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.

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.

were increased 48% and 115%, respectively, in SCP-2-expressing cells. Concomitantly, the level of the lipid droplet-specific adipose differentiation-related protein decreased 70%. Overall, HDL-mediated sterol efflux from L-cell fibroblasts reflected that of the cytoplasmic rather than lipid droplet compartment. SCP-2 differentially modulated sterol efflux from the two cytoplasmic pools. However, net efflux was determined primarily by inhibition of the slowly effluxing pool rather than by acceleration of the rapid protein-mediated pool. Finally, SCP-2 expression also inhibited sterol efflux from lipid droplets, an effect related to decreased adipose differentiation-related protein, a lipid droplet surface protein that binds cholesterol with high affinity.Although the HDL-mediated 1 steps of cholesterol transfer from the cell surface membrane and subsequent fate of cholesterol in the vasculature have been extensively studied, much less is known about intracellular components of cholesterol efflux (reviewed in Refs. 1-5). Plasma membrane cholesterol is distributed into multiple pools or domains (reviewed in Refs. 6 and 7). It is now recognized that there may be a connection between such domains and HDL receptor-mediated reverse cholesterol transport (reviewed in Refs. 7-9). The transbilayer distribution of cholesterol in plasma membranes is asymmetric, with the cholesterol enriched 400% in the cytofacial leaflet versus exofacial leaflet (reviewed in Refs. 10 -14). Transbilayer movement of cholesterol across the plasma membrane appears fast (t1 ⁄2 ϭ 1-6 min; reviewed in Ref. 10). Plasma membrane cholesterol is also distributed into lateral cholesterol-rich and -poor membrane domains (reviewed in Refs. 6 and 10). Most of the cholesterol in the plasma membrane is localized in lateral domains that are, for the most part, relatively inert in terms of transfer kinetics (i.e. t1 ⁄2 ϭ hours to days), and movement between such domains is also slow. However, a small pool of plasma membrane cholesterol appears highly dynamic (reviewed in Ref. 6) and is associated with cholesterol-rich, HDL receptor containing microdomains called caveolae (reviewed in Refs. 8, 9, 15, and 16). Molecular details of cholesterol entry/ exit, cholesterol organization, and mechanism(s) of cholesterol transbilayer movement in caveolae remain to be determined. Likewise, the relationships between caveolae, "rafts," and other cholesterol-rich plasma membrane microdomains is not yet clear (reviewed in Ref. 9).The intracellular steps preceding cellular cholesterol efflux include transfer of cholesterol from the Golgi, endoplasmic reticulum, and lipid droplets to the plasma membrane (reviewed in Refs. 1, 9, and 17). The time frame of bidirectional vesicular transfer of cholesterol between plasma membranes and Golgi has a t1 ⁄2 of 10 -20 min (reviewed in Refs. 8,18,and 19). Alternately, molecular cholesterol transfer, mediated by cholesterol-binding proteins in the cytoplasm, occurs much faster (t1 ⁄2 near 1-2 min) from the lysosome (exogenous cholesterol) ...