Liver fatty acid binding protein (L-FABP) is the major fatty acid binding/"chaperone" protein in hepatic cytosol. Although fatty acids can be derived from the breakdown of dietary fat and glucose, relatively little is known regarding the impact of L-FABP on phenotype in the context of high dietary glucose. Potential impact was examined in wild-type (WT) and Lfabp gene ablated (LKO) female mice fed either a control or pair-fed high glucose diet (HGD). WT mice fed HGD alone exhibited decreased whole body weight gain and weight gain/kcal food consumed-both as reduced lean tissue mass (LTM) and fat tissue mass (FTM). Conversely, LKO alone increased weight gain, lean tissue mass, and fat tissue mass while decreasing serum β-hydroxybutyrate (indicative of hepatic fatty acid oxidation)-regardless of diet. Both LKO alone and HGD alone significantly altered the serum lipoprotein profile and increased triacylglycerol (TG), but in HGD mice the LKO did not further exacerbate serum TG content. HGD had little effect on hepatic lipid composition in WT mice, but prevented the LKO-induced selective increase in hepatic phospholipid, free-cholesterol and cholesteryl-ester. Taken together, these findings suggest that high glucose diet diminished the effects of LKO on the whole body and lipid phenotype of these mice.

Although serum Δ9-tetrahydrocannabinol (Δ9-THC) undergoes rapid hepatic clearance and metabolism, almost nothing is known regarding the mechanism(s) whereby this highly lipophilic phytocannabinoid is transported for metabolism/excretion. A novel NBD-arachidonoylethanolamide (NBD-AEA) fluorescence displacement assay showed that liver fatty acid binding protein (FABP1), the major hepatic endocannabinoid (EC) binding protein, binds the first major metabolite of Δ9-THC (Δ9-THC-OH) as well as Δ9-THC itself. Circular dichroism (CD) confirmed that not only Δ9-THC and Δ9-THC-OH but also downstream metabolites Δ9-THC-COOH and Δ9-THC-CO-glucuronide directly interact with FABP1. Δ9-THC and metabolite interaction differentially altered the FABP1 secondary structure, increasing total α-helix (all), decreasing total β-sheet (Δ9-THC-COOH, Δ9-THC-CO-glucuronide), increasing turns (Δ9-THC-OH, Δ9-THC-COOH, Δ9-THC-CO-glucuronide), and decreasing unordered structure (Δ9-THC, Δ9-THC-OH). Cultured primary hepatocytes from wild-type (WT) mice took up and converted Δ9-THC to the above metabolites. Fabp1 gene ablation (LKO) dramatically increased hepatocyte accumulation of Δ9-THC and even more so its primary metabolites Δ9-THC-OH and Δ9-THC-COOH. Concomitantly, rtPCR and Western blotting indicated that LKO significantly increased Δ9-THC's ability to regulate downstream nuclear receptor transcription of genes important in both EC ( Napepld > Daglb > Dagla, Naaa, Cnr1) and lipid ( Cpt1A > Fasn, FATP4) metabolism. Taken together, the data indicated that FABP1 may play important roles in Δ9-THC uptake and elimination as well as Δ9-THC induction of genes regulating hepatic EC levels and downstream targets in lipid metabolism.

Liver fatty-acid-binding protein (FABP1, L-FABP) is the major cytosolic binding/chaperone protein for both precursor arachidonic acid (ARA) and the endocannabinoid (EC) products N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG). Although FABP1 regulates hepatic uptake and metabolism of ARA, almost nothing is known regarding FABP1's impact on AEA and 2-AG uptake, intracellular distribution, and targeting of AEA and 2-AG to degradative hepatic enzymes. In vitro assays revealed that FABP1 considerably enhanced monoacylglycerol lipase hydrolysis of 2-AG but only modestly enhanced AEA hydrolysis by fatty-acid amide hydrolase. Conversely, liquid chromatography-mass spectrometry of lipids from Fabp1 gene-ablated (LKO) hepatocytes confirmed that loss of FABP1 markedly diminished hydrolysis of 2-AG. Furthermore, the real-time imaging of novel fluorescent NBD-labeled probes (NBD-AEA, NBD-2-AG, and NBD-ARA) resolved FABP1's impact on uptake vs intracellular targeting/hydrolysis. FABP1 bound NBD-ARA with 2:1 stoichiometry analogous to ARA, but bound NBD-2-AG and NBD-AEA with 1:1 stoichiometry-apparently at different sites in FABP1's binding cavity. All three probes were taken up, but NBD-2-AG and NBD-AEA were targeted to lipid droplets. LKO reduced the uptake of NBD-ARA as expected, significantly enhanced that of NBD-AEA, but had little effect on NBD-2-AG. These data indicated that FABP1 impacts hepatocyte EC levels by binding EC and differentially impacts their intracellular hydrolysis (2-AG) and uptake (AEA).

Phytocannabinoids, such as Δ9-tetrahydrocannabinol (THC), bind and activate cannabinoid (CB) receptors, thereby "piggy-backing" on the same pathway's endogenous endocannabinoids (ECs). The recent discovery that liver fatty acid binding protein-1 (FABP1) is the major cytosolic "chaperone" protein with high affinity for both Δ9-THC and ECs suggests that Δ9-THC may alter hepatic EC levels. Therefore, the impact of Δ9-THC or EC treatment on the levels of endogenous ECs, such as N-arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), was examined in cultured primary mouse hepatocytes from WT and Fabp1 gene-ablated (LKO) mice. Δ9-THC alone or 2-AG alone significantly increased AEA and especially 2-AG levels in WT hepatocytes. LKO alone markedly increased AEA and 2-AG levels. However, LKO blocked/diminished the ability of Δ9-THC to further increase both AEA and 2-AG. In contrast, LKO potentiated the ability of exogenous 2-AG to increase the hepatocyte level of AEA and 2-AG. These and other data suggest that Δ9-THC increases hepatocyte EC levels, at least in part, by upregulating endogenous AEA and 2-AG levels. This may arise from Δ9-THC competing with AEA and 2-AG binding to FABP1, thereby decreasing targeting of bound AEA and 2-AG to the degradative enzymes, fatty acid amide hydrolase and monoacylglyceride lipase, to decrease hydrolysis within hepatocytes.

While prior studies focusing on male mice suggest a role for sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x; DKO) on hepatic phytol metabolism, its role in females is unresolved. This issue was addressed using female and male wild-type (WT) and DKO mice fed a phytoestrogen-free diet without or with 0.5% phytol. GC/MS showed that hepatic: i) phytol was absent and its branched-chain fatty acid (BCFA) metabolites were barely detectable in WT control-fed mice; ii) accumulation of phytol as well as its peroxisomal metabolite BCFAs (phytanic acid » pristanic and 2,3-pristenic acids) was increased by dietary phytol in WT females, but only slightly in WT males; iii) accumulation of phytol and BCFA was further increased by DKO in phytol-fed females, but much more markedly in males. Livers of phytol-fed WT female mice as well as phytol-fed DKO female and male mice also accumulated increased proportion of saturated straight-chain fatty acids (LCFA) at the expense of unsaturated LCFA. Liver phytol accumulation was not due to increased SCP-2 binding/transport of phytol since SCP-2 bound phytanic acid, but not its precursor phytol. Thus, the loss of Scp-2/Scp-x contributed to a sex-dependent hepatic accumulation of dietary phytol and BCFA.

The endocannabinoid (eCB) neurotransmitter system regulates diverse neurological functions including stress and anxiety, pain, mood, and reward. Understanding the mechanisms underlying eCB regulation is critical for developing targeted pharmacotherapies to treat these and other neurologic disorders. Cellular studies suggest that the arachidonate eCBs, N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), are substrates for intracellular binding and transport proteins, and several candidate proteins have been identified. Initial evidence from our laboratory indicates that the lipid transport protein, sterol carrier protein 2 (SCP-2), binds to the eCBs and can regulate their cellular concentrations. Here, we present methods for evaluating SCP-2 binding of eCBs and their application to the discovery of the first inhibitor lead molecules. Using a fluorescent probe displacement assay, we found SCP-2 binds the eCBs, AEA (K-i = 0.68 +/- 0.05 mu M) and 2-AG (K-i = 0.37 +/- 0.02 mu M), with moderate affinity. A series of structurally diverse arachidonate analogues also bind SCP-2 with K-i values between 0.82 and 2.95 mu M, suggesting a high degree of tolerance for arachidonic acid head group modifications in this region of the protein. We also report initial structure-activity relationships surrounding previously reported inhibitors of Aedis aegypti SCP-2, and the results of an in silico high-throughput screen that identified structurally novel SCP-2 inhibitor leads. The methods and results reported here provide the basis for a robust probe discovery effort to fully elucidate the role of facilitated transport mediated by SCP-2 in eCB regulation and function.

Studies in vitro have suggested that both sterol carrier protein-2/sterol carrier protein-x (Scp-2/Scp-x) and liver fatty acid binding protein [Fabp1 (L-FABP)] gene products facilitate hepatic uptake and metabolism of lipotoxic dietary phytol. However, interpretation of physiological function in mice singly gene ablated in the Scp-2/Scp-x has been complicated by concomitant upregulation of FABP1. The work presented herein provides several novel insights: i) An 8-anilino-1-naphthalenesulfonic acid displacement assay showed that neither SCP-2 nor L-FABP bound phytol, but both had high affinity for its metabolite, phytanic acid; ii) GC-MS studies with phytol-fed WT and Fabp1/Scp-2/SCP-x gene ablated [triple KO (TKO)] mice showed that TKO exacerbated hepatic accumulation of phytol metabolites in vivo in females and less so in males. Concomitantly, dietary phytol increased hepatic levels of total long-chain fatty acids (LCFAs) in both male and female WT and TKO mice. Moreover, in both WT and TKO female mice, dietary phytol increased hepatic ratios of saturated/unsaturated and polyunsaturated/monounsaturated LCFAs, while decreasing the peroxidizability index. However, in male mice, dietary phytol selectively increased the saturated/unsaturated ratio only in TKO mice, while decreasing the peroxidizability index in both WT and TKO mice. These findings suggested that: 1) SCP-2 and FABP1 both facilitated phytol metabolism after its conversion to phytanic acid; and 2) SCP-2/SCP-x had a greater impact on hepatic phytol metabolism than FABP1.

The endocannabinoid (eCB) neurotransmitter system regulates diverse neurological functions including stress and anxiety, pain, mood, and reward. Understanding the mechanisms underlying eCB regulation is critical for developing targeted pharmacotherapies to treat these and other neurologic disorders. Cellular studies suggest that the arachidonate eCBs, N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), are substrates for intracellular binding and transport proteins, and several candidate proteins have been identified. Initial evidence from our laboratory indicates that the lipid transport protein, sterol carrier protein 2 (SCP-2), binds to the eCBs and can regulate their cellular concentrations. Here, we present methods for evaluating SCP-2 binding of eCBs and their application to the discovery of the first inhibitor lead molecules. Using a fluorescent probe displacement assay, we found SCP-2 binds the eCBs, AEA (Ki=0.68±0.05μM) and 2-AG (Ki=0.37±0.02μM), with moderate affinity. A series of structurally diverse arachidonate analogues also bind SCP-2 with Ki values between 0.82 and 2.95μM, suggesting a high degree of tolerance for arachidonic acid head group modifications in this region of the protein. We also report initial structure-activity relationships surrounding previously reported inhibitors of Aedis aegypti SCP-2, and the results of an in silico high-throughput screen that identified structurally novel SCP-2 inhibitor leads. The methods and results reported here provide the basis for a robust probe discovery effort to fully elucidate the role of facilitated transport mediated by SCP-2 in eCB regulation and function.

Using recombinant human wild-type fatty acid binding protein 1 (WT FABP1 T94T) and a variant (FABP1 T94A) protein, fluorescence binding assays, and circular dichroism, it was shown for the first time that WT FABP1 and the T94A variant each have a single, relatively hydrophobic site for binding fluorescent NBD-labeled analogues of N-arachidonoylethanolamide and 2-arachidonoylglycerol with high affinity. Most native N-acylethanolamides (NAEs) but only one 2-monoacylglycerol [i.e., 2-arachidonoylglycerol (2-AG)] displaced WT FABP1-bound fluorescently labeled endocannabinoids (ECs). While the T94A variant did not differ in affinity for AEA and most other NAEs, it exhibited a modestly higher affinity for OEA, as well as a higher affinity for 2-AG. Binding of AEA and 2-AG altered WT FABP1's secondary structure more extensively than any other previously examined ligand did. The T94A variant without a ligand was more susceptible to temperature-induced unfolding. While the T94A variant was much less sensitive to ligand (i.e., AEA or 2-AG)-induced conformational change, nevertheless binding of AEA and 2-AG significantly stabilized the T94A structure to thermal unfolding. These data provide the first evidence that ECs not only bind to but also alter the secondary structure of the human FABP1, with the latter markedly impacted by the T94A substitution, a variant strongly associated with hepatic accumulation of lipids and non-alcoholic fatty liver disease (NAFLD). Importantly, NAFLD has been associated with elevated hepatic levels of ECs and FABP1.

Endocannabinoids (ECs) 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, and -7) serve this function in brain, relatively little is known about how such hydrophobic ECs and cannabinoids are transported within the liver. The most prominent hepatic FABP, liver fatty acid binding protein (FABP1 or L-FABP), has high affinity for arachidonic acid (ARA) and ARA-CoA, suggesting that FABP1 may also bind ARA-derived ECs (AEA and 2-AG). Indeed, FABP1 bound ECs 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, and phytocannabinoids and less so for synthetic cannabinoid receptor (CBR) agonists and antagonists. The physiological impact was examined with liver from wild-type (WT) versus FABP1 gene-ablated (LKO) male mice. As shown by liquid chromatography and mass spectrometry, 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 and DAGL) or a decreased level of EC degradative enzyme (FAAH) but correlated with complete loss of FABP1, a decreased level of SCP2 (8-fold less prevalent than FABP1, but also binds ECs), and a decreased level of degradative enzymes (NAAA and MAGL). These data indicated that FABP1 not only is the most prominent endocannabinoid and cannabinoid binding protein but also impacts hepatic endocannabinoid levels.

Liver fatty acid-binding protein (FABP1, L-FABP) has high affinity for and enhances uptake of arachidonic acid (ARA, C20:4, n-6) which, when esterified to phospholipids, is the requisite precursor for synthesis of endocannabinoids (EC) such as arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG). The brain derives most of its ARA from plasma, taking up ARA and transporting it intracellularly via cytosolic fatty acid-binding proteins (FABPs 3,5, and 7) localized within the brain. In contrast, the much more prevalent cytosolic FABP1 is not detectable in the brain but is instead highly expressed in the liver. Therefore, the possibility that FABP1 outside the central nervous system may regulate brain AEA and 2-AG was examined in wild-type (WT) and FABP1 null (LKO) male mice. LKO increased brain levels of AA-containing EC (AEA, 2-AG), correlating with increased free and total ARA in brain and serum. LKO also increased brain levels of non-ARA that contain potentiating endocannabinoids (EC*) such as oleoyl ethanolamide (OEA), PEA, 2-OG, and 2-PG. Concomitantly, LKO decreased serum total ARA-containing EC, but not non-ARA endocannabinoids. LKO did not elicit these changes in the brain EC and EC* as a result of compensatory up-regulation of brain protein levels of enzymes in EC synthesis (NAPEPLD, DAGL) or cytosolic EC chaperone proteins (FABPs 3, 5, 7, SCP-2, HSP70), or cannabinoid receptors (CB1, TRVP1). These data show for the first timethat the non-CNS fatty acid-binding protein FABP1 markedly affected brain levels of both ARA-containing endocannabinoids (AEA, 2-AG) as well as their non-ARA potentiating endocannabinoids.

Unlike the genomes of many mammals that have a single NK-lysin gene, the cattle genome contains a family of four genes, one of which is expressed preferentially in the lung. In this study, we compared the expression of the four bovine NK-lysin genes in healthy animals to animals challenged with pathogens known to be associated with bovine respiratory disease (BRD) using transcriptome sequencing (RNA-seq). The expression of several NK-lysins, especially NK2C, was elevated in challenged relative to control animals. The effects of synthetic peptides corresponding to functional region helices 2 and 3 of each gene product were tested on both model membranes and bio-membranes. Circular dichroism spectroscopy indicated that these peptides adopted a more helical secondary structure upon binding to an anionic model membrane and liposome leakage assays suggested that these peptides disrupt membranes. Bacterial killing assays further confirmed the antimicrobial effects of these peptides on BRD-associated bacteria, including both Pasteurella multocida and Mannhemia haemolytica and an ultrastructural examination of NK-lysin-treated P. multocida cells by transmission electron microscopy revealed the lysis of target membranes. These studies demonstrate that the expanded bovine NK-lysin gene family is potentially important in host defense against pathogens involved in bovine respiratory disease.

The first discovered member of the mammalian FABP family, liver fatty acid binding protein (FABP1, L-FABP), occurs at high cytosolic concentration in liver, intestine, and in the case of humans also in kidney. While the rat FABP1 is well studied, the extent these findings translate to human FABP1 is not clear—especially in view of recent studies showing that endocannabinoids and cannabinoids represent novel rat FABP1 ligands and FABP1 gene ablation impacts the hepatic endocannabinoid system, known to be involved in non-alcoholic fatty liver (NAFLD) development. Although not detectable in brain, FABP1 ablation nevertheless also impacts brain endocannabinoids. Despite overall tertiary structure similarity, human FABP1 differs significantly from rat FABP1 in secondary structure, much larger ligand binding cavity, and affinities/specificities for some ligands. Moreover, while both mouse and human FABP1 mediate ligand induction of peroxisome proliferator activated receptor-α (PPARα), they differ markedly in pattern of genes induced. This is critically important because a highly prevalent human single nucleotide polymorphism (SNP) (26–38 % minor allele frequency and 8.3 ± 1.9 % homozygous) results in a FABP1 T94A substitution that further accentuates these species differences. The human FABP1 T94A variant is associated with altered body mass index (BMI), clinical dyslipidemias (elevated plasma triglycerides and LDL cholesterol), atherothrombotic cerebral infarction, and non-alcoholic fatty liver disease (NAFLD). Resolving human FABP1 and the T94A variant’s impact on the endocannabinoid and cannabinoid system is an exciting challenge due to the importance of this system in hepatic lipid accumulation as well as behavior, pain, inflammation, and satiety.

Although expression of the human liver fatty acid binding protein (FABP1) T94A variant alters serum lipoprotein cholesterol levels in human subjects, nothing is known whereby the variant elicits these effects. This issue was addressed by in vitro cholesterol binding assays using purified recombinant wild-type (WT) FABP1 T94T and T94A variant proteins and in cultured primary human hepatocytes expressing the FABP1 T94T (genotyped as TT) or T94A (genotyped as CC) proteins. The human FABP1 T94A variant protein had 3-fold higher cholesterol-binding affinity than the WT FABP1 T94T as shown by NBD-cholesterol fluorescence binding assays and by cholesterol isothermal titration microcalorimetry (ITC) binding assays. CC variant hepatocytes also exhibited 30% higher total FABP1 protein. HDL- and LDL-mediated NBD-cholesterol uptake was faster in CC variant than TT WT human hepatocytes. VLDL-mediated uptake of NBD-cholesterol did not differ between CC and TT human hepatocytes. The increased HDL- and LDL-mediated NBD-cholesterol uptake was not associated with any significant change in mRNA levels of SCARB1, LDLR, CETP, and LCAT encoding the key proteins in lipoprotein cholesterol uptake. Thus, the increased HDL- and LDL-mediated NBD-cholesterol uptake by CC hepatocytes may be associated with higher affinity of T94A protein for cholesterol and/or increased total T94A protein level.

Although human liver fatty acid-binding protein (FABP1) T94A variant has been associated with nonalcoholic fatty liver disease and reduced ability of fenofibrate to lower serum triglycerides (TG) to target levels, molecular events leading to this phenotype are poorly understood. Cultured primary hepatocytes from female human subjects expressing the FABP1 T94A variant exhibited increased neutral lipid (TG, cholesteryl ester) accumulation associated with (1) upregulation of total FABP1, a key protein stimulating mitochondrial glycerol-3-phosphate acyltransferase (GPAM), the rate-limiting enzyme in lipogenesis; (2) increased mRNA expression of key enzymes in lipogenesis (GPAM, LPIN2) in heterozygotes; (3) decreased mRNA expression of microsomal triglyceride transfer protein; (4) increased secretion of ApoB100 but not TG; (5) decreased long-chain fatty acid (LCFA) β-oxidation. TG accumulation was not due to any increase in LCFA uptake, de novo lipogenesis, or the alternate monoacylglycerol O-acyltransferase pathway in lipogenesis. Despite increased expression of total FABP1 mRNA and protein, fenofibrate-mediated FABP1 redistribution to nuclei and ligand-induced peroxisome proliferator-activated receptor (PPAR-α) transcription of LCFA β-oxidative enzymes (carnitine palmitoyltransferase 1A, carnitine palmitoyltransferase 2, and acyl-coenzyme A oxidase 1, palmitoyl) were attenuated in FABP1 T94A hepatocytes. Although the phenotype of FABP1 T94A variant human hepatocytes exhibits some similarities to that of FABP1-null or PPAR-α-null hepatocytes and mice, expression of FABP1 T94A variant did not abolish or reduce ligand binding. Thus the FABP1 T94A variant represents an altered/reduced function mutation resulting in TG accumulation.

The human liver fatty acid‐binding protein (L‐ FABP ) T94A variant, the most common in the FABP family, has been associated with elevated liver triglyceride levels. How this amino acid substitution elicits these effects is not known. This issue was addressed using human recombinant wild‐type ( WT ) and T94A variant L‐ FABP proteins as well as cultured primary human hepatocytes expressing the respective proteins (genotyped as TT , TC and CC ). The T94A substitution did not alter or only slightly altered L‐ FABP binding affinities for saturated, monounsaturated or polyunsaturated long chain fatty acids, nor did it change the affinity for intermediates of triglyceride synthesis. Nevertheless, the T94A substitution markedly altered the secondary structural response of L‐ FABP induced by binding long chain fatty acids or intermediates of triglyceride synthesis. Finally, the T94A substitution markedly decreased the levels of induction of peroxisome proliferator‐activated receptor α‐regulated proteins such as L‐ FABP , fatty acid transport protein 5 and peroxisome proliferator‐activated receptor α itself meditated by the polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid in cultured primary human hepatocytes. Thus, although the T94A substitution did not alter the affinity of human L‐ FABP for long chain fatty acids, it significantly altered human L‐ FABP structure and stability, as well as the conformational and functional response to these ligands.

Although the human liver fatty acid binding protein (L-FABP) T94A variant arises from the most commonly occurring single-nucleotide polymorphism in the entire FABP family, there is a complete lack of understanding regarding the role of this polymorphism in human disease. It has been hypothesized that the T94A substitution results in the complete loss of ligand binding ability and function analogous to that seen with L-FABP gene ablation. This possibility was addressed using the recombinant human wild-type (WT) T94T and T94A variant L-FABP and cultured primary human hepatocytes. Nonconservative replacement of the medium-sized, polar, uncharged T residue with a smaller, nonpolar, aliphatic A residue at position 94 of the human L-FABP significantly increased the L-FABP α-helical structure content at the expense of β-sheet content and concomitantly decreased the thermal stability. T94A did not alter the binding affinities for peroxisome proliferator-activated receptor α (PPARα) agonist ligands (phytanic acid, fenofibrate, and fenofibric acid). While T94A did not alter the impact of phytanic acid and only slightly altered that of fenofibrate on the human L-FABP secondary structure, the active metabolite fenofibric acid altered the T94A secondary structure much more than that of the WT T94T L-FABP. Finally, in cultured primary human hepatocytes, the T94A variant exhibited a significantly reduced extent of fibrate-mediated induction of PPARα-regulated proteins such as L-FABP, FATP5, and PPARα itself. Thus, while the T94A substitution did not alter the affinity of the human L-FABP for PPARα agonist ligands, it significantly altered the human L-FABP structure, stability, and conformational and functional response to fibrate.

Candida albicans is an opportunistic human pathogen. Emergence of drug-resistant strains makes it necessary to develop new anti- Candida drugs. Vps4 is an AAA ATPase critical for the function of the ESCRT system. Studies have shown that the deletion of Vps4 is sufficient to abolish the pathogenicity of Candida albicans . In this work, we performed a virtual screening study on the homology model of the Candida albicans Vps4. Our results provide a list of compounds that can be later validated experimentally. The simulation found that the aromatic rings of small molecules can penetrate into a tunnel at the purine binding pocket that is not fully occupied by ATP. This tunnel also shows marked structural variations between the human and Candida albicans proteins. Therefore, by exploring this tunnel, we may develop potent competitive inhibitors that can distinguish the human and Candida albicans Vps4.

Although liver fatty acid binding protein (L-FABP) binds fibrates and PPARα in vitro and enhances fibrate induction of PPARα in transformed cells, the functional significance of these findings is unclear, especially in normal hepatocytes. Studies with cultured primary mouse hepatocytes show that: 1) At physiological (6 mM) glucose, fibrates (bezafibrate, fenofibrate) only weakly activated PPARα transcription of genes in LCFA β-oxidation; 2) High (11–20 mM) glucose, but not maltose (osmotic control), significantly potentiated fibrate-induction of mRNA of these and other PPARα target genes to increase LCFA β-oxidation. These effects were associated with fibrate-mediated redistribution of L-FABP into nuclei—an effect prolonged by high glucose—but not with increased de novo fatty acid synthesis from glucose; 3) Potentiation of bezafibrate action by high glucose required an intact L-FABP/PPARα signaling pathway as shown with L-FABP null, PPARα null, PPARα inhibitor-treated WT, or PPARα-specific fenofibrate-treated WT hepatocytes. High glucose alone in the absence of fibrate was ineffective. Thus, high glucose potentiation of PPARα occurred through FABP/PPARα rather than indirectly through other PPARs or glucose induced signaling pathways. These data indicated L-FABP's importance in fibrate-induction of hepatic PPARα LCFA β-oxidative genes, especially in the context of high glucose levels.

While TOFA (acetyl CoA carboxylase inhibitor) and C75 (fatty acid synthase inhibitor) prevent lipid accumulation by inhibiting fatty acid synthesis, the mechanism of action is not simply accounted for by inhibition of the enzymes alone. Liver fatty acid binding protein (L-FABP), a mediator of long chain fatty acid signaling to peroxisome proliferator-activated receptor-α (PPARα) in the nucleus, was found to bind TOFA and its activated CoA thioester, TOFyl-CoA, with high affinity while binding C75 and C75-CoA with lower affinity. Binding of TOFA and C75-CoA significantly altered L-FABP secondary structure. High (20 mM) but not physiological (6 mM) glucose conferred on both TOFA and C75 the ability to induce PPARα transcription of the fatty acid β-oxidative enzymes CPT1A, CPT2, and ACOX1 in cultured primary hepatocytes from wild-type (WT) mice. However, L-FABP gene ablation abolished the effects of TOFA and C75 in the context of high glucose. These effects were not associated with an increased cellular level of unesterified fatty acids but rather by increased intracellular glucose. These findings suggested that L-FABP may function as an intracellular fatty acid synthesis inhibitor binding protein facilitating TOFA and C75-mediated induction of PPARα in the context of high glucose at levels similar to those in uncontrolled diabetes.