No abstract

While low levels of unesterified long chain fatty acids (LCFAs) are normal metabolic intermediates of dietary and endogenous fat, LCFAs are also potent regulators of key receptors/enzymes, and at high levels become toxic detergents within the cell. Elevated levels of LCFAs are associated with diabetes, obesity, and metabolic syndrome. Consequently, mammals evolved fatty acid binding proteins (FABPs) that bind/sequester these potentially toxic free fatty acids in the cytosol and present them for rapid removal in oxidative (mitochondria, peroxisomes) or storage (endoplasmic reticulum, lipid droplets) organelles. Mammals have a large (15 member) family of FABPs with multiple members occurring within a single cell type. The first described FABP, liver-FABP (L-FABP, or FABP1), is expressed in very high levels (2-5% of cytosolic protein) in liver as well as intestine and kidney. Since L-FABP facilitates uptake and metabolism of LCFAs in vitro and in cultured cells, it was expected that abnormal function or loss of L-FABP would reduce hepatic LCFA uptake/ oxidation and thereby increase LCFAs available for oxidation in muscle and/or storage in adipose. This prediction was confirmed in vitro with isolated liver slices and cultured primary hepatocytes from L-FABP gene-ablated mice. Despite unaltered food consumption when fed a control diet ad libitum, the L-FABP null mice exhibited age-and sex-dependent weight gain and increased fat tissue mass. The obese phenotype was exacerbated in L-FABP null mice pair-fed a high fat diet. Taken together with other findings, these data suggest that L-FABP could have an important role in preventing age-or diet-induced obesity.

Abnormal energy regulation may significantly contribute to the pathogenesis of obesity, diabetes mellitus, cardiovascular disease, and cancer. For rapid control of energy homeostasis, allosteric and posttranslational events activate or alter activity of key metabolic enzymes. For longer impact, transcriptional regulation is more effective, especially in response to nutrients such as long chain fatty acids (LCFA). Recent advances provide insights into how poorly water-soluble lipid nutrients [LCFA; retinoic acid (RA)] and their metabolites (long chain fatty acyl Coenzyme A, LCFA-CoA) reach nuclei, bind their cognate ligand-activated receptors, and regulate transcription for signaling lipid and glucose catabolism or storage: (i) while serum and cytoplasmic LCFA levels are in the 200 mircroM-mM range, real-time imaging recently revealed that LCFA and LCFA-CoA are also located within nuclei (nM range); (ii) sensitive fluorescence binding assays show that LCFA-activated nuclear receptors [peroxisome proliferator-activated receptor-alpha (PPARalpha) and hepatocyte nuclear factor 4alpha (HNF4alpha)] exhibit high affinity (low nM KdS) for LCFA (PPARalpha) and/or LCFA-CoA (PPARalpha, HNF4alpha)-in the same range as nuclear levels of these ligands; (iii) live and fixed cell immunolabeling and imaging revealed that some cytoplasmic lipid binding proteins [liver fatty acid binding protein (L-FABP), acyl CoA binding protein (ACBP), cellular retinoic acid binding protein-2 (CRABP-2)] enter nuclei, bind nuclear receptors (PPARalpha, HNF4alpha, CRABP-2), and activate transcription of genes in fatty acid and glucose metabolism; and (iv) studies with gene ablated mice provided physiological relevance of LCFA and LCFA-CoA binding proteins in nuclear signaling. This led to the hypothesis that cytoplasmic lipid binding proteins transfer and channel lipidic ligands into nuclei for initiating nuclear receptor transcriptional activity to provide new lipid nutrient signaling pathways that affect lipid and glucose catabolism and storage.

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...

Endocannabinoids (EC) and cannabinoids are very lipophilic molecules requiring the presence of cytosolic binding proteins that chaperone these molecules to intracellular targets. While three different fatty acid binding proteins (FABP3, 5, 7) serve this function in brain, relatively little is known about how such hydrophobic EC and cannabinoids are transported within the liver. The most prominent hepatic FABP, liver fatty acid binding protein (FABP1, L-FABP), has high affinity for arachidonic acid (ARA) and ARA-CoA—suggesting that FABP1 may also bind ARA-derived ECs (AEA, 2-AG). Indeed, FABP1 bound EC with high affinity as shown by displacement of FABP1-bound fluorescent ligands and by quenching of FABP1 intrinsic tyrosine fluorescence. FABP1 also had high affinity for most non-ARA containing ECs, FABP1 inhibitors, EC uptake/hydrolysis inhibitors, phytocannabinoids, and less so synthetic cannabinoid receptor (CBR) agonists and antagonists. Physiological impact was examined with liver from wild-type (WT) versus FABP1 gene ablated (LKO) male mice. As shown by LC/MS, FABP1 gene ablation significantly increased hepatic levels of AEA, 2-AG, and 2-OG. These increases were not due to increased protein levels of EC synthetic enzymes (NAPEPLD, DAGL) or decreased level of EC degradative enzyme (FAAH), but correlated with complete loss of FABP1, decreased SCP2 (8-fold less prevalent than FABP1, but also binds ECs), and decreased degradative enzymes (NAAA, MAGL). These data indicated that FABP1 is not only the most prominent endocannabinoid and cannabinoid binding protein, but also impacts hepatic endocannabinoid levels.