Peroxisome proliferator-activated receptors (PPAR) are a family of ligand-activated nuclear receptors that induce transcription of multiple genes encoding proteins involved in fatty acid and glucose metabolism, as well as cell differ-

Journal of Lipid Research Volume 50, 2009 1663 Copyright © 2009 by the American Society for Biochemistry and Molecular Biology, Inc. Peroxisome proliferator-activated receptors (PPAR) are a family of ligand-activated nuclear receptors that induce transcription of multiple genes encoding proteins involved in fatty acid and glucose metabolism, as well as cell differentiation ( 1, 2 ). Because abnormal regulation of peroxisome proliferator-activated receptor(PPAR ), the major PPAR isoform found in liver, is associated with chronic diseases such as diabetes, obesity, and hyperlipidemia, considerable effort extends to understanding how endogenous ligands regulate PPAR transcriptional control ( 2, 3 ). Although a broad range of synthetic substances including hypolipidemic agents, plasticizers, and herbicides are known PPAR activators ( 4 ), until recently the identity of endogenous, high-affi nity PPAR ligands remained elusive. While both saturated and unsaturated long-chain fatty acids (LCFA) enhance PPAR -activated gene expression ( 5, 6 ), only unsaturated LCFA bind to PPAR with high affi nity ( 7 ). These discrepancies were later explained by studies showing that the activated form of LCFA, longchain fatty acyl-CoA (LCFA-CoA), could function as highaffi nity, endogenous PPAR ligands ( 6, 8 ). Binding of both saturated and unsaturated LCFA-CoA induce a conformational change in PPAR , enhance interaction with coactivator steroid receptor coactivator-1 (SRC-1), and enhance PPAR transactivation in cultured cells ( 6, 8, 9 ). These effects are not due to hydrolysis of LCFA-CoA, as the nonhydrolyzable S -hexadecyl-CoA is also bound with high affi nity, alters PPAR conformation, and alters interaction with coactivators ( 6, 10 ). In addition, the high affi nity (i.e., nM K d s) binding of LCFA-CoA is in the same range as that of LCFA-CoA concentrations in the nucleoplasm of living cells ( 11, 12 ). Abstract Although studies with liver type fatty acid binding protein (L-FABP) gene ablated mice demonstrate a physiological role for L-FABP in hepatic fatty acid metabolism, little is known about the mechanisms whereby L-FABP elicits these effects. Studies indicate that L-FABP may function to shuttle lipids to the nucleus, thereby increasing the availability of ligands of nuclear receptors, such as peroxisome proliferator-activated receptor(PPAR ). The data herein suggest that such mechanisms involve direct interaction of L-FABP with PPAR . L-FABP was shown to directly interact with PPAR in vitro through co-immunoprecipitation (co-IP) of pure proteins, altered circular dichroic (CD) spectra, and altered fl uorescence spectra. In vitro fl uorescence resonance energy transfer (FRET) between Cy3-labeled PPAR and Cy5-labeled L-FABP proteins showed that these proteins bound with high affi nity (K d approximately 156 nM) and in close proximity (intermolecular distance of 52Å). This interaction was further substantiated by co-IP of both proteins from liver homogenates of wild-type mice. Moreover, double immunogold electron microscopy and FRET confocal microscopy of cultured primary hepatocytes showed that L-FABP was in close proximity to PPAR (intermolecular distance 40–49Å) in vivo. Taken together, these studies were consistent with L-FABP regulating PPAR transcriptional activity in hepatocytes through direct interaction with PPAR . Our in vitro and imaging experiments demonstrate high affi nity, structural molecular interaction of L-FABP with PPAR and suggest a functional role for L-FABP interaction with PPAR in long chain fatty acid (LCFA) metabolism.— Hostetler, H. A., A. L. McIntosh, B. P. Atshaves, S. M. Storey, H. R. Payne, A. B. Kier, and F. Schroeder. L-FABP directly interacts with PPAR in cultured primary hepatocytes. J. Lipid Res. 2009. 50: 1663–1675.