Ectopic expression of GIP in pancreatic β-cells maintains enhanced insulin secretion in mice with complete absence of proglucagon-derived peptides

Abstract

Glucagon and glucagon-like peptide-1 (GLP-1) are produced in pancreatic α-cells and enteroendocrine L-cells, respectively, in a tissue-specific manner from the same precursor, proglucagon, that is encoded by glucagon gene (Gcg), and play critical roles in glucose homeostasis. Here, we studied glucose homeostasis and β-cell function of Gcg-deficient mice that are homozygous for a Gcg-GFP knock-in allele (Gcg(gfp/gfp)). The Gcg(gfp/gfp) mice displayed improved glucose tolerance and enhanced insulin secretion, as assessed by both oral glucose tolerance test (OGTT) and intraperitoneal glucose tolerance test (IPGTT). Responses of glucose-dependent insulinotropic polypeptide (GIP) to both oral and intraperitoneal glucose loads were unexpectedly enhanced in Gcg(gfp/gfp) mice, and immunohistochemistry localized GIP to pancreatic β-cells of Gcg(gfp/gfp) mice. Furthermore, secretion of GIP in response to glucose was detected in isolated islets of Gcg(gfp/gfp) mice. Blockade of GIP action in vitro and in vivo by cAMP antagonism and genetic deletion of the GIP receptor, respectively, almost completely abrogated enhanced insulin secretion in Gcg(gfp/gfp) mice. These results indicate that ectopic GIP expression in β-cells maintains insulin secretion in the absence of proglucagon-derived peptides (PGDPs), revealing a novel compensatory mechanism for sustaining incretin hormone action in islets.

FIG. 1.

Disruption of PGDPs improves glucose tolerance and enhances insulin secretion. Blood glucose levels (A) and plasma insulin levels (B) in the OGTT in 12- to 15-week-old control (Ctl, Gcg^+/+ and Gcg^gfp/+) and Gcg^gfp/gfp mice (n = 5–6 mice per group). Blood glucose levels (C) and plasma insulin levels (D) in the IPGTT in 11- to 22-week-old mice (n = 9–10 mice per group). E: Glucose-induced insulin secretion from isolated islets. Pancreatic islets were isolated from 5- to 7-month-old control (Gcg^+/+ and Gcg^gfp/+, n = 13–18 in each group) and Gcg^gfp/gfp (n = 8–9 in each group) mice and incubated with the indicated concentration of glucose for 30 min. Insulin secretion is expressed as the ratio of insulin released into the medium relative to insulin content. Values are expressed as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. Ctl. ††P < 0.01. †††P < 0.001 vs. 4.2 mmol/L glucose. #P < 0.05; ##P < 0.01; ###P < 0.001 vs. 2.8 mmol/L glucose.

FIG. 2.

Gcg^gfp/gfp mice exhibit α-cell hyperplasia. A: Representative pancreatic sections from 9-week-old Gcg^+/+ (left) and Gcg^gfp/gfp (right) mice. In the left panel, sections were immunostained for glucagon (green) and insulin (red). In the right panel, sections were immunostained for insulin (red) and shown with autofluorescence of GFP. Scale bar, 100 µm. B–F: Morphometric analysis of islets. B: Islet area shown relative to a 10³-µm² sectional area. C: Islet number shown relative to a 10⁶-µm² sectional area. D: The α-cell area shown as glucagon-positive or GFP-positive area relative to total pancreas area. E: The β-cell area shown as insulin-positive area relative to total pancreas area. F: The α-cell/β-cell area ratio shown as a ratio of glucagon-positive or GFP-positive area relative to insulin-positive area. G: Electron microscopy of islets. Scale bar, 500 nm. Values are expressed as means ± SEM. ***P < 0.001. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Gcg^gfp/gfp mice display enhanced GIP secretion and GIP expression in pancreatic islets, and enhancement of glucose-induced insulin secretion is blocked by Rp-cAMP in Gcg^gfp/gfp mice. Plasma GIP levels at 0 and 15 min after oral (A) or intraperitoneal (B) glucose administration in 12- to 17-week-old control (Ctl, Gcg^+/+, and Gcg^gfp/+) and Gcg^gfp/gfp mice (n = 6–11 mice per group). C: mRNA expression of GIP in the intestine (n = 6–8). The expression level of GIP mRNA was normalized to that of glyceraldehyde-3-phosphate dehydrogenase mRNA. D: mRNA expression of GIP and GIP receptor (GIPR) in isolated islets (n = 4). The expression levels normalized to that of insulin 1 are shown. E: GIP content in isolated islets (n = 5–7). GIP content was normalized to insulin content. F: GIP secretion from islets (n = 5–8). Isolated islets were incubated with 16.7 mmol/L glucose for 16 h. G: Effect of Rp-cAMP on glucose-induced insulin secretion. Isolated islets from control (n = 14–22 in each group) and Gcg^gfp/gfp (n = 13–22 in each group) mice were incubated with the indicated concentration of glucose and 500 μmol/L Rp-cAMP for 30 min. Insulin secretion is expressed as the ratio of insulin released into the medium relative to insulin content. Values are expressed as means ± SEM. *P < 0.05; **P < 0.01. #P < 0.05; ###P < 0.001 vs. 0 min. u.d., undetectable.

FIG. 4.

GIP is expressed in pancreatic β-cells in Gcg^gfp/gfp mice, but not in Gcgr^−/−, Glp1r^−/−, or Gcgr^−/−Glp1r^−/− mice. A: Representative immunostaining for GIP (red) and insulin (green), and GFP fluorescence (green) of islets from Gcg^gfp/gfp mice and Gcg^gfp/+ mice. B: Representative immunostaining for GIP (red) and glucagon (green) of islets from Gcgr^−/−, Glp1r^−/−, and Gcgr^−/−Glp1r^−/− mice. Scale bar, 100 µm. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Lack of GIP receptor signaling abrogates enhanced insulin secretion in Gcg^gfp/gfp mice. A: Representative immunostaining for GIP (red) and insulin (green), and GFP fluorescence (green) of islets from Gcg^gfp/gfpGipr^−/− mice. Scale bar, 100 µm. B: Blood glucose levels during the OGTT in 15- to 24-week-old control (Ctl), Gcg^gfp/gfp, and Gcg^gfp/gfpGipr^−/− mice (n = 5–7 mice per group). C: Plasma insulin levels at 0 and 15 min after oral glucose loading. D: Blood glucose levels during the IPGTT in 13- to 22-week-old mice (n = 6–8 mice per group). E: Plasma insulin levels 0 and 15 min after intraperitoneal glucose administration. F: Glucose-induced insulin secretion from isolated islets. Pancreatic islets were isolated from 5- to 7-month-old control (Ctl), Gcg^gfp/gfp, and Gcg^gfp/gfpGipr^−/− mice, and then incubated with the indicated concentration of glucose for 30 min. Insulin secretion is expressed as the ratio of insulin released into the medium relative to insulin content. Values are expressed as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. Ctl. †P < 0.05; †††P < 0.001 vs. Gcg^gfp/gfp, Ctl, Gcg^gfp/+Gipr^+/−; Gcg^gfp/gfp, Gcg^gfp/gfpGipr^+/+, and Gcg^gfp/gfpGipr^+/−. (A high-quality digital representation of this figure is available in the online issue.)