Liver Fatty Acid-binding Protein Gene Ablation Inhibits Branched-chain Fatty Acid Metabolism in Cultured Primary Hepatocytes

Barbara P Atshaves,Avery M Mcintosh,Olga I Lyuksyutova,Warren Zipfel,Watt W Webb,Friedhelm Schroeder

doi:10.1074/jbc.m313571200

Abstract

Whereas the role of liver fatty acid-binding protein (L-FABP) in the uptake, transport, mitochondrial oxidation, and esterification of normal straight-chain fatty acids has been studied extensively, almost nothing is known regarding the function of L-FABP in peroxisomal oxidation and metabolism of branched-chain fatty acids. Therefore, phytanic acid (most common dietary branched-chain fatty acid) was chosen to address these issues in cultured primary hepatocytes isolated from livers of L-FABP gene-ablated (-/-) and wild type (+/+) mice. These studies provided three new insights: First, L-FABP gene ablation reduced maximal, but not initial, uptake of phytanic acid 3.2-fold. Initial uptake of phytanic acid uptake was unaltered apparently due to concomitant 5.3-, 1.6-, and 1.4-fold up-regulation of plasma membrane fatty acid transporter/translocase proteins (glutamic-oxaloacetic transaminase, fatty acid transport protein, and fatty acid translocase, respectively). Second, L-FABP gene ablation inhibited phytanic acid peroxisomal oxidation and microsomal esterification. These effects were consistent with reduced cytoplasmic fatty acid transport as evidenced by multiphoton fluorescence photobleaching recovery, where L-FABP gene ablation reduced the cytoplasmic, but not membrane, diffusional component of NBD-stearic acid movement 2-fold. Third, lipid analysis of the L-FABP gene-ablated hepatocytes revealed an altered fatty acid phenotype. Free fatty acid and triglyceride levels were decreased 1.9- and 1.6-fold, respectively. In summary, results with cultured primary hepatocytes isolated from L-FABP (+/+) and L-FABP (-/-) mice demonstrated for the first time a physiological role of L-FABP in the uptake and metabolism of branched-chain fatty acids.

Highlights

The role of liver fatty acid-binding protein (L-FABP)1 in the uptake, intracellular transport, and esterification of normal
Effect of L-FABP Gene Ablation on Maximal Uptake and Specificity of Branched-chain Fatty Acid Uptake in Cultured Primary Hepatocytes Isolated from Male Mice—Since L-FABP binds branched-chain phytanic acid with high affinity [14], the possibility that L-FABP gene ablation decreases phytanic acid uptake was examined in cultured primary hepatocytes isolated from wild type L-FABPϩ/ϩ and L-FABPϪ/Ϫ mice
Effect of L-FABP Gene Ablation on Expression of Intracellular Fatty Acid Transport Proteins in Cultured Primary Hepatocytes Isolated from Male Mice—To assure that the reduced cytoplasmic transport/diffusion of NBD-stearic acid in hepatocytes isolated from livers of L-FABPϪ/Ϫ mice was associated with loss of L-FABP rather than concomitant down-regulation of other intracellular fatty acid or fatty acyl-CoA-binding proteins, Western blot analysis of cultured primary hepatocytes was performed to determine levels of several cytosolic proteins involved in fatty acid diffusion (e.g. sterol carrier protein-2 (SCP-2) [45] and sterol carrier protein-x (SCP-x) [47]) and fatty acid metabolism (SCP-x [47] and ACBP [48])

Summary

The abbreviations used are

L-FABP, liver fatty acid-binding protein; SCP-2, sterol carrier protein-2; SCP-x, sterol carrier protein-x; ACBP, acylCoA binding protein; GOT, glutamic-oxaloacetic transaminase; FAT, fatty acid translocase ( called CD-36); FATP, fatty acid transport protein; BSA, bovine serum albumin; MPFPR, multiphoton fluorescence photobleaching recovery; NBD, 7-nitrobenz-2-oxa-1,3-distraight-chain fatty acids (LCFA) has been examined extensively in vitro (reviewed in Refs. 1–3), in transfected cells (reviewed in Ref. 1), and cultured hepatocytes [4, 5]. Other in vitro studies measuring fatty acid oxidation and ␤-hydroxybutyrate production in liver homogenates showed that L-FABP gene ablation had no effect on oxidation of straight-chain, radiolabeled palmitic acid at high levels (1 mM) [9]. When fatty acid oxidation and ␤-hydroxybutyrate production were measured with hepatocyte suspensions freshly isolated from female mice, L-FABP gene ablation reduced oxidation of high levels (1 mM) of straight-chain palmitic acid by about 30% [9]. Whereas the above results appear contradictory, the intact hepatocyte and in vivo data suggest that L-FABP gene ablation does not affect oxidization of straight-chain fatty acids under normal fed conditions when serum fatty acid levels are low but may do so when serum fatty acids are high as in starvation. The data presented with cultured primary hepatocytes isolated from male L-FABP (Ϫ/Ϫ) mice yielded fundamental new insights indicating for the first time that L-FABP gene-ablation (i) reduces maximal but not initial uptake of phytanic acid; (ii) Reduces cytoplasmic fatty acid transport, independent of the contribution of membrane fatty acid diffusion; (iii) inhibits peroxisomal oxidation of phytanic acid; and (iv) reduces phytanic acid esterification

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION