The Peroxisomal Proliferator Clofibrate Enhances the Hepatic Cytoplasmic Movement of Fatty Acids in Rats

Abstract

The role of cytosolic fatty acid binding protein (FABP) in cellular fatty acid metabolism remains poorly defined. The intracellular movement of fatty acids is thought to be facilitated through codiffusion with FABP. Peroxisomal proliferators like clofibrate induce FABP and may stimulate fatty acid use by increasing cytoplasmic diffusion rates. Our aim was to determine if induction of FABP by clofibrate increases the cytoplasmic transport of a fluorescent fatty acid NBD-stearate. Male rats were fed clofibrate for 9 days to increase the hepatic concentration of FABP. Hepatocytes were isolated using collagenase perfusion. Cytosolic FABP was measured by enzyme-linked immunosorbent assay (ELISA) using a specific antibody to hepatic FABP. Two-dimensional laser photobleaching (FRAP) was used to measure the cytoplasmic movement of NBD-stearate in hepatocytes. Cytoplasmic transport of NBD-stearate occurred by isotropic diffusion with no evidence for convective or directed transport. Treatment with clofibrate increased FABP levels, the fraction of NBD-stearate found in cytosol, and the observed cytoplasmic diffusion rate. The increase in cytoplasmic movement exceeded that predicted from FABP binding changes suggesting that clofibrate also enhances fatty acid diffusion within intracellular membranes. Nonspecific effects of clofibrate on cytoplasmic viscosity or pore size were not observed. These data suggest that Small cytosolic proteins capable of binding hydrophobic molecules like nonesterified long chain fatty acids, bile acids, and bilirubin have been studied in hepatocytes, enterocytes, and myocytes for nearly 2 decades. 1,2 These proteins have been studied with regard to their binding affinity for a variety of ligands, 3-6 tissue localization, 7,8 and subcellular distribution. 9 However, despite an enormous body of literature, their precise function remains an enigma. 1,2 It has been hypothesized that the intracellular transport and solubility of hydrophobic molecules like fatty acids are facilitated by binding to a group of proteins called fatty acid binding proteins (FABPs). 3,5 After fatty acids enter the cell, they must be transported through the cytoplasm before they become available to metabolic enzymes. This transport has long been thought to occur by simple diffusion and to be facilitated by binding to FABP. Indirect evidence has suggested that FABP promotes cytoplasmic transport by increasing the amount of fatty acid that is mobile (bound to soluble proteins) and decreasing the amount that is relatively immobile (bound to fixed cytoplasmic membranes).We previously used the technique of fluorescence recovery after photobleaching (FRAP) to measure the intracellular movement of a fluorescent fatty acid NBD-stearate in rat hepatocytes from male and female rats. 10,11 NBD-stearate transport occurred by diffusion at a rate that was much slower than expected based on the molecular weight of NBD-stearate molecule or the NBD-stearate-FABP complex. We hypothesized that this marked retardation of the observed diffusion...

Alcoholic Liver Injury in the Rat Is Associated with Reduced Expression of Peroxisome Proliferator-α (PPARα)-Regulated Genes and Is Ameliorated by PPARα Activation

“…5). Although we did not directly evaluate PPAR␣-regulated gene expression in the clofibratetreated rats, it is reasonable to assume in light of our previous and other studies (Bass et al, 1989;Kaikaus et al, 1993a;Luxon et al, 2000;Milliano and Luxon, 2001) that clofibrate treatment restored the parameters such as L-FABP and FACO that are down-regulated by ethanol.…”

“…Based on the observation in the present study that decreased levels of PPAR␣-responsive genes contribute or are at least permissive to alcoholic liver injury, it was of interest to determine whether administration of clofibrate, a potent activator of PPAR␣, would ameliorate the adverse effects of alcohol on the liver. Clofibrate, in addition to being a PPAR␣ ligand, also stimulates the transport of fatty acids in hepatocytes (Milliano and Luxon, 2001) and increases levels of L-FABP (Kaikaus et al, 1993c). Clofibrate administration to ethanol-fed rats led to a marked decrease in the degree of fatty liver (Table 2; Fig.…”

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

“…With MPFPR, it was shown for the first time that L-FABP gene ablation specifically reduced by 2-fold the cytoplasmic, but not membrane, component of fatty acid transport/diffusion in cultured primary hepatocytes. Although earlier studies of NBDstearic acid diffusion in transfected cells, hepatocytes from drug-treated animals, and hepatocytes of male versus female mice suggested that L-FABP influenced the intracellular diffusion of NBD-stearic acid (1,42,43,45), these studies utilized confocal FPR to obtain an average diffusion coefficient of NBDstearic acid (i.e. membranes ϩ cytoplasm) and did not resolve the cytoplasmic from the membrane components of NBD-stearic acid diffusion.…”

“…Stearic Acid-The effective diffusion coefficient (D eff ) of NBD-stearic acid is an established method for determining intracellular fatty acid transport/diffusion (1,(42)(43)(44)(45). To determine whether L-FABP gene ablation altered the intracellular transport/diffusion of fatty acid, the effective diffusion coefficient (D eff ) of NBD-stearic acid was determined in cultured hepatocytes by MPFPR.…”

Transfer of fatty acids between intracellular membranes: roles of soluble binding proteins, distance, and time

“…More than 20 years ago, Tipping and Ketterer (32) proposed that soluble binding proteins may stim-ulate cytoplasmic transport of their ligands by increasing their aqueous solubility. FABP has been shown to stimulate intracellular fatty acid mobility in cultured cells (18,20,22,23), perfused rat liver (16,19), and artificial cytoplasm (29,38), whereas knock out of the gene for heart FABP was found to greatly impair cardiac fatty acid metabolism (3). Weisiger and co-workers (15,16,20) have shown that the rate of fatty acid diffusion within liver cells is directly proportional to the concentration of liver FABP, and they have proposed that soluble binding proteins act as an aqueous carrier system that reduces binding of fatty acids and other molecules to immobile cytoplasmic membranes (20,37).…”