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

Human G protein-coupled receptor 40 (hGPR40), with medium- and 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

Voxel placement and representative ¹H short-TE and GABA-edited spectra with LCModel fitting.

<p>(A) Volume of interest (VOI) for the thalamus (TH); (B) VOI for the posterior cingulate cortex (PCC); (C) a representative sagittal T1-weighted MRI brain image of a smelter, depicting the hyperintense signal associated with brain manganese deposition, especially in the globus pallidus; (D) a representative short-TE spectrum with LCModel fitting; (E) a representative MEGA-PRESS difference spectrum with LCModel fitting. CC: corpus callosum, Cho: choline, CN: caudate nucleus, Cr: creatine, Glx: combination of glutamate and glutamine, GP: globus pallidus, HCN: head of caudate nucleus, mI: myo-inositol, NAA: N-Acetyl-aspartate, PU: putamen,</p

Histograms of GABA+/tCr levels in the thalamus and myo-inositol (the ratio of myo-inositol to total creatine (mI/tCr)) levels in the thalamus and posterior cingulated cortex (PCC).

<p>(A) Mean and standard deviation for GABA+/tCr in the thalamus of Mn-exposed smelters and controls (p = 0.009); (B) Mean and standard deviation for the ratio of myo-inositol to total creatine (mI/tCr) in the thalamus of Mn-exposed smelters and controls (p = 0.030); (C) Mean and standard deviation for mI/tCr in the PCC of Mn-exposed smelters and controls (p = 0.009).</p