Liver Fatty Acid-Binding Protein Colocalizes with Peroxisome Proliferator Activated Receptor α and Enhances Ligand Distribution to Nuclei of Living Cells

Abstract

Although it is hypothesized that long-chain fatty acyl CoAs (LCFA-CoAs) and long-chain fatty acids (LCFAs) regulate transcription in the nucleus, little is known regarding factors that determine the distribution of these ligands to nuclei of living cells. Immunofluorescence colocalization showed that liver fatty acid-binding protein (L-FABP; binds LCFA-CoA as well as LCFA) significantly colocalized with PPARalpha in nuclei of transfected L-cell fibroblasts. Colocalization with a DNA binding dye (SYTO59) revealed that, within the nucleus of control L-cells, the nonhydrolyzable fluorescent LCFA-CoA (BODIPY-C16-S-S-CoA) was distributed primarily in a punctate pattern throughout the nucleoplasm, while nonmetabolizable fluorescent LCFAs (BODIPY-C16 and BODIPY-C12) were localized primarily near the nuclear envelope membranes. L-FABP overexpression selectively increased the targeting of BODIPY-C16-S-S-CoA by 1.9- and 2.7-fold into the nuclear membrane and nucleoplasm, respectively. L-FABP also increased the targeting of fluorescent LCFAs (especially long-chain-length BODIPY-C16) by 1.7-fold to the nuclear membrane and 7.4-fold into the nucleoplasm. A cis-parinaric acid displacement assay showed that L-FABP bound BODIPY-C12 and BODIPY-C16 with K(i)s of 10.1 +/- 2.5 and 20.7 +/- 1.5 nM, respectively, in the same range as naturally occurring LCFAs. Finally, solid-phase extraction and HPLC analysis revealed that, depending on the fatty acid content of the culture medium, L-FABP expression also increased the cellular LCFA-CoA pool size and altered the LCFA-CoA acyl chain composition. Thus, L-FABP may function as a carrier for selectively enhancing the distribution of LCFA-CoA, as well as LCFA, to nuclei for potential interaction with nuclear receptors.

Very-Long-Chain and Branched-Chain Fatty Acyl-CoAs Are High Affinity Ligands for the Peroxisome Proliferator-Activated Receptor α (PPARα)

“…First, the current work presented evidence that the free acid forms of BCFA and only one free acid form of VLCFA bind to PPARα with high affinity. PPARα bound phytanic acid, pristanic acid, and some 20-carbon VLCFA with K d s of 19-76nM, well within the physiological range of free fatty acids detected in the nucleoplasm of living cells (16,17). Further, these data showed for the first time that phytanic acid did not need to be metabolized to pristanic acid to be a high affinity PPARα ligand.…”

“…The high affinities (i.e. very low nM K d s) were in the range of fatty acyl-CoA concentrations observed in nucleoplasm of living cells (16,17). Furthermore, the high affinity binding of these ligands correlated with ligandinduced conformational changes in PPARα.…”

“…These data suggested that affinities of PPARα for these ligands are very weak, i.e. K d s in the μM range, much lower than the concentrations of free fatty acids in nuclei of living cells (16,17), and thus the free acid forms of the VLCFA and BCFA may not represent the endogenous ligands. Since unesterified fatty acids are rapidly activated/metabolized to their CoA thioesters in cells and tissues (rev.…”

“…It must be noted that most of these assays utilized very high concentrations of VLCFA (i.e. 30-100 μM) and BCFA (20 μM)--several orders of magnitude higher than concentrations of unesterified fatty acids detected in nuclei of living cells (16,17). These data suggested that affinities of PPARα for these ligands are very weak, i.e.…”

Tryptophan Insertion Mutagenesis of Liver Fatty Acid-binding Protein

“…A recent study involving liver FABP knock-out mice has highlighted the significant role of this protein in binding fatty acyl CoA in vivo while the relative contributions of liver FABP and acyl CoA binding protein under varying conditions were discussed (8). Overexpression of the liver FABP gene enhanced the targeting of both fluorescent fatty acids and acyl CoAs to the nucleus (33).…”