Binding Of Long-chain Fatty Acids Research Articles

Identification of Nine mRNA Signatures for Sepsis Using Random Forest.

Sepsis has high fatality rates. Early diagnosis could increase its curating rates. There were no reliable molecular biomarkers to distinguish between infected and uninfected patients currently, which limit the treatment of sepsis. To this end, we analyzed gene expression datasets from the GEO database to identify its mRNA signature. First, two gene expression datasets (GSE154918 and GSE131761) were downloaded to identify the differentially expressed genes (DEGs) using Limma package. Totally 384 common DEGs were found in three contrast groups. We found that as the condition worsens, more genes were under disorder condition. Then, random forest model was performed with expression matrix of all genes as feature and disease state as label. After which 279 genes were left. We further analyzed the functions of 279 important DEGs, and their potential biological roles mainly focused on neutrophil threshing, neutrophil activation involved in immune response, neutrophil-mediated immunity, RAGE receptor binding, long-chain fatty acid binding, specific granule, tertiary granule, and secretory granule lumen. Finally, the top nine mRNAs (MCEMP1, PSTPIP2, CD177, GCA, NDUFAF1, CLIC1, UFD1, SEPT9, and UBE2A) associated with sepsis were considered as signatures for distinguishing between sepsis and healthy controls. Based on 5-fold cross-validation and leave-one-out cross-validation, the nine mRNA signature showed very high AUC.

0.1 THz exposure affects primary hippocampus neuron gene expression via alternating transcription factor binding.

In recent years, many studies have been conducted to investigate the influence of terahertz (THz) radiation on the gene expression in various cell types, but the underling molecular mechanism has not yet been fully revealed. In this study, we explored the effects of 0.1 THz radiation on the gene expression in primary neuron cells through RNA-seq analysis. 111 up-regulated and 54 down-regulated genes were identified. Several biomolecule binding related categories such as "long-chain fatty acid binding", "tropomyosin binding", "BMP receptor binding", as well as "GTPase binding" and "phospholipid binding" were enriched by GO analysis. Moreover, the GSEA analysis indicated that genes encoding protein biosynthetic machinery ribosome were up-regulated by 0.1 THz irradiation. In addition, we demonstrated that the binding efficiency of a transcription factor (TF) AP-1 with its transcription factor binding site (TFBS) in DNA was reduced by THz irradiation, which suggested that THz irradiation might affect the interaction between TFs with DNA and consequently regulate the gene expression. Our results provide new insights into the biological effects of terahertz irradiation.

Cellular and Molecular Mechanisms of Fat Taste Perception.

During the last couples of years, a number of studies have increasingly accumulated on the gustatory perception of dietary fatty acids in rodent models and human beings in health and disease. There is still a debate to coin a specific term for the gustatory perception of dietary fatty acids either as the sixth basic taste quality or as an alimentary taste. Indeed, the psycho-physical cues of orosensory detection of dietary lipids are not as distinctly perceived as other taste qualities like sweet or bitter. The cellular and molecular pharmacological mechanisms, triggered by the binding of dietary long-chain fatty acids (LCFAs) to tongue taste bud lipid receptors like CD36 and GPR120, involve Ca2+ signaling as other five basic taste qualities. We have not only elucidated the role of Ca2+ signaling but also identified different components of the second messenger cascade like STIM1 and MAP kinases, implicated in fat taste perception. We have also demonstrated the implication of Calhm1 voltage-gated channels and store-operated Ca2+ (SOC) channels like Orai1, Orai1/3, and TRPC3 in gustatory perception of dietary fatty acids. We have not only employed siRNA technology in vitro and ex vivo on tissues but also used animal models of genetic invalidation of STIM1, ERK1, Orai1, Calhm1 genes to explore their implications in fat taste signal transduction. Moreover, our laboratory has also demonstrated the importance of LCFAs detection dysfunction in obesity in animal models and human beings.

Structural models of mitochondrial uncoupling proteins obtained in DPC micelles are not functionally relevant.

Uncoupling protein 1 (UCP1) is found in the inner mitochondrial membrane of brown adipocytes. In the presence of long-chain fatty acids (LCFAs), UCP1 increases the proton conductance, which, in turn, increases fatty acid oxidation and energy release as heat. Atomic models of UCP1 and UCP2 have been generated based on the NMR backbone structure of UCP2 in dodecylphosphocholine (DPC), a detergent known to inactivate UCP1. Based on NMR titration experiments on UCP1 with LCFA, it has been proposed that K56 and K269 are crucial for LCFA binding and UCP1 activation. Given the numerous controversies on the use of DPC for structure-function analyses of membrane proteins, we revisited those UCP1 mutants in a more physiological context by expressing them in the mitochondria of Saccharomycescerevisiae. Mitochondrial respiration, assayed on permeabilized spheroplasts, enables the determination of UCP1 activation and inhibition. The K56S, K269S, and K56S/K269S mutants did not display any default in activation, which shows that the NMR titration experiments in DPC detergent are not relevant to UCP1 function.

Nitro-oleic acid, a ligand of CD36, reduces cholesterol accumulation by modulating oxidized-LDL uptake and cholesterol efflux in RAW264.7 macrophages

Macrophages play a pivotal role in the early stages of atherosclerosis development; they excessively accumulate cholesterol in the cytosol in response to modified Low Density Lipoprotein (mLDL). The mLDL are incorporated through scavenger receptors. CD36 is a high-affinity cell surface scavenger receptor that facilitates the binding and uptake of long-chain fatty acids and mLDL into the cell. Numerous structurally diverse ligands can initiate signaling responses through CD36 to regulate cell metabolism, migration, and angiogenesis. Nitro-fatty acids are endogenous electrophilic lipid mediators that react with and modulate the function of multiple enzymes and transcriptional regulatory proteins. These actions induce the expression of several anti-inflammatory and cytoprotective genes and limit pathologic responses in experimental models of atherosclerosis, cardiac ischemia/reperfusion, and inflammatory diseases. Pharmacological and genetic approaches were used to explore the actions of nitro-oleic acid (NO2-OA) on macrophage lipid metabolism. Pure synthetic NO2-OA dose-dependently increased CD36 expression in RAW264.7 macrophages and this up-regulation was abrogated in BMDM from Nrf2-KO mice. Ligand binding analysis revealed that NO2-OA specifically interacts with CD36, thus limiting the binding and uptake of mLDL. Docking analysis shows that NO2-OA establishes a low binding energy interaction with the alpha helix containing Lys164 in CD36. NO2-OA also restored autophagy flux in mLDL-loaded macrophages, thus reversing cholesterol deposition within the cell. In aggregate, these results indicate that NO2-OA reduces cholesterol uptake by binding to CD36 and increases cholesterol efflux by restoring autophagy.

The ligand-mediated affinity of brain-type fatty acid-binding protein for membranes determines the directionality of lipophilic cargo transport

The intracellular transport of lipophilic cargoes is a highly dynamic process. In eukaryotic cells, the uptake and release of long-chain fatty acids (LCFAs) are executed by fatty-acid binding proteins. However, how these carriers control the directionality of cargo trafficking remains unclear. Here, we revealed that the unliganded archetypal Drosophila brain-type fatty acid-binding protein (dFABP) possesses a stronger binding affinity than its liganded counterpart for empty nanodiscs (ND). Titrating unliganded dFABP and nanodiscs with LCFAs rescued the broadening of FABP cross-peak intensities in HSQC spectra from a weakened protein–membrane interaction. Two out of the 3 strongest LCFA contacting residues in dFABP identified by NMR HSQC chemical shift perturbation (CSP) are also part of the 30 ND-contacting residues (out of the total 130 residues in dFABP), revealed by attenuated TROSY signal in the presence of lipid ND to apo-like dFABP. Our crystallographic temperature factor data suggest enhanced αII helix dynamics upon LCFA binding, compensating for the entropic loss in the βC-D/βE-F loops. The aliphatic tail of bound LCFA impedes the charge-charge interaction between dFABP and the head groups of the membrane, and dFABP is prone to dissociate from the membrane upon ligand binding. We therefore conclude that lipophilic ligands participate directly in the control of the functionally required membrane association and dissociation of FABPs.

Fatty-Acid Receptor CD36 Functions As a Hydrogen Sulfide-Targeting Receptor with Its Cys333-Cys272 Disulfide Bond Serving As a Specific Molecular Switch to Accelerate Gastric Cancer Metastasis

Background: There is a large body of evidence suggesting that hydrogen sulfide (H2S) plays an important role in cancer metastasis; however, the molecular mechanisms of H2S-mediated acceleration of cancer metastasis remain unknown. Methods: We used RNA-sequencing to identify potential differences in gene expression in vitro. Using mass spectrometry, we identified a disulfide complex within CD36 that was labile to H2S-mediated modification. Targeting CD36 in vivo blocked H2S-promoted gastric cancer (GC) metastasis and improved animal survival. Tumor patients' samples were used for clinical translational significance evaluation. Findings: H2S triggered lipid metabolism reprogramming by significantly up-regulating the expression of the fatty-acid receptor CD36 and directly activating CD36 in GC cells. Mechanistically, a disulfide bond located between cysteine (Cys)333 and Cys272 within the CD36 protein structure that was labile to H2S-mediated modification. The long chain-fatty acid (LC-FA) binding pocket was capped by a turn in the CD36 protein, located between helical and sheet structures that were stabilized by the Cys333-Cys272. This limited the secondary binding between LC-FAs and lysine (Lys)334. Breaking the Cys333-Cys272 disulfide bond restored the second LC-FA binding conformation of CD36. Interpretation: CD36 gene transcription and CD36 subcellular recycling formed a positive feedback loop, and this disulfide bond within CD36 acted as a special molecular for this positive feedback. H2S opened the switch and induced LC-FA-mediated signaling, thus accelerating the metastasis of GC. Genetic and pharmacological blockade of CD36 function could be harnessed as an anti-metastasis therapeutic strategy. Therefore, we propose an effective strategy for anti-metastasis by blocking CD36. Funding: This work was supported by the Strategic Priority Research Program of the Science and Technology Commission of Shanghai (1312JC1408402 for Zhirong Wang). Declaration of Interest: All authors have no potential conflicts of interest. Ethical Approval: This animal study was conducted in accordance with the rules and regulations of the Institutional Animal Care and Use Committee (IACUC) at the Department of Laboratory Animal Science, Fudan University (Shanghai, P.R. China). Informed consent was approved by the Ethics Committees of Tongji Hospital and all subjects gave written informed consent.

Fatty-acid receptor CD36 functions as a hydrogen sulfide-targeted receptor with its Cys333-Cys272 disulfide bond serving as a specific molecular switch to accelerate gastric cancer metastasis.

BackgroundHydrogen Sulfide (H2S), a third member of gasotransmitter family along with nitric oxide (NO) and carbon monoxide (CO), exerts a wide range of cellular and molecular actions in our body. There is a large body of evidence suggesting that H2S plays an important role in cancer metastasis; however, the molecular mechanisms of H2S-mediated acceleration of cancer metastasis remain unknown.MethodsWe examined the promote effects of H2S on phenotype of gastric cancer (GC) cells (including those of express wild type CD36 and mutant CD36) in vitro and in vivo. GC patients' samples were used for clinical translational significance evaluation.FindingsH2S triggered lipid metabolism reprogramming by significantly up-regulating the expression of the fatty-acid receptor CD36 (CD36) and directly activating CD36 in GC cells. Mechanistically, a disulfide bond located between cysteine (Cys)333 and Cys272 within the CD36 protein structure that was labile to H2S-mediated modification. The long chain-fatty acid (LC-FA) binding pocket was capped by a turn in the CD36 protein, located between helical and sheet structures that were stabilized by the Cys333-Cys272. This limited the secondary binding between LC-FAs and lysine (Lys)334. Breaking the Cys333-Cys272 disulfide bond restored the second LC-FA binding conformation of CD36. Targeting CD36 in vivo blocked H2S-promoted metastasis and improved animal survival.InterpretationThese findings identify that the Cys333-Cys272 disulfide bond disrupted the integrity of the second LC-FA binding conformation of CD36. Therefore, CD36 can directly activate LC-FA access to the cytoplasm by acting as a direct target molecule for H2S.

112 - CD36 a new cellular target of electrophilic nitro-fatty acids

CD36 is a high affinity receptor that facilitates the binding and uptake of long-chain fatty acids and modified LDL into the cell. These CD36 ligands have also been described to trigger cell signaling with different effects on cell metabolism, migration and viability. Since nitro-fatty acids are electrophilic lipid mediators which exhibit anti-inflammatory and cytoprotective actions in experimental models of atherosclerosis, cardiac isquemia/reperfusion and other inflammatory diseases, we have hypothesized that nitrolipids may bind and signal through CD36 receptor. In order to this, herein we demonstrated that Nitro-Oleic Acid (NO2-OA) displays CD36-mediated intracellular signaling via Src/ERK and AMPK pathways. Pharmacological strategies to inhibit both ligands binding to CD36 (SSO) and CD36 downstream signaling pathway for ERK (PD98059) and Src (PP1), allowed us to elucidate the specificity of the pathway involved. Therefore, to study their interaction, an in vitro assay was developed using recombinant CD36 (rCD36) and biotinylated-NO2-OA. The products of this reaction exhibited that NO2-OA interacts with CD36 revealed by Western blot using HRP-streptavidin. Competition assays with increasing molar concentration of OA, GSH and unbiotinylated NO2-OA (1:1, 1:10 and 1:100) exposed the reversibility of this interaction. Similar experimental settings showed that NO2OA impairs modified-LDL binding to rCD36, which was supplemented with experiments in RAW264.7 macrophages cell line, where pre-incubation with NO2-OA showed a decrease in cholesterol accumulation after modified-LDL treatment. This result indicates that NO2-OA may bind to the same site as modified-LDL, on the ligand binding domain, and alter its incorporation into the macrophages. Altogether this data suggests that NO2-OA acts as a CD36 ligand and triggers its downstream signaling to modulate fatty acid metabolism in macrophages.

CD36 in chronic kidney disease: novel insights and therapeutic opportunities.

CD36 (also known as scavenger receptor B2) is a multifunctional receptor that mediates the binding and cellular uptake of long-chain fatty acids, oxidized lipids and phospholipids, advanced oxidation protein products, thrombospondin and advanced glycation end products, and has roles in lipid accumulation, inflammatory signalling, energy reprogramming, apoptosis and kidney fibrosis. Renal CD36 is mainly expressed in tubular epithelial cells, podocytes and mesangial cells, and is markedly upregulated in the setting of chronic kidney disease (CKD). As fatty acids are the preferred energy source for proximal tubule cells, a reduction in fatty acid oxidation in CKD affects kidney lipid metabolism by disrupting the balance between fatty acid synthesis, uptake and consumption. The outcome is intracellular lipid accumulation, which has an important role in the pathogenesis of kidney fibrosis. In experimental models, antagonist blockade or genetic knockout of CD36 prevents kidney injury, suggesting that CD36 could be a novel target for therapy. Here, we discuss the regulation and post-translational modification of CD36, its role in renal pathophysiology and its potential as a biomarker and as a therapeutic target for the prevention of kidney fibrosis.

Long chain fatty acids alter the interactive binding of ligands to the two principal drug binding sites of human serum albumin.

A wide variety of drugs bind to human serum albumin (HSA) at its two principal sites, namely site I and site II. A number of reports indicate that drug binding to these two binding sites are not completely independent, and that interactions between ligands of these two discrete sites can play a role. In this study, the effect of the binding of long-chain fatty acids on the interactive binding between dansyl-L-asparagine (DNSA; site I ligand) and ibuprofen (site II ligand) at pH6.5 was examined. Binding experiments showed that the binding of sodium oleate (Ole) to HSA induces conformational changes in the molecule, which, in turn, changes the individual binding of DNSA and ibuprofen, as well as the mode of interaction between these two ligands from a ‘competitive-like’ allosteric interaction in the case of the defatted HSA conformer to a ‘nearly independent’ binding in the case of non-defatted HSA conformer. Circular dichroism measurements indicated that ibuprofen and Ole are likely to modify the spatial orientation of DNSA at its binding site. Docking simulations suggest that the long-distance electric repulsion between DNSA and ibuprofen on defatted HSA contributes to a ‘competitive-like’ allosteric interaction, whereas extending the distance between ligands and/or increasing the flexibility or size of the DNSA binding site in fatted HSA evokes a change in the interaction mode to ‘nearly independent’ binding. The present findings provide further insights into the structural dynamics of HSA upon the binding of fatty acids, and its effects on drug binding and drug-drug interactions that occur on HSA.

Recombinant expression, purification and crystallographic studies of the mature form of human mitochondrial aspartate aminotransferase.

Mitochondrial aspartate aminotransferase (mAspAT) was recognized as a moonlighting enzyme because it has not only aminotransferase activity but also a high-affinity long-chain fatty acids (LCFA) binding site. This enzyme plays a key role in amino acid metabolism, biosynthesis of kynurenic acid and transport of the LCFA. Therefore, it is important to study the structure-function relationships of human mAspAT protein. In this work, the mature form of human mAspAT was expressed to a high level in Escherichia coli periplasmic space using pET-22b vector, purified by a combination of immobilized metal-affinity chromatography and cation exchange chromatography. Optimal activity of the enzyme occurred at a temperature of 47.5ºC and a pH of 8.5. Crystals of human mAspAT were grown using the hanging-drop vapour diffusion method at 277K with 0.1 M HEPES pH 6.8 and 25%(v/v) Jeffamine(®) ED-2001 pH 6.8. The crystals diffracted to 2.99 Å and belonged to the space group P1 with the unit-cell parameters a =56.7, b = 76.1, c = 94.2 Å, α =78.0, β =85.6, γ = 78.4º. Elucidation of mAspAT structure can provide a molecular basis towards understanding catalysis mechanism and substrate binding site of enzyme.

The Effects of Steroid Hormones on Long-Chain 3-Hydroxyacyl- CoA Dehydrogenase (LCHAD) Enzyme Expression in LCHAD Deficient Cells and Induction of Long-Chain Fatty Acid Binding Proteins on Long-Chain Fatty Acid-Induced Hepatotoxicity

Background: Long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency has been implicated in the pathogenesis of acute fatty liver of pregnancy (AFLP) and the Hemolysis Elevated Liver Enzyme, Low Platelet (HELLP) syndrome. Yet not all pregnant women with LCHAD deficiency develop these conditions and whether induction of long-chain fatty acid binding proteins (L-FABP) might be of therapeutic value remains to be determined. Objectives: To document: (1) the effects of pregnancy associated steroid hormones on LCHAD expression and (2) induction of L-FABP on long-chain fatty acid (LCFA)-induced hepatotoxicity. Methods: LCHAD deficient B2325 cells were exposed to various concentrations of β-estradiol and/or progesterone in vitro and LCHAD mRNA and protein expression documented by RT-PCR and Western Blot analysis for 24-72 hours thereafter. In addition, Huh-7 hepatocytes exposed to toxic concentrations of LCFA (a mixture of oleic and palmitic acid) for 24 hours were treated with L-FABP inducers clofibrate or simvastatin (6.25-100 mM) for a subsequent 48 hours prior to documenting cell toxicity. Results: Neither β-estradiol, progesterone or a combination thereof consistently decreased LCHAD mRNA or protein expression. Moreover, hepatocyte survival was not altered by either clofibrate or simvastatin. Conclusions: Increases in steroid hormones associated with pregnancy are unlikely to contribute to LCFA-induced hepatotoxicity in LCHAD deficient women. Induction of L-FABP does not hold promise as a therapeutic strategy for pregnant women with AFLP or HELLP.

A single amino acid change humanizes long-chain fatty acid binding and activation of mouse peroxisome proliferator-activated receptor α

Peroxisome proliferator-activated receptor α (PPARα) is an important regulator of hepatic lipid metabolism which functions through ligand binding. Despite high amino acid sequence identity (>90%), marked differences in PPARα ligand binding, activation and gene regulation have been noted across species. Similar to previous observations with synthetic agonists, we have recently reported differences in ligand affinities and extent of activation between human PPARα (hPPARα) and mouse PPARα (mPPARα) in response to long chain fatty acids (LCFA). The present study was aimed to determine if structural alterations could account for these differences. The binding of PPARα to LCFA was examined through in silico molecular modeling and docking simulations. Modeling suggested that variances at amino acid position 272 are likely to be responsible for differences in saturated LCFA binding to hPPARα and mPPARα. To confirm these results experimentally, LCFA binding, circular dichroism, and transactivation studies were performed using a F272I mutant form of mPPARα. Experimental data correlated with in silico docking simulations, further confirming the importance of amino acid 272 in LCFA binding. Although the driving force for evolution of species differences at this position are yet unidentified, this study enhances our understanding of ligand-induced regulation by PPARα and demonstrates the efficacy of molecular modeling and docking simulations.

The effect of sodium salicylate on the binding of long-chain fatty acids to plasma proteins.

Abstract Salicylate causes a release of palmitic, stearic, oleic and linolenic acids from their binding sites on human plasma proteins and bovine albumin. The implications of this finding with respect to other effects of salicylate on fatty acid metabolism are discussed.

Albumin as a zinc carrier: properties of its high-affinity zinc-binding site

Although details of the molecular mechanisms for the uptake of the essential nutrient zinc into the bloodstream and its subsequent delivery to zinc-requiring organs and cells are poorly understood, it is clear that in vertebrates the majority of plasma zinc (9-14 microM; approx. 75-85%) is bound to serum albumin, constituting part of the so-called exchangeable pool. The binding of metal ions to serum albumins has been the subject of decades of studies, employing a multitude of techniques, but only recently has the identity and putative structure of the major zinc site on albumin been reported. Intriguingly, this site is located at the interface between two domains, and involves two residues from each of domains I and II. Comparisons of X-ray crystal structures of free and fatty-acid bound human serum albumin suggest that zinc binding to this site and fatty acid binding to one of the five major sites may be interdependent. Interactive binding of zinc and long-chain fatty acids to albumin may therefore have physiological implications.

Hepatic Fatty Acid Transporter Cd36 Is a Common Target of LXR, PXR, and PPARγ in Promoting Steatosis

Liver X receptor (LXR) is known to promote hepatic lipogenesis by activating the lipogenic transcriptional factor sterol regulatory element-binding protein (Srebp). Pregnane X receptor (PXR), a previously known "xenobiotic receptor," could mediate a Srebp-independent lipogenic pathway by activating the free fatty acid uptake transporter Cd36. The goal of this study is to investigate further the role of Cd36 in hepatic steatosis. Wild-type, LXR transgenic, PXR transgenic, and Cd36 null mice were used to study the regulation of Cd36 and other hepatic lipogenic genes and the implication of this regulation in hepatic steatosis. Promoter sequences of Cd36 and peroxisome proliferator-activated receptor (PPAR) gamma were cloned, and their respective regulation by LXR and PXR was investigated by combinations of receptor-DNA binding and reporter gene assays. We showed that genetic (transgene) or pharmacologic (ligands) activation of LXR induced Cd36. Promoter analysis established Cd36 as a novel transcription target of LXRalpha. Moreover, the hepatic steatosis induced by LXR agonists was largely abolished in Cd36 null mice. We also showed that PPARgamma, a positive regulator of Cd36, is a transcriptional target of PXR, suggesting that PXR can regulate Cd36 directly or through its activation of PPARgamma. Interestingly, both LXR-mediated Cd36 regulation and PXR-mediated PPARgamma regulation are liver specific. We conclude that Cd36 is a shared target of LXR, PXR, and PPARgamma. The network of CD36 regulation by LXR, PXR, and PPARgamma establishes this free fatty acid transporter as a common target of orphan nuclear receptors in their mediation of lipid homeostasis.

Fatty acid metabolism in adipocytes: functional analysis of fatty acid transport proteins 1 and 4

The role of fatty acid transport protein 1 (FATP1) and FATP4 in facilitating adipocyte fatty acid metabolism was investigated using stable FATP1 or FATP4 knockdown (kd) 3T3-L1 cell lines derived from retrovirus-delivered short hairpin RNA (shRNA). Decreased expression of FATP1 or FATP4 did not affect preadipocyte differentiation or the expression of FATP1 (in FATP4 kd), FATP4 (in FATP1 kd), fatty acid translocase, acyl-coenzyme A synthetase 1, and adipocyte fatty acid binding protein but did lead to increased levels of peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha. Both FATP1 and FATP4 kd adipocytes exhibited reduced triacylglycerol deposition and corresponding reductions in diacylglycerol and monoacylglycerol levels compared with control cells. FATP1 kd adipocytes displayed an approximately 25% reduction in basal (3)H-labeled fatty acid uptake and a complete loss of insulin-stimulated (3)H-labeled fatty acid uptake compared with control adipocytes. In contrast, FATP4 kd adipocytes as well as HEK-293 cells overexpressing FATP4 did not display any changes in fatty acid influx. FATP4 kd cells exhibited increased basal lipolysis, whereas FATP1 kd cells exhibited no change in lipolytic capacity. Consistent with reduced triacylglycerol accumulation, FATP1 and FATP4 kd adipocytes exhibited enhanced 2-deoxyglucose uptake compared with control adipocytes. These findings define unique and distinct roles for FATP1 and FATP4 in adipose fatty acid metabolism.

Type 2 diabetes-associated fatty acid binding protein 2 promoter haplotypes are differentially regulated by GATA factors

The human intestinal fatty acid binding protein 2 (FABP2) mediates fat absorption by binding and intracellular trafficking of long-chain free fatty acids. Studies with knockout mice and association analysis of polymorphisms revealed that FABP2 is a susceptibility gene for type 2 diabetes (noninsulin dependent diabetes mellitus [NIDDM]) and related traits. Relevant FABP2 promoter polymorphisms c.-80_-79insT (rs5861422), c.-136_-132delAGTAG (rs5861423), c.-168_-166delAAGinsT (rs1973598), c.-260G>A (rs6857641), c.-471G>A (rs2282688), and c.-778G>T (rs10034579) result in two haplotypes A and B, whereby B possesses two- to three-fold lower transcriptional activity than A. We show in luciferase reporter gene assays by a series of chimeric FABP2 promoter constructs in intestinal Caco-2 cells that polymorphism c.-80_-79insT essentially determines different activities of the FABP2 promoter. In accordance, in electrophoretic mobility shift assays (EMSAs), transcriptional factors GATA-5 and -6 bind with higher binding affinities to the FABP2 promoter region containing the -80A allele compared to B. As functional consequence, haplotype A is twice as much more activated by GATA factors than haplotype B in liver Huh7 cells. Additionally, a construct bearing the -80B allele in the background of haplotype A reversed the activity from A to B. Thus, the GATA mediated differential activation of FABP2 haplotypes depends on polymorphism c.-80_-79insT. This provides the molecular basis for the variant specific transcriptional regulation of the diabetes type 2-associated FABP2 gene.

Applications of Tailored Ferrocenyl Molecules as Electrochemical Probes of Biochemical Interactions

The development of electrochemical probes useful for investigating the occupancy by other molecules of sites on complex proteins such as human serum albumin (HSA) is described. Ferrocenyl-(oxoethylene)-fatty acid compounds of different fatty acid chain length probed different binding sites on HSA. The interaction could be changed from one primarily with a drug binding site, when the probe was ferrocene methanol, to one predominantly with medium-chain fatty acid binding sites, by adding an (oxoethylene)-fatty acid substituents. Finally, the interaction could be changed to one interacting primarily with high-affinity long-chain fatty acid binding sites, as the fatty acid chain length in ferrocene-(oxoethylene)-fatty acid molecules increased. These results strongly implied that the binding could be further tailored by relatively simple modifications to the probe, for example, by changing the balance of hydrophobicity and hydrophilicity. The possibility of a procedure using mass-produced electrochemical cells to determine the fractional occupancy of different sites on HSA is demonstrated.