Abstract
Adipose lipolysis is mediated, in part, via interaction of fatty acid-binding protein (FABP) with hormone-sensitive lipase (HSL). Mice with reduced FABP content in fat (adipocyte FABP null) exhibit diminished fat cell lipolysis, whereas transgenic mice with increased FABP content in fat (epithelial FABP transgenic) exhibit enhanced lipolysis. To examine the relationship between the binding of FABP to HSL and activation of catalytic activity, isothermal titration microcalorimetry as well as kinetic analysis using a variety of FABP isoforms have been employed. In the absence of fatty acids, no FABP-HSL association could be demonstrated for any FABP form. However, in the presence of 10 microm oleate, A-FABP and E-FABP each bound to HSL with high affinity (Kd of 0.5 and 3 nM, respectively) in a approximately 1:1 molar stoichiometry, whereas liver FABP and intestinal FABP did not exhibit any association. To compare binding to catalysis, each FABP isoform was incubated with HSL in vitro, and enzymatic activity was assessed. Importantly, each FABP form stimulated HSL activity approximately 2-fold using cholesteryl oleate as substrate but exhibited no activation using p-nitrophenyl butyrate. The activation by A-FABP was dependent upon its fatty acid binding properties because a non-fatty acid binding mutant, R126Q, failed to activate HSL. These results suggest that binding and activation of HSL by FABPs are separate and distinct functions and that HSL contains a site for fatty acid binding that allows for FABP association.
Highlights
Adipose lipolysis is mediated, in part, via interaction of fatty acid-binding protein (FABP) with hormone-sensitive lipase (HSL)
We describe that in addition to A-FABP, HSL associates with epithelial FABP (E-FABP), but not with FABPs from liver (L-FABP) or intestine (I-FABP) and the surprising finding that the binding is dependent upon fatty acids
Previous studies have demonstrated that A-FABP forms a physical complex with hormone-sensitive lipase and that coincubation results in an increase of HSL activity ϳ2-fold [9]
Summary
Materials—Oleate and purified fatty acid standards used for gas chromatography analysis were purchased from Nu Chek Prep (Elysian, MN), and 1,8-anilinonaphthalene-8-sulfonic acid (1,8-ANS) was purchased from Molecular Probes (Eugene, OR). Pooled fractions containing purified His6-HSL protein were dialyzed against buffer containing 50 mM NaPO4, pH 7.0, 20% glycerol, and 0.5% Nonidet P-40 and stored at Ϫ80 °C until use. For fatty acid binding assays, each FABP form was dialyzed into a buffer containing 50 mM NaPO4, pH 7.0, and stored at Ϫ80 °C. Any fatty acids bound endogenously to FABP were removed when isolated in the presence of detergent. To remove any fatty acids bound to the FABPs when purified from non-detergent-containing buffers, each purified protein was incubated at 37 °C for 30 min and subjected to chromatography through Lipidx 1000 (hydroxyalkoxypropyl derivative of Sephadex G-25) resin at the same temperature. Lipid Extraction and Analysis—Fatty acids bound to bacterially derived His-FABPs were extracted using a mixture of chloroform-methanol-water (2:1:1.5 v/v/v) in a volume five times the sample volume after the addition of C15:0 as an internal standard. Fatty Acid Binding Studies—The fatty acid binding properties of each protein were evaluated using the fluorescent probe 1,8-ANS as a surrogate ligand as described by Jenkins et al [21]
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