Interleukin-6 regulates pancreatic alpha-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 beta-cell function and mass, an increased proportion of alpha-cells relative to beta-cells, and alpha-cell dysfunction. Here we show that the alpha 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 alpha-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 alpha-cell proliferation, prevents apoptosis due to metabolic stress, and regulates alpha-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 alpha-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 alpha-cell mass display decreased fasting glucagon levels. However, despite these alpha-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 alpha-cell mass in response to high-fat diet feeding, and we suggest that this expansion may be needed for functional beta-cell compensation to increased metabolic demand.

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 RT-PCR on RNA from FACS sorted rat α-cells and β-cells (purity ≈90% as assessed by insulin and glucagon staining respectively) normalized for 18S (n = 3). (E) Western blot analysis of the IL-6R in HeLa cell extracts (+ control), purified rat α-cells and β-cells, and whole mouse islets (representative of n = 3). (F) Western blot of pSTAT3 and total STAT3 in mouse and human islets after 15 min. exposure to 100 ng/ml IL-6 (representative of n = 3). *, P < 0.05.

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 in culture medium of human islets after exposure to 200 ng/ml IL-6. (E) Glucagon secretion from human islets during 1 h static incubation in the presence of 20 mM glucose (white bars), 2 mM glucose (hatched bars), and 10 mM Arginine (black bars). Islets were pretreated with 200 ng/ml IL-6 for the indicated times (n = 4). (F) Circulating glucagon levels 2 h after 100 ng bolus IL-6 injection in mice during fed and fasted state (n = 3–5). All secretion experiments were performed on 20 islets per well in triplicate with the number of independent experiments indicated above. *, P < 0.05 vs. respective controls.

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). (B) Ki67-positive human islet-cells per islet after 4 days treatment with the IL-6R antagonist, Sant7 (200 ng/ml; n = 3). (C and D) Percent BrdU-positive mouse α- and β-cells (glucagon and insulin positive, respectively) of total number of cells. Cells were treated in the absence (Ctrl) and presence of 100 ng/ml IL-6 for 24 and 96 h with BrdU present during the entire experiment (n = 3). (E and F) Percent TUNEL-positive mouse α-cells and β-cells after 12 h treatment with 33.3 mM glucose and 0.5 mM palmitate (gluc + palm), in the absence (Ctrl) and presence of 100 ng/ml IL-6 (n = 3). (G) Representative image of mouse islets on extracellular matrix coated dishes stained for BrdU after 4 days in the absence (control) and presence of 100 ng/ml IL-6, with BrdU present during the entire experiment. *, P < 0.05.

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 18 weeks (n = 8 WT, n = 9 IL-6 KO). (B) Fasting blood glucose, (C) fasting plasma glucagon, (D) fasting plasma insulin, (E) glucagon during ipGTT, (F) fed blood glucose, (J) HOMA-IR, (K) a-cell mass, and (L) b-cell mass in WT (white bars) and IL-6 KO (black bars) mice after 18 weeks on HF diet (chow WT n = 5, chow IL-6 KO n = 8, HF WT n = 8, HF IL-6 KO n = 9) *, P < 0.05.