GPR40 partial agonist MK-2305 lower fasting glucose in the Goto Kakizaki rat via suppression of endogenous glucose production

Abstract

GPR40 (FFA1) is a fatty acid receptor whose activation results in potent glucose lowering and insulinotropic effects in vivo. Several reports illustrate that GPR40 agonists exert glucose lowering in diabetic humans. To assess the mechanisms by which GPR40 partial agonists improve glucose homeostasis, we evaluated the effects of MK-2305, a potent and selective partial GPR40 agonist, in diabetic Goto Kakizaki rats. MK-2305 decreased fasting glucose after acute and chronic treatment. MK-2305-mediated changes in glucose were coupled with increases in plasma insulin during hyperglycemia and glucose challenges but not during fasting, when glucose was normalized. To determine the mechanism(s) mediating these changes in glucose metabolism, we measured the absolute contribution of precursors to glucose production in the presence or absence of MK-2305. MK-2305 treatment resulted in decreased endogenous glucose production (EGP) driven primarily through changes in gluconeogenesis from substrates entering at the TCA cycle. The decrease in EGP was not likely due to a direct effect on the liver, as isolated perfused liver studies showed no effect of MK-2305 ex vivo and GPR40 is not expressed in the liver. Taken together, our results suggest MK-2305 treatment increases glucose stimulated insulin secretion (GSIS), resulting in changes to hepatic substrate handling that improve glucose homeostasis in the diabetic state. Importantly, these data extend our understanding of the underlying mechanisms by which GPR40 partial agonists reduce hyperglycemia.

Fig 1. In vitro and ex-vivo pharmacology of MK-2305.

(A) Structure of the synthetic GPR40 partial agonist MK-2305. (B) Dose-response curves for MK-2305 were generated monitoring IP accumulation in CHO cells expressing rat GPR40. Data are expressed as a percentage of the control response of an in-house partial agonist, and fitted to a standard 4-parameter non-linear regression model. EC₅₀’s were determined for each test compound using a custom in-house developed software package. Each experiment was multiple times with a representative graph shown. The mean parameters of these and other individual experiments are shown in Table 1. (C) Effect of MK-2305 on GSIS in isolated GPR40 WT and KO islets under high (15 mM) and not basal (2 mM) glucose. Data provided are means +/- SEM. Data were analyzed via ANOVA followed by Bonferroni multiple comparisons test. **p<0.01compared to DMSO treated islets under 15 mM glucose.

Fig 2. Effects of MK-2305 on oral glucose tolerance in GK rats.

(A) Blood glucose time course in GK rats treated acutely with MK-2305, followed by an OGTT. (B) AUC data for glucose vs. time post-challenge data in (A). (C) Plasma insulin vs. time data for the OGTT shown. (D) Plasma insulin AUC data for the OGTT. Statistical comparisons of AUC for glucose or insulin across treatment groups were compared via ANOVA followed by Dunnetts post hoc analysis where MK-2305 treated groups were compared to vehicle. ***p<0.001.

Fig 3. Effects of chronic treatment with MK-2305 in the GK rat.

(A) Morning blood glucose levels in GK rats treated with vehicle, 10, or 30 mg/kg of MK-2305, or 10 mg/kg rosiglitazone for 20 days in feed. (B) Fasted blood glucose levels on days 7 and 14 of the study were significantly reduced with MK-2305 and rosiglitazone treatment compared to vehicle controls. (C) Fasted plasma insulin levels on days 7 and 14. (D) Effects on food intake and (E) body weight during the chronic study. (F) Plasma insulin levels during a OGTT in chronically treated rats on day 13. Changes in blood glucose, food intake or body weight over time with MK-2305 or rosiglitazone vs. vehicle were analyzed by two-way ANOVA with repeated measures followed by Tukeys post hoc analysis. Changes in fasted glucose or insulin of glucose AUC were analyzed by one way ANOVA comparing MK-2305 or rosiglitazone treatments with vehicle followed by Dunnetts post hoc analysis. *p<0.05, **p<0.01.

Fig 4. Effects of chronic treatment with MK-2305 on lipids and HBA1c in the GK rat.

Effects of vehicle, Rosiglitazone, and MK-2305 on rat (A) NEFA, (B) liver TG, and (C) HbA1c following 20 days of treatment. Data were analyzed via ANOVA followed by Dunnets test comparing Rosiglitazone or MK-2305 treatments with vehicle. *p<0.05, ***p<0.001.

Fig 5. Effects of MK-2305 on glucose metabolism in perfused mouse livers.

Effect of 10 μM MK-2305 or DMSO on the conversion of [2-¹³C] pyruvate to ¹³C-glucose, ¹³C-glycogen, and ¹³C-lactate in perfused db/db mouse livers. MK-2305 treatments were compared to vehicle for each endpoint via students ttest.

Fig 6. Effect of (A) acute treatment and (B) chronic treatment with 10 mg/kg MK-2305 or vehicle on endogenous glucose production (EGP) from various substrates in the GK or WKY rats.

Data were analyzed via ANOVA followed by Tukey’s multiple comparisons test comparing MK-2305 treatment to vehicle within GK or WKY rats or comparing WKY rats treated with vehicle to GK rats treated with vehicle. *p< 0.05, **p<0.01, ***p<0.005, ****p<0.001.

Fig 7. Uptake and metabolic conversion of [1-¹³C] glucose in liver (A) and muscle (B) following acute treatment with MK-2305 or vehicle.

Data were analyzed via students ttest comparing MK-2305 treatment vs. vehicle for each metabolite in each tissue measured.