Activation of Transmembrane Bile Acid Receptor TGR5 Modulates Pancreatic Islet α Cells to Promote Glucose Homeostasis

Abstract

The physiological role of the TGR5 receptor in the pancreas is not fully understood. We previously showed that activation of TGR5 in pancreatic β cells by bile acids induces insulin secretion. Glucagon released from pancreatic α cells and glucagon-like peptide 1 (GLP-1) released from intestinal L cells regulate insulin secretion. Both glucagon and GLP-1 are derived from alternate splicing of a common precursor, proglucagon by PC2 and PC1, respectively. We investigated whether TGR5 activation in pancreatic α cells enhances hyperglycemia-induced PC1 expression thereby releasing GLP-1, which in turn increases β cell mass and function in a paracrine manner. TGR5 activation augmented a hyperglycemia-induced switch from glucagon to GLP-1 synthesis in human and mouse islet α cells by GS/cAMP/PKA/cAMP-response element-binding protein-dependent activation of PC1. Furthermore, TGR5-induced GLP-1 release from α cells was via an Epac-mediated PKA-independent mechanism. Administration of the TGR5 agonist, INT-777, to db/db mice attenuated the increase in body weight and improved glucose tolerance and insulin sensitivity. INT-777 augmented PC1 expression in α cells and stimulated GLP-1 release from islets of db/db mice compared with control. INT-777 also increased pancreatic β cell proliferation and insulin synthesis. The effect of TGR5-mediated GLP-1 from α cells on insulin release from islets could be blocked by GLP-1 receptor antagonist. These results suggest that TGR5 activation mediates cross-talk between α and β cells by switching from glucagon to GLP-1 to restore β cell mass and function under hyperglycemic conditions. Thus, INT-777-mediated TGR5 activation could be leveraged as a novel way to treat type 2 diabetes mellitus.

Keywords: G protein-coupled receptor; bile acid; cell signaling; metabolic syndrome; pancreatic islet; reprogramming.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.

INT-777 up-regulates PC1 expression in human and mouse islets and αTC1-6 cells under hyperglycemic conditions. Expression of PC1 mRNA after 48 h of incubation with or without INT-777 (25 μm) in αTC1-6 cells (A), control mouse islets (B), and human islets (C) cultured under low (5 mm) or high (25 mm) glucose (G) conditions for 7 days. D, PC1 mRNA expression in db/db mouse islets treated with or without INT-777 (25 μm) for 48 h. Whole cell lysates were prepared, and immunoblot analysis was performed for PC1 in αTC1-6 cells (E) and db/db mouse islets (F) treated with or without INT-777. Bar graphs show the densitometric values calculated after normalization to loading control (β-actin). Data are expressed as the mean ± S.E. of three or four experiments. ##, p < 0.001, compared with 5 mm glucose basal or control basal; *, p < 0.05, or **, p < 0.001, compared with 25 mm glucose basal or db/db basal.

FIGURE 2.

Effect of INT-777 on PC2 expression. PC2 mRNA expression in αTC1-6 cells (A), control mouse islets (B), and human islets (C) cultured for 7 days under low (5 mm) or high (25 mm) glucose (G) conditions in the presence or absence of INT-777 (25 μm) for 48 h. D, PC2 mRNA expression in db/db mouse islets treated with or without INT-777 (25 μm) for 48 h. Data are expressed as the mean ± S.E. of three experiments.

FIGURE 3.

Effect of hyperglycemia and INT-777 on TGR5 expression. TGR5 protein (top panel) and mRNA expression in αTC1-6 cells (A), control mouse islets (B), and MIN6 cells (C) cultured for 7 days under low (5 mm) or high (25 mm) glucose (G) conditions in the presence or absence of INT-777 (25 μm) for 48 h. D, TGR5 protein (top panel) and mRNA expression in db/db mouse islets treated with or without INT-777 (25 μm) for 48 h. Data are expressed as the mean ± S.E. of three experiments.

FIGURE 4.

Signaling mechanism involved in INT-777-induced increased PC1 expression in α cells.A, PC1 promoter activity in αTC1-6 cells treated with or without INT-777 in the presence (+) of Gαs inhibitor (NF449, 10 μm) or PKA inhibitor (Myr-PKI, 1 μm). Immunoblot analysis was performed for phosphorylated and total CREB (pCREB and CREB) in αTC1-6 cells (B) and db/db mouse islets (C) treated with or without INT-777. Bar graphs show the densitometric values calculated after normalization to CREB. G, glucose. Data are expressed as the mean ± S.E. of three or four experiments. *, p < 0.05, compared with 5 mm glucose basal; **, p < 0.001, compared with 25 mm glucose basal.

FIGURE 5.

INT-777 increases GLP-1 release in human and mouse islets and αTC1-6 cells under hyperglycemic conditions via Epac in a PKA-independent mechanism. GLP-1 release after 24 h of incubation with or without INT-777 (25 μm) or LCA (25 μm) in αTC1-6 cells (A), control mouse islets (B), and human islets (C) cultured under low (5 mm) or high (25 mm) glucose (G) condition for 7 days. D, GLP-1 release from db/db mouse islets treated with or without INT-777 (25 μm) for 24 h. Data are expressed as the mean ± S.E. of three experiments. ##, p < 0.001 compared with 5 mm glucose basal or control basal; *, p < 0.05, or **, p < 0.001, compared with 25 mm glucose basal or db/db basal. E, PI hydrolysis measured by ion exchange chromatography in αTC1-6 cells labeled with myo-[3H]inositol for 24 h and treated with INT-777(25 μm) or Epac ligand (10 μm) with or without inhibitors of Gαs (NF449,10 μm), PI hydrolysis (U73122, 10 μm), or Epac2 (ESI-05, 10 μm). Data are expressed as mean ± S.E. of three experiments. *, p < 0.05, or **, p < 0.001, significant inhibition in PI hydrolysis compared with INT-777 or Epac ligand. F, signaling pathway involved in TGR5-mediated up-regulation of PC1 expression and GLP-1 release from αTC1-6 cells under hyperglycemia.

FIGURE 6.

A, INT-777 induces GLP-1 release in α cells via Epac. GLP-1 release from αTC1-6 cells cultured under low (5 mm) or high (25 mm) glucose conditions for 7 days treated with INT-777 (25 μm) or Epac ligand (10 μm) with or without inhibitors of Gαs (NF449, 10 μm), PI hydrolysis (U73122, 10 μm), or Epac2 (ESI-05, 10 μm). Data are expressed as mean ± S.E. of four experiments. *, p < 0.05, or **, p < 0.001, compared with 25 mm glucose basal. B, effect of exendin(9–39) (Ex(9–39)) on insulin secretion in human islets. Human islets were cultured in high (25 mm) glucose for 7 days. Glucose-stimulated insulin secretion after 1 h of incubation with or without INT-777 in the presence or absence of GLP-1 receptor antagonist, exendin(9–39) (0.25 μm). Data are expressed as mean ± S.E. of three experiments. **, p < 0.001 compared with 25 mm glucose.

FIGURE 7.

INT-777 improves insulin sensitivity and glucose tolerance in db/db mice. Wild type and db/db mice were treated with vehicle (Veh) (DMSO) or INT-777 (30 mg/kg/day) intraperitoneally for 7 weeks (n = 10 per group). Body weights (A), fasting blood glucose concentration (B), fasting plasma insulin (C), and HOMA-IR values (D) were calculated based on the following formula: (fasting insulin (milliunits/liter) × fasting glucose (mmol/liter))/22.5. E, blood glucose concentration (mg/dl) after intraperitoneal injection of 1 g/kg glucose. F, blood glucose concentration (mg/dl) after administration of 0.75 units/kg insulin. Inset depicts the area under curve (AUC) with or without treatment of INT-777 in db/db mice. Data are expressed as the mean ± S.E. of three experiments. #, p < 0.05, or ##, p < 0.001, compared with wild type mice; *, p < 0.05, or **, p < 0.001, compared with db/db mice. HOMA-IR, Homeostatic Model Assessment for Insulin Resistance. AU, arbitrary units.

FIGURE 8.

Effect of INT-777 on plasma glucagon in db/db mice. Fasting plasma glucagon in db/db mice treated with vehicle (DMSO) or INT-777 intraperitoneally for 7 weeks. Data are expressed as the mean ± S.E. (n = 5 mice per group).

FIGURE 9.

INT-777 up-regulates PC1 expression and GLP-1 production in db/db mice. Wild type (Wt) and db/db mice were treated with vehicle (Veh) (DMSO) or INT-777 (30 mg/kg/day) intraperitoneally for 7 weeks (n = 10 per group). A, representative immunohistochemistry images (n = 5) of pancreatic tissue sections using antibodies against glucagon (red) and PC1 (green). Arrows indicate glucagon/PC1 colocalization. Scale bars, 100 μm. B, PC1/glucagon colocalization expressed as Pearson's coefficient of colocalization. Expression of PC1 mRNA (C), GLP-1 release over a 24-h incubation (D), and TGR5 protein (top panel) and mRNA expression (E) in pancreatic islets from wild type and db/db mice after treatment with or without INT-777 for 7 weeks. Data are expressed as the mean ± S.E. of three experiments. ##, p < 0.001, compared with wild type mice; *, p < 0.05, or **, p < 0.001, compared with db/db mice.

FIGURE 10.

INT-777 increases pancreatic β cell area in db/db mice.A, representative immunohistochemistry images (n = 5) of pancreatic tissue sections using antibodies against insulin (green) and glucagon (red). Effect of INT-777 on β cell area, expressed as % of pancreas area (B), pancreas area, expressed as μm2 (C), α cell area, expressed as % of pancreas (D), and β cell area per islet, expressed as μm2 (E) in wild type (Wt) and db/db mice with or without INT-777 treatment for 7 weeks. Data are expressed as the mean ± S.E. for 5 mice per group; *, p < 0.05, or **, p < 0.001, compared with db/db mice.

FIGURE 11.

INT-777 increases pancreatic β cell proliferation in db/db mice.A, immunohistochemistry of pancreatic tissue sections using antibodies against Ki67 (green) and insulin (red). Representative image of n = 5; arrows indicate Ki67-positive cells. Scale bars, 100 μm. B, effect of INT-777 on β cell proliferation, expressed as % of β cells analyzed. C, INT-777 increases insulin staining intensity of β cells in db/db mice. Representative immunohistochemistry images of pancreatic tissue sections using antibody against insulin (green) in db/db mice treated with vehicle (DMSO) or INT-777 (30 mg/kg/d) intraperitoneally for 7 weeks and quantitation of the intensity of insulin staining. Data are expressed as mean fluorescence intensity (n = 6). *, p < 0.05, compared with db/db mice. Scale bars, 100 μm.

FIGURE 12.

A, INT-777 does not affect MIN6 cell proliferation. MTT assay for cell proliferation of MIN6 cells with or without INT-777 (10, 25, and 50 μm) for 24, 48, or 72 h. Data are expressed as the mean ± S.E. of four experiments. C, untreated. B, mechanism of TGR5-mediated regulation of pancreatic α cells to promote glucose homeostasis. Under hyperglycemia or diabetes, activation of TGR5 receptors on α cells augments glucose-induced PC1 expression leading to GLP-1 secretion that mediates trophic effects on β cells. This results in increased insulin secretion, β cell proliferation, and increased insulin biosynthesis providing the evidence of improvement in glucose homeostasis in db/db mice treated with INT-777, a TGR5-specific agonist.