4-day treatment of human and mouse islets with elevated IL-6 does impair glucose-stimulated insulin secretion, an effect that is reversed by Sant 7 (Fig. S3). Finally, in 10 independent human islet preparations we found a negative correlation between glucose-stimulated insulin secretion and the amount of IL-6 released into the culture media (Fig. S3).

Interestingly, comparison of IL-6R mRNA expression in isolated islets from fed versus 18 h fasted mice indicated a consistent up-regulation of the receptor during fasting: fed islets 0.025 ± 0.001 vs. fasted islets 0.037 ± 0.005 IL-6R mRNA/β-actin (n = 4, P < 0.05). Therefore, we administered a bolus injection of IL-6 (100 ng) into conscious mice and monitored circulating glucagon levels over time. Injection of IL-6 increased glucagon levels after 2 h only during fasting conditions, with no effect during the fed state (Fig. 2F). Thus, in summary IL-6 influences α-cell function by regulating both pro-glucagon mRNA and glucagon secretion. The latter effects are most prominent in the presence of low glucose conditions.

Interleukin-6 Increases α-Cell and β-Cell Proliferation, and Has Distinct Effects on α-Cell and β-Cell Apoptosis.

Interleukin-6 regulates proliferation and/or apoptosis of various cell types, including immune cells, neuronal cells, blood cells, hepatocytes, and carcinomas (7). We investigated human islet cell proliferation using both BrdU incorporation (data not shown) and Ki67 antigen labeling. The same doses of IL-6 that regulated glucagon secretion strongly induced islet-cell proliferation in human islets (Fig. 3A). Further, locally produced IL-6 (inhibited using the receptor antagonist, Sant7) also contributed to basal human islet cell proliferation (Fig. 3B).

Fig. 3.
Interleukin-6 increases pancreatic α-cell proliferation and prevents α-cell apoptosis in vitro. (A) Ki67-positive human islet-cells per islet after 4 days' treatment in the absence (Ctrl) and presence of 200 ng/ml IL-6 (n = 3–5). ...

Given the α-cell expression of the IL-6R and our α-cell specific effects, we investigated the kinetics of α- and β-cell proliferation in mouse islet cells. Indeed, IL-6 induced α-cell proliferation already after 24 h (Fig. 3C). In contrast, IL-6-induced β-cell proliferation was not detected after 24 h, however, was evident after 96 h (Fig. 3D). The identical experiment was conducted on purified rat α- and β-cells. Under these conditions, IL-6 increased both α- and β-cell proliferation after 48 h (Fig. S4). Thus, in whole mouse islets, IL-6 stimulated islet-cell proliferation after 4 days (Fig. 3G) is likely due to a combination of both α- and β-cell proliferation.

To examine whether IL-6 regulates apoptosis in the presence of a type 2 diabetic milieu, mouse single islet cells were treated for 12 h with elevated glucose and palmitate in combination, in the presence and absence of IL-6. The presence of IL-6 almost completely protected α-cells from glucolipotoxicity-induced apoptosis (Fig. 3E), whereas β-cell apoptosis was exaggerated in the presence of IL-6 (Fig. 3F). Thus, IL-6 exerts distinct effects on α-cell and β-cell apoptosis.

To gain insight into these proliferative and anti-apoptotic mechanisms, islets were incubated with IL-6 for 12 and 24 h. After 12 h of IL-6 treatment, expression levels of c-myc and bcl-2 were increased, while the cell cycle inhibitor p27 was decreased. No differences in D cyclins were detected on an mRNA level due to IL-6 treatment (Fig. S5).

Interleukin-6 Is Necessary for Pancreatic α-Cell Mass Expansion in Response to HF Diet Feeding.

We went on to investigate IL-6 regulation of islet-cell mass in vivo. IL-6 KO mice are known to become obese and glucose intolerant after 6–9 months of age due to lack of IL-6 central nervous system effects (10). Thus, we investigated mice younger than 22 weeks of age. At 10–12 weeks of age, IL-6 KO mice and WT mice displayed no differences in glucose tolerance, insulin sensitivity, or islet morphology (Fig. S6). Furthermore, IL-6 KO islets displayed normal insulin secretion in response to glucose and showed normal proliferative responses to IL-6 in vitro (Fig. S6). Thus, IL-6 does not appear to be necessary for normal α-cell or β-cell development and function.

It is known that obesity increases systemic IL-6 levels (9). Therefore, we investigated the ability of IL-6 KO mice to expand α-cell mass in vivo, by placing WT and IL-6 KO mice on a HF diet for 8 weeks.

Similar to obesity and type 2 diabetes pathology in humans (9, 12), HF diet increased circulating IL-6; in WT mice IL-6 was increased from 3.0 ± 0.9 pg/ml in chow controls to 9.1 ± 2.5 pg/ml (P < 0.05, n = 5) in HF diet fed mice, with no detectable IL-6 in IL-6 KO mice. Body weight increased significantly due to the HF diet, with no significant difference between genotypes (WT: 27.7 ± 0.1 and 31.8 ± 0.7 g, IL-6 KO: 28.2 ± 0.9 and 30.6 ± 0.6 g, chow and HF diet respectively) (Fig. S7). There were no differences between genotypes with respect to fasting glycemia, fasting glucagon, or insulin levels (Fig. S7). Further, there were no differences in fed blood glucose, or glucose and insulin tolerance between genotypes (Fig. S7). However, HF diet did induce glucose intolerance and mild insulin resistance compared with normal chow in both genotypes (data not shown).

Assessment of pancreatic α- and β-cell mass indicated a dramatic increase in α-cell mass in WT mice on HF diet compared with chow fed animals (Fig. S7). This effect was IL-6-dependent, as it was not present in IL-6 KO mice on HF diet. There were no differences between genotypes on chow diet (Fig. S7). In contrast, at this time point β-cell mass showed no differences due to HF diet in WT mice (Fig. S7). There were also no differences in β-cell mass between genotypes. The increase in α-cell mass due to HF diet was due to α-cell hyperplasia within individual islets, yielding a greater percent α-cell area/section, as islet density and pancreas mass were unchanged (Fig. S8). Thus, these data support the above in vitro data with respect to an overall positive effect of IL-6 on α-cell fate, and demonstrate that IL-6/IL-6R signaling is necessary for the HF diet-induced increase in α-cell mass.

To investigate if a lack of IL-6 during long-term HF diet feeding has more dramatic effects on glucose homeostasis, WT and IL-6 KO mice were placed on HF diet for 18 weeks. At 22 weeks, body weight was similar between genotypes in HF groups (Fig. 4A), and systemic IL-6 was significantly elevated only in WT mice (19.0 ± 6.8 pg/ml; n = 9 vs. 99.9 ± 32.4 pg/ml, in chow versus HF respectively, n = 8, P < 0.05). While HF diet increased fasting glycemia, there were no differences between genotypes (Fig. 4B). However, IL-6 KO mice had decreased systemic fasting glucagon and insulin levels in response to HF diet feeding (Fig. 4 C and D). Further, high glucose suppression of glucagon secretion was unchanged between genotypes, despite being impaired relative to chow controls (Fig. 4E).

Fig. 4.
Impaired glucose tolerance in IL-6 KO mice after 18 weeks on HF diet. (A) Body weight, (G) ipGTT, (H) glucose-stimulated insulin secretion, and (I) ipITT in WT (solid line, open squares) and IL-6 KO (dashed line, closed circles) mice fed an HF diet for ...

Despite reduced fasting glucagon levels, fed blood glucose was paradoxically increased only in IL-6 KO mice on HF diet (Fig. 4F), suggestive of islet dysfunction. In support of this, when assessing glucose tolerance, IL-6 KO mice were unable to clear blood glucose as rapidly as WT mice (Fig. 4G), while chow fed mice showed no differences between genotypes (data not shown). This was paralleled by significantly reduced insulin secretion in HF diet fed IL-6 KO mice during a glucose tolerance test (Fig. 4H). Further, insulin sensitivity was unchanged or enhanced in IL-6 KO HF diet fed mice, as shown by an insulin tolerance test (ITT) or calculated by HOMA-IR (Homeostasis model for assessment of insulin resistance), supporting the notion of a β-cell defect (Fig. 4 I and J).

Finally, islet morphologic assessment showed no α-cell mass expansion in IL-6 KO mice (Fig. 4K), whereas β-cell mass indicated no differences between genotypes (Fig. 4L). As after 8 weeks, the increase in α-cell mass due to HF diet after 18 weeks was due to α-cell hyperplasia within individual islets, yielding a greater percent α-cell area/section, as islet density and pancreas mass were unchanged (Fig. S9).

Therefore, long-term HF diet feeding of IL-6 KO mice leads to no increase in α-cell mass and reduced fasting glucagon levels relative to controls. Paradoxically, the dominant phenotype of the IL-6 KO mice on HF diet with respect to glucose homeostasis is a β-cell defect, resulting in reduced insulin secretion.

Discussion

In the present study, we have examined the regulation of the pancreatic islet by IL-6 and identified IL-6 as a regulator of the pancreatic α-cell. The pancreatic islet, specifically the α-cell, expresses a high amount of IL-6R message compared with other rodent tissues. In support of α-cell specific IL-6 effects, IL-6 regulates α-cell pro-glucagon production and glucagon secretion, increases α-cell proliferation, and inhibits α-cell apoptosis induced by elevated glucose and the free fatty acid palmitate in vitro. Further, in vivo data support the concept that elevated systemic IL-6 levels regulate glucagon secretion and α-cell mass. Remarkably, IL-6 enhanced β-cell apoptosis in the presence of elevated glucose and palmitate, while effects on β-cell proliferation were secondary to α-cell effects kinetically in vitro. Thus, IL-6 is a positive regulator of α-cell glucagon secretion and α-cell fate, and displays distinct effects on the α- versus β-cell fate.

Interestingly, α-cell development in IL-6 KO mice is normal under chow fed conditions, and these mice do not show any metabolic disturbances with respect to glucose homeostasis at 10–12 weeks of age. This is supported by previously published data (24). Further, islet insulin secretion and architecture in chow fed IL-6 KO mice was identical to WT animals, and fasting glucagon levels were identical in WT and KO mice at 16 weeks and 22 weeks of age. High glucose suppression of glucagon secretion was also normal in 22-week-old chow fed WT and IL-6 KO animals. Thus, it appears that IL-6 signaling is not necessary for normal α-cell development, but for adaptation under specific metabolic stress conditions.

To investigate islet IL-6 effects in the context of metabolic stress in vivo, short term and long term HF diet feeding of WT and IL-6 KO mice was performed. The influence of elevated IL-6 levels on pancreatic α-cell fate was already apparent after 8 weeks of HF feeding. In contrast to chow fed animals displaying normal islet morphology, IL-6 KO mice on HF diet were unable to increase their α-cell mass in response to HF diet feeding, likely due to the absence of proliferative and/or protective effects of IL-6 on the α-cell. High-fat diet feeding increases circulating free fatty acids such as palmitate in addition to elevating systemic glucose. Thus, elevated IL-6 levels during HF diet feeding may normally stimulate α-cell proliferation and prevent glucolipotoxicity-induced α-cell apoptosis. Therefore, expansion of α-cell mass in response to HF diet feeding is IL-6-dependent in vivo, supporting the in vitro data and suggesting α-cell-specific IL-6 effects.

Also interesting to note is the appearance of an increased α-cell mass in response to HF feeding as an early morphologic event detectable before any change in β-cell mass, implicating it as an important adaptive process. After 18 week HF diet feeding IL-6 KO animals presented with decreased fasting glucagon levels, while exhibiting fed hyperglycemia and decreased insulin secretion in response to glucose compared with WT controls. One previous report of IL-6 KO mice on HF diet supports this observation (24). This is suggestive of β-cell failure, in the absence of differences in insulin resistance between genotypes. Further, there was no defect in β-cell mass in IL-6 KO mice, suggesting that α-cell mass expansion and glucagon may regulate β-cell secretory function. In vitro, we found that IL-6 increases pro-glucagon expression in addition to regulating α-cell fate. It is known that the pancreatic α-cell helps to maintain β-cell glucose competence via glucagon (25), and the glucagon receptor KO mice has impaired β-cell function (26). Recently, establishment of a β-cell overexpressing glucagon receptor transgenic mouse confirmed this paradigm, as these mice have improved glucose tolerance and increased insulin secretion in response to glucose (27). Thus, we suggest that β-cell glucose-competence is impaired in IL-6 KO animals due to reduced α-cell derived glucagon regulating the pancreatic β-cell. However, we cannot exclude the possibility that other α-cell derived factors could also be contributing to this lack of β-cell glucose competence.

Interleukin-6 exerts its effects on a number of metabolically active tissues, and the topic of IL-6 as a “good-guy” or “bad-guy” is a matter of ongoing debate (28, 29). Thus, with respect to its contribution to type 2 diabetes pathophysiology, the overall consequences of tissue-specific IL-6 effects need to be considered. Pancreatic islet pathology in type 2 diabetes is characterized not only by reduced β-cell function and mass, but also by a disproportionate number of α-cells. Further, in obesity and type 2 diabetes, plasma IL-6 levels are chronically elevated (9, 12) and reports suggest that elevated systemic IL-6 levels are a risk factor for type 2 diabetes development (9, 12). Our data suggest the potential relevance of these elevated IL-6 levels with respect to the pancreatic α-cell and its role in regulating β-cell function. We propose that elevated IL-6 levels during obesity drive α-cell mass expansion and glucagon expression, which may be required for functional β-cell compensation in response to HF diet induced insulin resistance. However, prolonged elevated IL-6 may lead to the observed pathologic glucagon secretion at onset and during progression of diabetes (30).

Materials and Methods

For detailed materials and methods, please refer to SI Materials and Methods.

Animals.

Male C57BL/6J mice (Harlan) and Wistar Kyoto rats (Janvier) were used for all rodent islet experiments. Only C57BL/6J wild-type and B6;129S2-Il6tm1Kopf/J (IL-6 KO) mice backcrossed for 11 generations and maintained on a C57BL/6J background were used (Jackson Laboratory). Guidelines for the use and care of laboratory animals at the University of Zurich were followed, and ethical approval was granted by the Zurich Cantonal Animal Experimentation Committee.

Islet Isolation, α- and β-Cell Isolation, and Cell Culture.

Human and rodent islets were cultured as described (31, 32). Islet α- and β-cells were purified from male Wistar rats as previously described (33, 34). Proliferation studies were performed in the presence of BrdU, IL-6, and the super antagonist 7 (Sant7, kindly provided by Sigma Tau) (35). Studies investigating apoptosis were performed on dispersed islet cells and apoptotic cells identified by TUNEL (Roche).

Gene Array.

Total RNA from mouse pancreatic islets, acini and whole tissues was extracted and mRNA quantification was performed using Affymetrix mouse 430 2.0 expression microarrays as described (36).

RNA Extraction, PCR and Quantitative RT-PCR.

Total human and rodent islet, and rat α- and β-cell RNA was extracted as described (32). Commercially available primers were used (Applied Biosystems). Conventional PCR primers are available upon request.

Histochemical Analysis.

Rabbit anti-IL-6R antibody (Santa Cruz), guinea-pig anti-glucagon antibody (Linco), and guinea-pig anti-insulin antibody (Dako) were used. For specific staining controls please refer to the online SI. For Western blot analysis proteins were electrically transferred to nitrocellulose filters and incubated with IL-6R (Santa Cruz), pSTAT3 and total STAT3 antibodies (Cell Signaling Technology). For proliferation studies, cells were stained with either a monoclonal Ki67 antibody (Zymed) or with a BrdU antibody (Roche). FACS sorted and dispersed cells were co-stained with a BrdU antibody (Roche), or a TUNEL kit (Roche), and either a rabbit anti-glucagon (Dako) or a guinea pig anti-insulin antibody (Dako).

Islet Morphometry.

Section area, and insulin and glucagon-positive cell area were determined from 3 pancreatic sections per animal, at 200-μm intervals, averaging 80–100 islets in total per animal using AxioVision (Zeiss) and Image J (National Institutes of Health).

Glucagon and Insulin Secretion.

Glucagon and insulin were assayed by RIA (Linco and CIS Biointernational). Glucose-stimulated insulin secretion was performed as described (23).

Intra-Peritoneal Glucose and Insulin Tolerance Test.

Glucose and insulin tolerance tests were performed as described (38). Homeostasis model for assessment of insulin resistance (HOMA-IR) was calculated as published (37).

Cytokines, Chemokines, and Hormones.

Circulating cytokines, chemokines, insulin, and glucagon were assayed using a mouse Luminex kit (Linco).

Statistics.

Data are expressed as means ± SEM. Significance was tested using Student's t test (2-tailed) and ANOVA with Bonferonni's post hoc test (P < 0.05) for multiple comparison analysis.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank A. Vervoort, N. Perriraz, S. Bencke, M. Borsig, I. Danneman, G. Seigfried-Kellenberger, and R. Prazak for technical assistance. This work was supported by grants from the University of Leuven, Swiss National Science Foundation, the European Foundation for the Study of Diabetes, the Juvenile Diabetes Research Foundation and the University Research Priority Program “Integrative Human Physiology” at the University of Zürich. J.A.E. is supported by a Juvenile Diabetes Research Foundation postdoctoral fellowship. Human islets were acquired via the European consortium for islet transplantation, supported by the Juvenile Diabetes Research Foundation.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0801059105/DCSupplemental.

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