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The hormone-sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP/aP2) form a physical complex that affects basal and hormone-stimulated adipocyte fatty acid efflux. Previous work has established that AFABP/aP2-HSL complex formation requires that HSL be in its activated, phosphorylated form and AFABP/aP2 have a bound fatty acid. To identify the HSL binding site of AFABP/aP2 a combination of alanine-scanning mutagenesis and fluorescence resonance energy transfer was used. Mutation of Asp17, Asp18, Lys21, or Arg30 (but not other amino acids in the helix-turn-helix region) to alanine inhibited interaction with HSL without affecting fatty acid binding. The cluster of residues on the helical domain of AFABP/aP2 form two ion pairs (Asp17-Arg30 and Asp18-Lys21) and identifies the region we have termed the charge quartet as the HSL interaction site. To demonstrate direct association, the non-interacting AFABP/aP2-D18K mutant was rescued by complementary mutation of HSL (K196E). The charge quartet is conserved on other FABPs that interact with HSL such as the heart and epithelial FABPs but not on non-interacting proteins from the liver or intestine and may be a general protein interaction domain utilized by fatty acid-binding proteins in regulatory control of lipid metabolism.

Adipocyte fatty acid-binding protein (AFABP/aP2) forms a physical complex with the hormone-sensitive lipase (HSL) and AFABP/aP2-null mice exhibit reduced basal and hormone-stimulated lipolysis. To identify the determinants affecting the interaction fluorescence resonance energy transfer (FRET) imaging was used in conjunction with a mutagenesis strategy to evaluate the roles AFABP/aP2 fatty acid binding and HSL phosphorylation have in complex formation as well as determine the HSL binding site on AFABP/aP2. The nonfatty acid binding mutant of AFABP/aP2 (R126Q) failed to form a FRET-competent complex with HSL either under basal or forskolin-stimulated conditions, indicating that lipid binding is required for association. Once bound to HSL and on the surface of the lipid droplet, YFP-AFABP/aP2 (but not YFP-HSL) exhibited energy transfer between the fusion protein and BODIPY-C12-labeled triacylglycerol. Serine to alanine mutations at the two PKA phosphorylation sites of HSL (659 and 660), or at the AMPK phosphorylation sites (565), blocked FRET between HSL and AFABP/aP2. Substitution of isoleucine for lysine at position 21 of AFABP/aP2 (K21I), but not 31 (K31I), resulted in a non-HSL-binding protein indicating that residues on helix αI of AFABP/aP2 define a component of the HSL binding site. These results indicate that the ligand-bound form of AFABP/aP2.interacts with the activated, phosphorylated HSL and that the association is likely to be regulatory; either delivering FA to inhibit HSL (facilitating feedback inhibition) or affecting multicomponent complex formation on the droplet surface.

Previous in vitro studies have established that hormone sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP) form a physical complex that presumably positions the FABP to accept a product fatty acid generated during catalysis. To assess AFABP-HSL interaction within a cellular context, we have used lipocytes derived from 293 cells (C8PA cells) and examined physical association using fluorescence resonance energy transfer. Transfection of C8PA cells with cyan fluorescent protein (CFP)-HSL, yellow fluorescent protein (YFP)-adipocyte FABP, or YFP-liver FABP revealed that under basal conditions each protein was cytoplasmic. In the presence of 20 μm forskolin, CFP-HSL translocated to the triacylglycerol droplet, coincident with BODIPY-FA labeled depots. Fluorescence resonance energy transfer analysis demonstrated that CFP-HSL associated with YFP-adipocyte FABP in both basal and forskolin-treated cells. In contrast, little if any fluorescence resonance energy transfer could be detected between CFP-HSL and YFP-liver FABP. These results suggest that a pre-lipolysis complex containing at least AFABP and HSL exists and that the complex translocates to the surface of the lipid droplet.

Fatty acid transport protein (FATP) was identified by expression cloning strategies (Schaffer, J. E., and Lodish, H. F. (1994) Cell 79, 427-436) and shown by transfection analysis to catalyze the transfer of long-chain fatty acids across the plasma membrane of cells. It is expressed highly in tissues exhibiting rapid fatty acid metabolism such as skeletal muscle, heart, and adipose. FATP mRNA levels are down-regulated by insulin in cultured 3T3-L1 adipocytes and up-regulated by nutrient depletion in murine adipose tissue (Man, M. Z., Hui, T. Y., Schaffer, J. E., Lodish, H. F., and Bernlohr, D. A. (1996) Mol. Endocrinol. 10, 1021-1028). To determine the molecular mechanism of insulin regulation of FATP transcription, we have isolated the murine FATP gene and its 5'-flanking sequences. The FATP gene spans approximately 16 kilobases and contains 13 exons, of which exon 2 is alternatively spliced. S1 nuclease and RNase protection assays revealed the presence of multiple transcription start sites; the DNA sequence upstream of the predominant transcription start sites lacks a typical TATA box. By transient transfection assays in 3T3-L1 adipocytes, the inhibitory action of insulin on FATP transcription was localized to a cis-acting element with the sequence 5'-TGTTTTC-3' from -1347 to -1353. This sequence is very similar to the insulin response sequence found in the regulatory region of other genes negatively regulated by insulin such as those encoding phosphoenolpyruvate carboxykinase, tyrosine aminotransferase, and insulin-like growth factor-binding protein 1. Fluorescence in situ hybridization analysis revealed that the murine FATP gene is localized to chromosome 8, band 8B3.3. Interestingly, this region of chromosome 8 contains a cluster of three other genes important for fatty acid homeostasis, lipoprotein lipase, the mitochondrial uncoupling protein 1 (UCP1) and sterol regulatory element-binding protein 1. These results characterize the murine FATP gene and its insulin responsiveness as well as present a framework for future studies of its role in lipid metabolism, obesity, and type II diabetes mellitus.