To further investigate if GPR was the functional
To further investigate if GPR40 was the functional target of these phenylpropiolic Sometimes analogs, GW-1100, a selective antagonist of GPR40-mediated Ca2+ elevations in HEK293 cells with an IC50 value equal to 1 μM, was used to inhibit agonist-induced intracellular calcium flux . HEK293-GPR40 cells were pretreated with GW-1100 (10 μM) for 15 min, then stimulated with TAK-875, compounds 9, 11, and 14 respectively. As presented in Fig. 2B, GW-1100 was capable of blocking the increase of Ca2+ level induced by TAK-875. Similarly, GW-1100 also reversed the agonist response elicited by compounds 9, 11, and 14. It has been well established that the activation of GPCR could often stimulate mitogen activated protein kinase (MAPK) signaling pathway. Specifically, the phosphorylation of ERK has been proven to be a frequent consequence of the activation of GPCR . Indeed, the activation of GPR40 by TAK875 resulted in the phosphorylation of ERK1/2 (Fig. 1C). In the same way, compounds 9, 11, and 14 can also induce the phosphorylation of ERK1/2 in HEK293-GPR40 cells which could be completely reversed by GPR40 antagonist GW-1100. In contrast, only epidermal growth factor (EGF) can activated ERK1/2 in HEK293 cells transfected with empty plasmid (Fig. 1D). Taken together, these results confirmed that the agonist activity of compounds 9, 11, and 14 was mediated directly through GPR40 receptor. It is well recognized that repeated administration of an agonist would result in the desensitization of a GPCR response and subsequent the loss of GPCR function . To determine whether compounds 9, 11, and 14 could induce GPR40 receptor desensitization, HEK293-GPR40 cells were firstly stimulated with tested compounds (10 μM) or DMSO solution (0.2%). As shown in Fig. 3, TAK875 and three synthetic compounds 9, 11, and 14 all produced a significant increase of intracellular Ca2+ level. After 10 min incubation, cells were washed with HBSS buffer and re-treated with 10 μM of TAK-875. Cells pretreated with 0.2% DMSO responded sufficiently to TAK-875 stimulation, while cells pretreated with TAK-875, compounds 9, 11, and 14 led to receptor desensitization and displayed a reduced calcium response at the second stimulation of TAK-875 (Fig. 3). Previous studies have reported that GPR40 is predominantly expressed in pancreatic β-cells, and activation of GPR40 by endogenous long-chain fatty acids or synthetic small-molecule compounds is capable of producing a glucose-dependent increase of insulin secretion in β-cells [15,18,19]. Thus, we evaluated the effects of compounds 9, 11, and 14 on glucose-stimulated insulin secretion using the MIN6 mouse insulinoma cell line. As shown in Fig. 4A, high concentration of glucose (25 mM) promoted insulin release in MIN6 cells. Similar to TAK-875, our compounds 9, 11, and 14 remarkably induced insulin secretion under high-glucose conditions, while they were less effective or had no effect under low-glucose conditions (2.5 mM). In addition, GPR40 antagonist GW-1100 (10 μM) could effectively block the effects of tested compounds on stimulating insulin release under high-glucose conditions indicating that these compounds can induce insulin secretion through activation of GPR40. Of note, compound 9 produced a dose-dependent (0.1–10 μM) increase in the glucose-stimulated insulin secretion at high glucose levels (25 mM), and the capability of compound 9 to stimulate insulin secretion was comparable to that found for positive control TAK-875 (Fig. 4B). Further in vivo efficacy of compound 9 was evaluated by an oral glucose tolerance test (OGTT) in C57BL/6 mice. As shown in Fig. 5A, oral administration of compound 9 at 3 or 10 mg/kg can both significantly reduce plasma glucose concentrations at 15, 30, and 60 min respectively. Notably, the capacity of compound 9 to decrease glucose levels was similar to that of TAK-875 (Fig. 5B): 21% at 3 mg/kg vs vehicle (9, p < 0.001), 34% at 10 mg/kg vs vehicle (9, p < 0.001), 24% at 3 mg/kg vs vehicle (TAK-875, p < 0.001), respectively. Taken together, these results indicated that compound 9 was a potential therapeutic agent for type 2 diabetes by inducing insulin secretion through the activation of GRP40.