Deletion of GPR40 impairs glucose-induced insulin secretion in vivo in mice without affecting intracellular fuel metabolism in islets

Abstract

Objective: The G-protein-coupled receptor GPR40 mediates fatty acid potentiation of glucose-stimulated insulin secretion, but its contribution to insulin secretion in vivo and mechanisms of action remain uncertain. This study was aimed to ascertain whether GPR40 controls insulin secretion in vivo and modulates intracellular fuel metabolism in islets.

Research design and methods: Insulin secretion and sensitivity were assessed in GPR40 knockout mice and their wild-type littermates by hyperglycemic clamps and hyperinsulinemic euglycemic clamps, respectively. Transcriptomic analysis, metabolic studies, and lipid profiling were used to ascertain whether GPR40 modulates intracellular fuel metabolism in islets.

Results: Both glucose- and arginine-stimulated insulin secretion in vivo were decreased by approximately 60% in GPR40 knockout fasted and fed mice, without changes in insulin sensitivity. Neither gene expression profiles nor intracellular metabolism of glucose and palmitate in isolated islets were affected by GPR40 deletion. Lipid profiling of isolated islets revealed that the increase in triglyceride and decrease in lyso-phosphatidylethanolamine species in response to palmitate in vitro was similar in wild-type and knockout islets. In contrast, the increase in intracellular inositol phosphate levels observed in wild-type islets in response to fatty acids in vitro was absent in knockout islets.

Conclusions: These results indicate that deletion of GPR40 impairs insulin secretion in vivo not only in response to fatty acids but also to glucose and arginine, without altering intracellular fuel metabolism in islets, via a mechanism that may involve the generation of inositol phosphates downstream of GPR40 activation.

FIG. 1.

Hyperglycemic clamps in 24-h–fasted and ad libitum–fed GPR40 wild-type and knockout mice. Glucose (A and D) and insulin (B and E) levels during the course of the hyperglycemic clamp in fasted and fed GPR40 wild-type and knockout mice. Insulin levels (C and F) in response to an arginine bolus (1 mmol/kg). Values are expressed as means ± SE of seven to nine mice per group. □, Wild type; ●, knockout.

FIG. 2.

Hyperinsulinemic-euglycemic clamps in GPR40 wild-type and knockout mice. Glucose levels (A) and glucose infusion rate (GIR) (B) during the course of the hyperinsulinemic clamp in 5-h-food–restricted GPR40 wild-type and knockout mice. The insulin sentivity index (M/I) (C) was calculated as the glucose infusion rate (M) divided by the average insulinemia during the last 30 min of the clamp (I). Values are expressed as means ± SE of five to six mice per group. □, Wild type; ●, knockout.

FIG. 3.

Glucose and palmitate metabolism in GPR40 wild-type and knockout islets. Glucose utilization (A), glucose oxidation (B), palmitate oxidation (C), and palmitate incorporation into total lipids (D) in islets isolated from fasted or fed mice incubated at 2.8 (□) or 16.7 (■) mmol/l glucose for 2 h. Data are expressed as means ± SE of three to five independent experiments. *P < 0.05, **P < 0.01, or ***P < 0.001 vs. 2.8 mmol/l glucose. $P < 0.05, $$$P < 0.001 vs. fasted.

FIG. 4.

Palmitate esterification in GPR40 wild-type and knockout islets. Palmitate esterification into triacylglycerol (A), diacylglycerol (B), nonesterified fatty acid (C), and phospholipids (D) in islets incubated at 2.8 (□) or 16.7 (■) mmol/l glucose for 4 h. Data are expressed as means ± SE of five independent experiments. *P < 0.05 or **P < 0.01 vs. 2.8 mmol/l glucose.

FIG. 5.

Lipid profiles in GPR40 wild-type and knockout islets. Log2 relative abundances of triacylglycerols (A) and lyso-phospholipids (B) in islets incubated at 16.7 mmol/l glucose with or without 0.5 mmol/l palmitate for 1 h. Lipid extracts were subjected to liquid chromatography–mass spectrometry analysis (see supplementary research design and methods). Data are expressed as means ± SE of five independent experiments as relative abundance (i.e., comparative mass measurements across samples) after log2 transformation. *P < 0.05, **P < 0.01, or ***P < 0.001 vs. wild-type control. #P < 0.05, ##P < 0.01, or ###P < 0.001 vs. knockout control. □, Wild-type control; ■, wild-type palmitate; , knockout control; , knockout palmitate.

FIG. 6.

Arachidonic acid release and InsPs accumulation in GPR40 wild-type and knockout islets. A: AA release in islets incubated during 30 min at 16.7 mmol/l glucose with or without 0.5 mmol/l palmitate and 100 μmol/l carbachol. AA efflux is expressed as the percentage release of total incorporated radioactivity. Data are expressed as means ± SE of three to four independent experiments. **P < 0.01 vs. control. B: InsPs accumulation in islets incubated during 20 min at 16.7 mmol/l glucose with or without 30 μmol/l oleate. InsPs accumulation is expressed as fold changes over control conditions (16.7 mmol/l glucose). Data are expressed as means ± SE of three independent experiments. *P < 0.05 vs. control. □, Wild type; ■, knockout.