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183

Although liver fatty acid-binding protein (L-FABP) isan important binding site for various hydrophobic ligands in hepatocytes, its in vivo significance is not understood. We have therefore created L-FABP null mice and report here their initial analysis, focusing on the impact of this mutation on hepatic fatty acid binding capacity, lipid composition, and expression of other lipid-binding proteins. Gel-filtered cytosol from L-FABP null liver lacked the main fatty acid binding peak in the fraction that normally comprises both L-FABP and sterol carrier protein-2 (SCP-2). The binding capacity for cis-parinaric acid was decreased >80% in this region. Molar ratios of cholesterol/cholesterol ester, cholesteryl ester/triglyceride, and cholesterol/phospholipid were 2-to 3-fold greater, reflecting up to 3-fold absolute increases in specific lipid classes in the order cholesterol > cholesterol esters > phospholipids. In contrast, the liver pool sizes of nonesterified fatty acids and triglycerides were not altered. However, hepatic deposition of a bolus of intravenously injected family (1-3), is found in the liver, intestine, and kidney, but only in liver is it not co-expressed with other members of its family. L-FABP is known to bind fatty acids and various other hydrophobic molecules, although its actual contribution to the lipid-binding capacity of liver cytosol is not known. Given that L-FABP is expressed at very high levels (2-5% of cytosolic protein) in the differentiated hepatocyte (4, 5) and that these levels correlate well with lipid metabolism (2), it can be speculated that L-FABP contributes considerably to hepatic lipidbinding and lipid metabolism. Work with cell-free systems and transfected cells has further strengthened this view. For example, in cell-free preparations L-FABP was shown to stimulate the esterification of oleic acid while inhibiting that of palmitic acid (6). L cells overexpressing L-FABP show increased rates of fatty acid uptake and esterification (7) as well as increased contents of phospholipid and cholesterol esters (8, 9). HepG2 hepatoma cells expressing an L-FABP antisense RNA showed a dose-dependent reduction of fatty acid uptake (10). Furthermore, overexpression of L-FABP in McA-RH7777 hepatoma cells incubated with palmitic acid decreased the synthesis and secretion of triglycerides while increasing beta oxidation and the secretion of apolipoprotein B100 (11). Thus, the various in vitro systems have allowed researchers to propose specific functions of L-FABP in vivo.However, in vitro studies of FABPs have inherent limitations. The only firmly established function of FABPs is the reversible binding of hydrophobic ligands, and these proteins do not exhibit any enzymatic function or energy requirement. This suggests that these proteins play passive (facilitative) roles that, almost by definition, are strongly dependent on the cellular context. One context of the highly expressed L-FABP is the highly differentiated hepatocyte, a cell type featuring an intense lipid metabolism that is not eas...

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Decreased Liver Fatty Acid Binding Capacity and Altered Liver Lipid Distribution in Mice Lacking the Liver Fatty Acid-binding Protein Gene

Martin

Danneberg

Kumar

et al. 2003

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157

183

“…Examination of multiple chambers of cultured hepatocytes with this dual stain assay revealed that cell viability was typically also Ͼ90%. Third, hepatocyte morphology was monitored as previously described (29). Briefly, primary hepatocytes were cultured in serum-free medium supplemented with BSA (4 M) and seeded onto chambered cover glass slides at a cell density of 5 ϫ 10 5 cells/chamber.…”

Section: Methodsmentioning

confidence: 99%

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

Atshaves

McIntosh

Lyuksyutova

et al. 2004

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155

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. , 7), specific effects of L-FABP gene ablation on liver uptake of straight-chain fatty acid and liver lipid distribution are complex, apparently depending on feeding status, gender, and age of the mice (6 -9).Although relatively little is known about the role of L-FABP in the oxidation of straight-chain fatty acids, which occurs primarily in mitochondria (reviewed in Refs. 1-3), recent studies with L-FABP gene-ablated mice suggest that L-FABP may affect liver oxidation of straight-chain fatty acids under high fatty acid load. Under fed conditions, serum free fatty acid levels were found to be low, and ␤-hydroxybutyrate levels were unaltered in L-FABP (Ϫ/Ϫ) male or female mice, suggesting that L-FABP may not play a role in fatty acid oxidation (8, 9). However, under starvation conditions, serum fatty acid levels were highly elevated, and serum ␤-hydroxybutyrate levels were reduced. These findings led to the conclusion that under fasting conditions L-FABP gene ablation reduces fatty acid oxidation (8, 9). However, 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). In contrast, 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.In contrast to the above studies with straight-chain fatty acids, almost nothing is known regarding potential roles of L-FABP in the uptake, oxidation, and esterification of branched-chain fatty acids. The most common dietary branched-chain fatty acid, phytanic acid, is produced by ruminants in the gut by bacterial cleavage of the side chain of chlorophyll to yield phytol, followed by conversion to phytanic acid (reviewed in Ref. 10). Consequently, levels of...

“…The high distribution to the fluorescent LCFAs to cytoplasm reflected the predominant localization of soluble proteins that bind LCFAs, i.e. FABPs (42,44,63). Mock-transfected control L-cells contain an endogenous FABP immunologically distinct from L-FABP (45).…”

Section: L-fabp Expression Stimulates Uptake and Nuclear Distributionmentioning

confidence: 99%

Liver Fatty Acid-binding Protein Targets Fatty Acids to the Nucleus

Huang¹,

Starodub²,

McIntosh³

et al. 2002

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131

Because the nuclear peroxisome proliferator-activated receptors (PPAR) 1 regulate expression of genes involved in fatty acid oxidation (PPAR␣) and adiposity (PPAR␥), it has been postulated that PPARs play important roles in diabetes, atherosclerosis, and obesity (reviewed in Refs. 1-6). In support of this hypothesis, fatty acid oxidation is diminished in PPAR␣ geneablated mice (7-9) accompanied by fat accumulation (heart (10), centrilobular regions of the liver (7, 8), adipose tissue (11)), hypercholesterolemia (12), and hypoglycemia (10). (13). These data strongly support the possibility that LCFAs may be natural ligands for PPARs and that LCFAs may be more specific ligands for PPAR␣ as compared with other PPARs. Unfortunately, physiological significance of these findings is not yet clear since it is not known whether unesterified, unbound LCFAs are present in the cell and that their concentration in the nucleus is at least in the nanomolar range.A variety of studies suggest that unesterified LCFAs are present within the cell at levels physiologically significant for regulating PPAR␣ activity. Based on the Michaelis constant of long chain fatty acyl-CoA synthetase, the total cellular unesterified LCFA concentration has been estimated near 20 M (18). However, because of the presence of intracellular fatty acid-binding proteins (FABPs) (reviewed in Ref. 19), the intracellular concentration of unbound, unesterified LCFAs is thought to be nearly 3 orders of magnitude lower, near 7-50 nM in liver, heart, and intestine (20, 21). Whether nuclear levels of unbound LCFAs are also in the nanomolar range is not known. Another key question is the origin of nuclear unesterified LCFAs. Nuclei do not synthesize LCFAs but must import them from the cytosol (22-24) by as yet unresolved transport systems. Nearly 20 and 4% of the plasma-derived unesterified LCFAs appear in nuclei isolated from hepatocytes (23) and cerebral cortex or leukemic lymphocytes (22, 24 -26), respectively. Despite data suggesting the presence of high (up to 40% of nuclear lipids) levels of unesterified LCFAs in isolated nuclei (27), subcellular fractionation studies may be complicated by LCFAs derived from lipolytic release, transfer from other intracellular sites, or cross-contamination with other mem-