Novel selective ligands for free fatty acid receptors GPR120 and GPR40.

GPR120 and GPR40 are G-protein-coupled receptors whose endogenous ligands are medium- and long-chain free fatty acids, and they are thought to play an important physiological role in insulin release. Despite recent progress in understanding their roles, much still remains unclear about their pharmacology, and few specific ligands for GPR120 and GPR40 besides medium- to long-chain fatty acids have been reported so far. To identify new selective ligands for these receptors, more than 80 natural compounds were screened, together with a reference compound MEDICA16, which is known to activate GPR40, by monitoring the extracellular regulated kinase (ERK) and [Ca(2+)](i) responses in inducible and stable expression cell lines for GPR40 and GPR120, respectively. MEDICA16 selectively activated [Ca(2+)](i) response in GPR40-expressing cells but not in GPR120-expressing cells. Among the natural compounds tested, grifolin derivatives, grifolic acid and grifolic acid methyl ether, promoted ERK and [Ca(2+)](i) responses in GPR120-expressing cells, but not in GPR40-expressing cells, and inhibited the alpha-linolenic acid (LA)-induced ERK and [Ca(2+)](i) responses in GPR120-expressing cells. Interestingly, in accordance with the pharmacological profiles of these compounds, similar profiles of glucagon-like peptide-1 secretion were seen for mouse enteroendocrine cell line, STC-1 cells, which express GPR120 endogenously. Taken together, these studies identified a selective GPR40 agonist and several GPR120 partial agonists. These compounds would be useful probes to further investigate the physiological and pharmacological functions of GPR40 and GPR120.

Distribution and regulation of protein expression of the free fatty acid receptor GPR120.

GPR120 is a G-protein-coupled receptor whose endogenous ligands have recently been identified as free fatty acids. It has been implicated as playing an important role in the control of lipid and glucose metabolism by regulating the secretion of glucagon-like peptide-1 and cholecystokinin. We have developed an antibody against the extracellular domain of GPR120. The specificity of the antibody was demonstrated by immunoprecipitation, Western blotting, flow cytometry, and immunocytochemistry using GPR120-transfected cells. Immunoreactivity for GPR120 was abundant in the mouse large intestine, lung, and adipose tissue. Furthermore, we found that the expression of GPR120 protein was up-regulated during the adipogenic differentiation of 3T3-L1 cells, which corresponded well with changes in mRNA expression. The anti-GPR120 antibody will be of value for the further study of the function of this nutrient-sensing receptor.

Flow cytometry-based binding assay for GPR40 (FFAR1; free fatty acid receptor 1).

GPR40 is a G protein-coupled receptor (GPCR) whose endogenous ligands have recently been identified as medium- and long-chain free fatty acids (FFAs), and it is thought to play an important role in insulin release. Despite recent research efforts, much still remains unclear in our understanding of its pharmacology, mainly because the receptor-ligand interaction has not been analyzed directly. To study the pharmacology of GPR40 in a more direct fashion, we developed a flow cytometry-based binding assay. FLAG-tagged GPR40 protein was expressed in Sf9 cells, solubilized, immobilized on immunomagnetic beads, and labeled with the fluorescent probe C1-BODIPY-C12. Flow cytometry analysis showed that C1-BODIPY-C12 specifically labels a single class of binding site in a saturable and reversible manner with an apparent dissociation constant of approximately 3 microM. The FFAs that activate GPR40 competed with C1-BODIPY-C12 binding; thus, medium- to long-chain FFAs could compete, whereas short-chain FFAs and methyl linoleate had no inhibitory effect. Furthermore, ligands that are known to activate GPR40 competed for binding in a concentration-dependent manner. All the ligands that inhibited the binding promoted phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in human embryonic kidney (HEK) 293 cells that expressed GPR40 and [Ca(2+)](i) responses in mouse insulinoma (MIN6) cells that natively express GPR40; however, pioglitazone, a thiazolidinedione that failed to compete for the binding, did not activate ERK or [Ca(2+)](i) response. This study showed that a flow cytometry-based binding assay can successfully identify direct interactions between GPR40 and its ligands. This approach would be of value in studying the pharmacology of GPCRs.

Production and characterization of a monoclonal antibody against GPR40 (FFAR1; free fatty acid receptor 1).

GPR40 is G protein-coupled receptor whose endogenous ligands have recently been identified as free fatty acids (FFAs), and it has been implicated to play an important role in FFA-mediated enhancement of glucose-stimulated insulin release. We have developed a monoclonal antibody against the extracellular domain of GPR40. Specificity of the antibody was demonstrated by immunoprecipitation and cell surface staining using GPR40-transfected cells. GPR40 immunoreactivity was highly abundant in mouse pancreatic beta-cells and splenocytes, THP-1 cells, and human peripheral blood mononuclear cells. The anti-GPR40 monoclonal antibody should prove valuable for further studying the function of this nutrient sensing receptor.