Overexpression of GPR40 in pancreatic beta-cells augments glucose-stimulated insulin secretion and improves glucose tolerance in normal and diabetic mice

Abstract

Objective: GPR40 is a G protein-coupled receptor regulating free fatty acid-induced insulin secretion. We generated transgenic mice overexpressing the hGPR40 gene under control of the mouse insulin II promoter and used them to examine the role of GPR40 in the regulation of insulin secretion and glucose homeostasis.

Research design and methods: Normal (C57BL/6J) and diabetic (KK) mice overexpressing the hGPR40 gene under control of the insulin II promoter were generated, and their glucose metabolism and islet function were analyzed.

Results: In comparison with nontransgenic littermates, hGPR40 transgenic mice exhibited improved oral glucose tolerance with an increase in insulin secretion. Although islet morphologic analysis showed no obvious differences between hGPR40 transgenic and nontransgenic mice, isolated islets from hGPR40 transgenic mice had enhanced insulin secretion in response to high glucose (16 mmol/l) compared with those from nontransgenic mice, and they both had similar low glucose (3 mmol/l)-stimulated insulin secretion. In addition, hGPR40 transgenic islets significantly increased insulin secretion against a naturally occurring agonist palmitate in the presence of 11 mmol/l glucose. hGPR40 transgenic mice were also found to be resistant to high-fat diet-induced glucose intolerance, and hGPR40 transgenic mice harboring KK background showed augmented insulin secretion and improved oral glucose tolerance compared with nontransgenic littermates.

Conclusions: Our results suggest that GPR40 may have a role in regulating glucose-stimulated insulin secretion and plasma glucose levels in vivo and that pharmacological activation of GPR40 may provide a novel insulin secretagogue beneficial for the treatment of type 2 diabetes.

FIG. 1.

Generation and characterization of hGPR40 transgenic mice. A: A DNA construct for microinjection to generate transgenic mice for the mouse insulin II promoter and hGPR40 gene. The box denotes the coding region of hGPR40 cDNA. B: Human and mouse GPR40 mRNA levels from hGPR40 transgenic (47M, 23F, n = 3) and nontransgenic mice (n = 3) by quantitative real-time PCR analyses. mRNA levels of actin were used as an internal control. A, subcutaneous adipose tissue; B, brain; Is, islet; K, kidney; L, liver; M, muscle; NonTg, nontransgenic; Tg, transgenic.

FIG. 2.

Improved glucose tolerance in hGPR40 transgenic mice harboring C57BL/6J background. Oral glucose tolerance testing was conducted in nontransgenic and hGPR40 transgenic on a regular diet. Glucose was administered orally at 1 g/kg body wt. Panels show plasma glucose and plasma insulin in the 47M line at 16 weeks of age (n = 8) (A and C) and in the 23F line at 15 weeks of age (n = 10) (B and D). Data in panel E represent the AUC_{0–120 min} of the plasma glucose shown in panels A and B, and data in panel F represent the AUC_{0–30 min} of the plasma insulin shown in panels C and D. G and H: Insulin tolerance test for nontransgenic and hGPR40 transgenic mice. Insulin was injected intraperitoneally at 0.5 units/kg. Panels show plasma glucose on regular diet at 14–15 weeks of 47M (n = 13–15) (G) and 23F (n = 5–7) (H), respectively. All values are the means ± SE. **P ≤ 0.01, *P ≤ 0.05 vs. nontransgenic mice by Student's t test. NonTg, nontransgenic; Tg, transgenic.

FIG. 2.

Improved glucose tolerance in hGPR40 transgenic mice harboring C57BL/6J background. Oral glucose tolerance testing was conducted in nontransgenic and hGPR40 transgenic on a regular diet. Glucose was administered orally at 1 g/kg body wt. Panels show plasma glucose and plasma insulin in the 47M line at 16 weeks of age (n = 8) (A and C) and in the 23F line at 15 weeks of age (n = 10) (B and D). Data in panel E represent the AUC_{0–120 min} of the plasma glucose shown in panels A and B, and data in panel F represent the AUC_{0–30 min} of the plasma insulin shown in panels C and D. G and H: Insulin tolerance test for nontransgenic and hGPR40 transgenic mice. Insulin was injected intraperitoneally at 0.5 units/kg. Panels show plasma glucose on regular diet at 14–15 weeks of 47M (n = 13–15) (G) and 23F (n = 5–7) (H), respectively. All values are the means ± SE. **P ≤ 0.01, *P ≤ 0.05 vs. nontransgenic mice by Student's t test. NonTg, nontransgenic; Tg, transgenic.

FIG. 3.

Islet structure and β-cell function of hGPR40 transgenic mice and nontransgenic mice. A: Islet morphology of hGPR40 transgenic (47M and 23F) and nontransgenic mice at 16 weeks of age. The sections were stained with anti-insulin, -glucagon, -GLUT2, and -proinsulin antibodies, respectively. B: β-Cell area was measured as the stained area for anti-insulin antibody. C and D: Glucose-stimulated insulin secretion in isolated islets from hGPR40 transgenic and nontransgenic mice. E: Palmitate-stimulated insulin secretion in isolated islets from hGRP40 transgenic (47M) mice and nontransgenic mice. Islets were isolated at 9 weeks from hGPR40 transgenic and nontransgenic mice fed regular diet. Five islets with similar sizes from each group (four batches in each group) were used. All values are the means ± SE. **P ≤ 0.01, *P ≤ 0.05 vs. nontransgenic mice by Student's t test. NonTg, nontransgenic; Tg, transgenic. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Effects of high-fat–diet feeding on glucose homeostasis in hGPR40 transgenic mice. The hGPR40 transgenic (47M) and nontransgenic mice were fed on 60 kcal% fat diet from 8 weeks of age. Oral glucose tolerance testing was used for hGPR40 transgenic mice and nontransgenic mice. Glucose was administered orally at 1 g/kg body wt. A and B: Plasma glucose (A) and plasma insulin (B) at 17 weeks of age, respectively. Data in panel C represent the AUC_{0–120 min} of plasma glucose shown in panel A, and data in panel D represent the AUC_{0–30 min} of plasma insulin shown in panel B. E: Insulin tolerance test for nontransgenic and hGPR40 transgenic mice. Insulin was injected intraperitoneally at 0.5 units/kg. Plasma glucose of high-fat–diet–fed mice at 20 weeks of age. F: Epididymal adipose tissue weight at 20 weeks of age. All values are the means ± SE (n = 10). **P ≤ 0.01 vs. nontransgenic mice by Student's t test. NonTg, nontransgenic; Tg, transgenic.

FIG. 5.

Improved glucose tolerance in hGPR40 transgenic mice harboring diabetic KK background on regular diet. A: Human and mouse GPR40 mRNA levels in islets from hGPR40 transgenic × KK and nontransgenic × KK mice (n = 3) by quantitative real-time PCR analyses. mRNA levels of actin were used as an internal control. B–E: Oral glucose tolerance test for hGPR40 transgenic mice and nontransgenic mice harboring hybrid background. Glucose was administered orally at 2 g/kg body wt. B and C: Plasma glucose (B) and plasma insulin (C) at 12 weeks of age on regular diet. Data in panel D represent the AUC_{0–120 min} of plasma glucose shown in panel B, and data in panel E represent the AUC_{0–30 min} of plasma insulin shown in panel C. F and G: Glucose- and palmitate-stimulated insulin secretion in isolated islets from hGPR40 transgenic × KK and nontransgenic × KK mice. Islets were isolated from regular diet–fed mice at 12 weeks. Five islets with similar sizes from each group (four batches in each group) were used. All values are means ± SE (n = 8–10). **P ≤ 0.01, *P ≤ 0.05 vs. nontransgenic mice by Student's t test. NonTg, nontransgenic; Tg, transgenic.

FIG. 6.

Gene expression profiles in islets from hGPR40 transgenic and nontransgenic mice. The hGPR40 transgenic (47M) and nontransgenic mice were fed on 60 kcal% fat diet for 9 weeks. In each group, the islets from more than three mice were collected, and mRNA levels of indicated genes were estimated by quantitative real-time PCR analyses in duplicate. mRNA levels of actin were used as an internal control. Ratios in hGPR40 transgenic mice were with respect to values in nontransgenic mice. All values are the means ± SE (n = 3–4). Gck; glucokinase; NonTg, nontransgenic; Tg, transgenic.