Despite the importance of peroxisomal oxidation in branched-chain lipid (phytol, cholesterol) detoxification, little is known regarding the factors regulating the peroxisomal uptake, targeting, and metabolism of these lipids. Although in vitro data suggest that sterol carrier protein (SCP)-x plays an important role in branched-chain lipid oxidation, the full physiological significance of this peroxisomal enzyme is not completely clear. To begin to resolve this issue, SCP-x-null mice were generated by gene ablation of SCP-x from the SCP-x/SCP-2 gene and fed a phytol-enriched diet to characterize the effects of lipid overload in a system with minimal 2/3-oxoacyl-CoA thiolytic activity. It was shown that SCP-x gene ablation 1) did not result in reduced expression of SCP-2 (previously thought to be derived in considerable part by posttranslational cleavage of SCP-x); 2) increased expression levels of key enzymes involved in alpha- and beta-oxidation; and 3) altered lipid distributions, leading to decreased hepatic fatty acid and triglyceride levels. In response to dietary phytol, lack of SCP-x resulted in 1) accumulation of phytol metabolites despite substantial upregulation of hepatic peroxisomal and mitochondrial enzymes; 2) reduced body weight gain and fat tissue mass; and 3) hepatic enlargement, increased mottling, and necrosis. In summary, the present work with SCP-x gene-ablated mice demonstrates, for the first time, a direct physiological relationship between lack of SCP-x and decreased ability to metabolize branched-chain lipids.

(L-FABP) in the metabolism of branched-chain fatty acids has been suggested based on data obtained with cultured cells, the physiological significance of this observation remains to be demonstrated. To address this issue, the lipid phenotype and metabolism of phytanic acid, a branched-chain fatty acid, were determined in L-FABP gene-ablated mice fed a diet with and without 1% phytol (a metabolic precursor to phytanic acid). In response to dietary phytol, L-FABP gene ablation exhibited a gender-dependent lipid phenotype. Livers of phytol-fed female L-FABPϪ/Ϫ mice had significantly more fatty lipid droplets than male L-FABPϪ/Ϫ mice, whereas in phytolfed wild-type L-FABPϩ/ϩ mice differences between males and females were not significant. Thus L-FABP gene ablation exacerbated the accumulation of lipid droplets in phytol-fed female, but not male, mice. These results were reflected in the lipid profile, where hepatic levels of triacylglycerides in phytol-fed female L-FABPϪ/Ϫ mice were significantly higher than in male L-FABPϪ/Ϫ mice. Furthermore, livers of phytol-fed female L-FABPϪ/Ϫ mice exhibited more necrosis than their male counterparts, consistent with the accumulation of higher levels of phytol metabolites (phytanic acid, pristanic acid) in liver and serum, in addition to increased hepatic levels of sterol carrier protein (SCP)-x, the only known peroxisomal enzyme specifically required for branched-chain fatty acid oxidation. In summary, L-FABP gene ablation exerted a significant role, especially in female mice, in branched-chain fatty acid metabolism. These effects were only partially compensated by concomitant upregulation of SCP-x in response to L-FABP gene ablation and dietary phytol. gene targeting; phytanic acid ALTHOUGH THE LIVER FATTY ACID binding protein (L-FABP) is the first known and perhaps most thoroughly studied member of the large family of intracellular transport proteins involved in long-chain fatty acid (LCFA) metabolism, its full physiological function is only now beginning to be resolved (reviewed in Ref. 40). Early studies focused on the structure of L-FABP, resolution of its ligand binding site, and determination of ligand specificity (reviewed in Refs. 15,17,40,48,64,72). Although L-FABP has high affinity for both straight (reviewed in Refs. 15, 17, 40)-and branched (16, 75)-chain LCFAs, as well as their CoA derivatives, most previous investigations to elucidate L-FABP's physiological function focused on its roles in the uptake and metabolism of straight-chain LCFAs. Cultured cell and in vitro studies suggest that L-FABP enhances multiple aspects of straight-chain LCFA metabolism, including 1) cellular LCFA uptake (9,37,40,44,45,(51)(52)(53)74), 2) cytoplasmic LCFA and LCFA-CoA binding capacity and pool size (37, 38), 3) intracellular LCFA transport and diffusion (35,40,44,69,70) and LCFA-CoA transport (54, 55), 4) LCFA targeting to microsomal LCFA-CoA synthase and LCFA-CoA pool size regulation (10,20,41,47), 5) LCFA-CoA targeting for microsomal transacylation to phosphatidic acid (7,27,...

Although liver fatty acid binding protein (L-FABP) is postulated to influence cholesterol homeostasis, the physiological significance of this hypothesis remains to be resolved. This issue was addressed by examining the response of young (7 wk) female mice to L-FABP gene ablation and a cholesterol-rich diet. In control-fed mice, L-FABP gene ablation alone induced hepatic cholesterol accumulation (2.6-fold), increased bile acid levels, and increased body weight gain (primarily as fat tissue mass). In cholesterol-fed mice, L-FABP gene ablation further enhanced the hepatic accumulation of cholesterol (especially cholesterol ester, 12-fold) and potentiated the effects of dietary cholesterol on increased body weight gain, again mainly as fat tissue mass. However, in contrast to the effects of L-FABP gene ablation in control-fed mice, biliary levels of bile acids (as well as cholesterol and phospholipids) were reduced. These phenotypic alterations were not associated with differences in food intake. In conclusion, it was shown for the first time that L-FABP altered cholesterol metabolism and the response of female mice to dietary cholesterol. While the biliary and lipid phenotype of female wild-type L-FABP+/+ mice was sensitive to dietary cholesterol, L-FABP gene ablation dramatically enhanced many of the effects of dietary cholesterol to greatly induce hepatic cholesterol (primarily cholesterol ester) and triacylglycerol accumulation as well as to potentiate body weight gain (primarily as fat tissue mass). Taken together, these data support the hypothesis that L-FABP is involved in the physiological regulation of cholesterol metabolism, body weight gain, and obesity.

Although the physiological roles of the individual bile acid synthetic enzymes have been extensively examined, relatively little is known regarding the function of intracellular bile acid-binding proteins. Male L-FABP (liver fatty-acid-binding protein) gene-ablated mice were used to determine a role for L-FABP, the major liver bile acid-binding protein, in bile acid and biliary cholesterol metabolism. First, in control-fed mice L-FABP gene ablation alone increased the total bile acid pool size by 1.5-fold, especially in gall-bladder and liver, but without altering the proportions of bile acid, cholesterol and phospholipid. Loss of liver L-FABP was more than compensated by up-regulation of: other liver cytosolic bile acid-binding proteins [GST (glutathione S-transferase), 3alpha-HSD (3alpha-hydroxysteroid dehydrogenase)], key hepatic bile acid synthetic enzymes [CYP7A1 (cholesterol 7alpha-hydroxylase) and CYP27A1 (sterol 27alpha-hydroxylase)], membrane bile acid translocases [canalicular BSEP (bile salt export pump), canalicular MRP2 (multidrug resistance associated protein 2), and basolateral/serosal OATP-1 (organic anion transporting polypeptide 1)], and positive alterations in nuclear receptors [more LXRalpha (liver X receptor alpha) and less SHP (short heterodimer partner)]. Secondly, L-FABP gene ablation reversed the cholesterol-responsiveness of bile acid metabolic parameters such that total bile acid pool size, especially in gall-bladder and liver, was reduced 4-fold, while the mass of biliary cholesterol increased 1.9-fold. The dramatically reduced bile acid levels in cholesterol-fed male L-FABP (-/-) mice were associated with reduced expression of: (i) liver cytosolic bile acid-binding proteins (L-FABP, GST and 3alpha-HSD), (ii) hepatic bile acid synthetic enzymes [CYP7A1, CYP27A1 and SCP-x (sterol carrier protein-x/3-ketoacyl-CoA thiolase)] concomitant with decreased positive nuclear receptor alterations (i.e. less LXRalpha and more SHP), and (iii) membrane bile acid transporters (BSEP, MRP2 and OATP-1). These are the first results suggesting a physiological role for the major cytosolic bile acid-binding protein (L-FABP) in influencing liver bile metabolic phenotype and gall-bladder bile lipids of male mice, especially in response to dietary cholesterol.

While a high-cholesterol diet induces hepatic steatosis, the role of intracellular sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x) proteins is unknown. We hypothesized that ablating SCP-2/SCP-x [double knockout (DKO)] would impact hepatic lipids (cholesterol and cholesteryl ester), especially in high-cholesterol-fed mice. DKO did not alter food consumption, and body weight (BW) gain decreased especially in females, concomitant with hepatic steatosis in females and less so in males. DKO-induced steatosis in control-fed wild-type (WT) mice was associated with 1) loss of SCP-2; 2) upregulation of liver fatty acid binding protein (L-FABP); 3) increased mRNA and/or protein levels of sterol regulatory element binding proteins (SREBP1 and SREBP2) as well as increased expression of target genes of cholesterol synthesis (Hmgcs1 and Hmgcr) and fatty acid synthesis (Acc1 and Fas); and 4) cholesteryl ester accumulation was also associated with increased acyl-CoA cholesterol acyltransferase-2 (ACAT2) in males. DKO exacerbated the high-cholesterol diet-induced hepatic cholesterol and glyceride accumulation, without further increasing SREBP1, SREBP2, or target genes. This exacerbation was associated both with loss of SCP-2 and concomitant downregulation of Ceh/Hsl, apolipoprotein B (ApoB), MTP, and/or L-FABP protein expression. DKO diminished the ability to secrete excess cholesterol into bile and oxidize cholesterol to bile acid for biliary excretion, especially in females. This suggested that SCP-2/SCP-x affects cholesterol transport to particular intracellular compartments, with ablation resulting in less to the endoplasmic reticulum for SREBP regulation, making more available for cholesteryl ester synthesis, for cholesteryl-ester storage in lipid droplets, and for bile salt synthesis and/or secretion. These alterations are significant findings, since they affect key processes in regulation of sterol metabolism.