GPR40 is necessary but not sufficient for fatty acid stimulation of insulin secretion in vivo

Abstract

Long-chain fatty acids amplify insulin secretion from the pancreatic beta-cell. The G-protein-coupled receptor GPR40 is specifically expressed in beta-cells and is activated by fatty acids; however, its role in acute regulation of insulin secretion in vivo remains unclear. To this aim, we generated GPR40 knockout (KO) mice and examined glucose homeostasis, insulin secretion in response to glucose and Intralipid in vivo, and insulin secretion in vitro after short- and long-term exposure to fatty acids. Our results show that GPR40 KO mice have essentially normal glucose tolerance and insulin secretion in response to glucose. Insulin secretion in response to Intralipid was reduced by approximately 50%. In isolated islets, insulin secretion in response to glucose and other secretagogues was unaltered, but fatty acid potentiation of insulin release was markedly reduced. The Galpha(q/11) inhibitor YM-254890 dose-dependently reduced palmitate potentiation of glucose-induced insulin secretion. Islets from GPR40 KO mice were as sensitive to fatty acid inhibition of insulin secretion upon prolonged exposure as islets from wild-type animals. We conclude that GPR40 contributes approximately half of the full acute insulin secretory response to fatty acids in mice but does not play a role in the mechanisms by which fatty acids chronically impair insulin secretion.

Figure 1. Generation of GPR40 KO mice

Structure of the targeting vector (A) and representative PCR analysis of tail DNA (B). Bp: base pairs; L: molecular weight marker; NTC: no-template control.

Figure 2. Metabolic characterization of GPR40 KO mice

A cohort of male (A, C, E, G) and female (B, D, F, H) GPR40 KO and WT littermates was followed from 5 to 24 weeks of age. Weight (A, B), Fasting blood glucose (C, D), plasma free-fatty acids (FFA) (E, F), and plasma insulin levels (G, H) were measured throughout the study. N= 6–8 animals per group.

Figure 3. Oral glucose tolerance and intraperitoneal insulin tolerance

Glucose (1g/kg) was administered orally to overnight-fasted male (A, C) and female (B, D)) mice at 6 (A, B) and 13 (C, D) weeks of age at time 0. Plasma glucose was measured at 0, 15, 30, 60, 90, and 120 min. N= 6–8 animals per group. Insulin (0.75 U/kg) was administered to overnight-fasted, 14-week old male (E) and female (F) mice by intraperitoneal injection at time 0. Plasma glucose was measured at times 0, 30, 60, and 120 min. Data are expressed as percentage of plasma glucose measured at time 0. N=6–8 animals per group.

Figure 4. Insulin response to intravenous glucose and Intralipid

Glucose (0.5g/kg) or Intralipid (100 μL of a 20% solution preceded by 20U of heparin) was administered to overnight-fasted, 13-week old male mice by intravenous injection at time 0. Plasma glucose (A), FFA (B), and insulin (C) levels were measured at times 0, 2.5, 5, 15, 30, 45, and 60 min. The insulinogenic indexes of glucose and Intralipid (D) were calculated as the ratio between the increment in secreted insulin from 0 to 30 min and the increment in plasma glucose during the same period. N=5–7 animals per group. *: P<0.05.

Figure 5. Insulin secretion in freshly isolated islets

A: Insulin secretion was assessed in 1-h static incubations in the presence of 2.8, 8.3, or 16.7 mmol/l glucose with or without 0.5 mmol/l palmitate. N=2–10 replicate experiments per condition. *: P<0.05; **: P<0.001. B: Insulin secretion was assessed in 1-h static incubations in the presence of 2.8 mmol/l glucose + 40 mmol/l KCl or 16.7 mmol/l glucose + 0.1 mmol/l IBMX. N=4 replicate experiments. C: Insulin secretion was assessed in 1-h static incubations in the presence of 2.8 mmol/l glucose, 16.7 mmol/l glucose, or 16.7 mmol/l glucose + 0.5 mmol/l palmitate, with increasing concentrations of YM-254890. Data are expressed as the percentage of secreted insulin / insulin content. N=2–9 replicate experiments per condition.

Figure 6. Effects of prolonged exposure to fatty acids on insulin secretion

Isolated islets were cultured for 72 h in the presence of 16.7 mmol/l glucose with or without 0.5 mmol/l palmitate or oleate. Insulin secretion was assessed in 1-h static incubations. Data are expressed as the ratio of insulin secreted at 16.7 mmol/l glucose / insulin secreted at 2.8 mmol/l glucose (fold response). N = 7 (WT) and 8 (KO) replicate experiments. *: P<0.01.