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Journal of Lipid Research, Vol. 50, 1663-1675, August 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

L-FABP directly interacts with PPAR in cultured primary hepatocytes

Heather A. Hostetler^*, Avery L. McIntosh^*, Barbara P. Atshaves^*, Stephen M. Storey^*, H. Ross Payne, Ann B. Kier and Friedhelm Schroeder^1,*

^* Department of Physiology and Pharmacology, Texas A&M University, College of Veterinary Medicine and Biomedical Sciences, College Station, TX 77843
Department of Pathobiology, Texas A&M University, College of Veterinary Medicine and Biomedical Sciences, College Station, TX 77843

¹ To whom correspondence should be addressed. e-mail: fschroeder{at}cvm.tamu.edu

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 fluorescence spectra. In vitro fluorescence resonance energy transfer (FRET) between Cy3-labeled PPAR and Cy5-labeled L-FABP proteins showed that these proteins bound with high affinity (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 affinity, 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.

Supplementary key words cytoplasmic lipid binding protein • fluorescence • FRET • liver fatty acid binding protein • nuclei • peroxisome proliferator activated receptor • transcription factor

Abbreviations: CD, circular dichroism; co-IP, co-immunoprecipitation; FRET, fluorescence resonance energy transfer; GR, glucocorticoid receptor; LCFA, long chain fatty acid; LCFA-CoA, long chain fatty acyl CoA; L-FABP, liver fatty acid binding protein; LSCM, laser scanning confocal microscopy; PPAR, peroxisome proliferator activated receptor-; SRC-1, steroid receptor coactivator-1; SREBP-1, sterol regulatory element-binding protein-1

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