Investigation of the Binding Interaction of Fatty Acids with Human G Protein-Coupled Receptor 40 Using a Site-Specific Fluorescence Probe by Flow Cytometry

Abstract

Human G protein-coupled receptor 40 (hGPR40), with mediumand long-chain free fatty acids (FFAs) as its natural ligands, plays an important role in the enhancement of glucose-dependent insulin secretion. To date, information about the direct binding of FFAs to hGPR40 is very limited, and how carbon-chain length affects the activities of FFAs on hGPR40 is not yet understood. In this study, a fluorescein-fasiglifam analogue (F-TAK-875A) conjugate was designed and synthesized as a site-specific fluorescence probe to study the interaction of FFAs with hGPR40. hGPR40 was expressed in human embryonic kidney 293 cells and labeled with F-TAK-875A. By using flow cytometry, competitive binding of FFA and F-TAK-875A to hGPR40-expressed cells was measured. Binding affinities of 18 saturated FFAs, with carbon-chain lengths ranging from C6 to C23, were analyzed. The results showed that the binding potencies of FFAs to hGPR40 were dependent on carbon length. There was a positive correlation between length and binding potency for seven FFAs (C9−C15), with myristic acid (C15) showing the highest potency, 0.2% relative to TAK-875. For FFAs with a length of fewer than C9 or more than C15, they had very weak or no binding. Molecular docking results showed that the binding pocket of TAK-875 in hGPR40 could enclose FFAs with lengths of C15 or fewer. However, for FFAs with lengths longer than C15, part of the alkyl chain extended out of the binding pocket. This study provided insights into the structural dependence of FFAs binding to and activation of hGPR40.H uman G protein-coupled receptor 40 (hGPR40), also known as free fatty acid receptor 1, is strongly expressed in humans in pancreatic β cells, brain, and endocrine cells of the gastrointestinal tract.1−3 Free fatty acids (FFAs), which are essential nutritional components, were identified as the natural substrates of GPR40.4,5 Medium-and long-chain FFAs activated Gq-coupled GPR40, leading to activation of phospholipase C and subsequent elevation of intracellular Ca 2+ levels. 6,7 Several in vitro and in vivo studies have demonstrated that FFAs augmented glucose-stimulated insulin secretion in pancreatic β cells by specifically activating GPR40. 8,9 Therefore, GPR40 is regarded as an attractive target for enhancing insulin secretion in patients with type 2 diabetes.10,11 Many potent GPR40 ligands, such as fasiglifam (TAK-875), GW9508, and AMG 837, have been synthesized as potential drugs. 12−18To date, most studies of activation of chemicals on GPR40, including activation of FFAs and synthetic chemicals, involved monitoring GPR40-mediated cellular Ca 2+ mobilization. 1,19 For example, Itoh et al. compared activities of nine saturated FFAs, with carbon-chain length ranging from C2 to C18, in hGRP40-expressing Chinese hamster ovary cells. FFAs with carbon lengths shorter than C8 had no agonist activity. Activity increased when the length increased from C8 to C16 but decreased as the length increased further from C16 to C18. FFAs with C12, C14, and C16 had similar activities....

Perfluoroalkyl Substances Stimulate Insulin Secretion by Islet β Cells via G Protein-Coupled Receptor 40

“…This may destabilize the binding between PFASs and human GPR40. This dependency of binding affinity on carbon-chain length is in good accordance with our previous study on the binding affinity of 18 FFAs (C5-C23) with human GPR40 40 . The above results obtained from the competitive binding assay and molecular docking demonstrated that some PFASs other than PFOS could also bind to human GPR40, suggesting the possibility of the disruption effect of these PFASs on the insulin secretion activity of β cells.…”

“…Human GPR40 Fluorescence Competitive Binding Assay. The binding affinities of PFASs with human GPR40 (NM_005303) were measured by using a fluorescence competitive binding assay according to our previous work 40 . Briefly, HEK293 cells were transfected with GPR40 plasmid by using lipofectamine 3000 transfection reagent (Life Technologies, USA).…”

Long-Chain Fatty Acid Receptors Mediate Relaxation of the Porcine Lower Esophageal Sphincter

“…Free fatty acid receptor 1 and FFA4 are G-protein coupled receptors activated by long-chain free fatty acids. FFA1 and FFA4 have been detected in the gastrointestinal tract and pancreas (Itoh et al, 2003; Hirasawa, 2015; Ren et al, 2016). FFA1 induces secretion of cholecystokinin in the duodenum in response to dietary fat (Liou et al, 2011).…”

Dairy Fat Consumption and the Risk of Metabolic Syndrome: An Examination of the Saturated Fatty Acids in Dairy

“…Myristic acid (14:0) is involved in the acetylation of at least 0.5% of total proteins [100]. In addition, myristic acid has been found to have the highest binding potency with the human G protein-coupled receptor 40 (GPR40), a major LCFA receptor expressed in enteroendocrine and β-cells, compared to 6:0–23:0 SFA in experimental conditions [101]. Human studies thus far have primarily focused on myristic acid’s unfavorable impact on blood lipid levels [102,103].…”