Biomedical subjects

David A Bernlohr

Publications and source records attributed to David A Bernlohr.

At least 19 recordsLinked to original sources

PubMed2007

Carbonylation of adipose proteins in obesity and insulin resistance: identification of adipocyte fatty acid-binding protein as a cellular target of 4-hydroxynonenal.

Obesity is a state of mild inflammation correlated with increased oxidative stress. In general, pro-oxidative conditions lead to production of reactive aldehydes such as trans-4-hydroxy-2-nonenal (4-HNE) and trans-4-oxo-2-nonenal implicated in the development of a variety of metabolic diseases. To investigate protein modification by 4-HNE as a consequence of obesity and its potential relationship to the development of insulin resistance, proteomics technologies were utilized to identify aldehyde-modified proteins in adipose tissue. Adipose proteins from lean insulin-sensitive and obese insulin-resistant C57Bl/6J mice were incubated with biotin hydrazide and detected using horseradish peroxidase-conjugated streptavidin. High carbohydrate, high fat feeding of mice resulted in a approximately 2-3-fold increase in total adipose protein carbonylation. Consistent with an increase in oxidative stress in obesity, the abundance of glutathione S-transferase A4 (GSTA4), a key enzyme responsible for metabolizing 4-HNE, was decreased approximately 3-4-fold in adipose tissue of obese mice. To identify specific carbonylated proteins, biotin hydrazide-modified adipose proteins from obese mice were captured using avidin-Sepharose affinity chromatography, proteolytically digested, and subjected to LC-ESI MS/MS. Interestingly enzymes involved in cellular stress response, lipotoxicity, and insulin signaling such as glutathione S-transferase M1, peroxiredoxin 1, glutathione peroxidase 1, eukaryotic elongation factor 1alpha-1 (eEF1alpha1), and filamin A were identified. The adipocyte fatty acid-binding protein, a protein implicated in the regulation of insulin resistance, was found to be carbonylated in vivo with 4-HNE. In vitro modification of adipocyte fatty acid-binding protein with 4-HNE was mapped to Cys-117, occurred equivalently using either the R or S enantiomer of 4-HNE, and reduced the affinity of the protein for fatty acids approximately 10-fold. These results indicate that obesity is accompanied by an increase in the carbonylation of a number of adipose-regulatory proteins that may serve as a mechanistic link between increased oxidative stress and the development of insulin resistance.

Adipose Tissue↗

PubMed2006

Gene expression profiling of the human maternal-fetal interface reveals dramatic changes between midgestation and term.

Human placentation entails the remarkable integration of fetal and maternal cells into a single functional unit. In the basal plate region (the maternal-fetal interface) of the placenta, fetal cytotrophoblasts from the placenta invade the uterus and remodel the resident vasculature and avoid maternal immune rejection. Knowing the molecular bases for these unique cell-cell interactions is important for understanding how this specialized region functions during normal pregnancy with implications for tumor biology and transplantation immunology. Therefore, we undertook a global analysis of the gene expression profiles at the maternal-fetal interface. Basal plate biopsy specimens were obtained from 36 placentas (14-40 wk) at the conclusion of normal pregnancies. RNA was isolated, processed, and hybridized to HG-U133A&B Affymetrix GeneChips. Surprisingly, there was little change in gene expression during the 14- to 24-wk interval. In contrast, 418 genes were differentially expressed at term (37-40 wk) as compared with midgestation (14-24 wk). Subsequent analyses using quantitative PCR and immunolocalization approaches validated a portion of these results. Many of the differentially expressed genes are known in other contexts to be involved in differentiation, motility, transcription, immunity, angiogenesis, extracellular matrix dissolution, or lipid metabolism. One sixth were nonannotated or encoded hypothetical proteins. Modeling based on structural homology revealed potential functions for 31 of these proteins. These data provide a reference set for understanding the molecular components of the dialogue taking place between maternal and fetal cells in the basal plate as well as for future comparisons of alterations in this region that occur in obstetric complications.

Female↗

PubMed2006

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.

3T3-L1 Cells↗

PubMed2006

FABP3 and FABP10 in Atlantic salmon (Salmo salar L.)--general effects of dietary fatty acid composition and life cycle variations.

The increased use of dietary plant oil supplementation combined with high dietary lipid loads challenges the lipid transport systems of cultivated fish species. Fatty acid binding proteins (FABPs) have been thoroughly studied as intracellular fatty acid transporters in vertebrates, but no data have been reported in Atlantic salmon. In the present study, comparative characterizations were performed, and dietary influence of plant oil supplementation on FABP3 and FABP10 expression was studied for several tissues in two separate dietary trials. In trial I, groups (6 fish each) were fed diets for 42 weeks (body mass 142+/-1 to 1463+/-83 g) (mean+/-S.D.), containing graded levels of rapeseed oil substituting for fish oil using a linear regression design. In trial II, groups (3 fish each) were fed 100% fish oil or 100% plant oil for 22 months (0.160+/-0.052 to 2523+/-590 g) (mean+/-S.D.) and sampled at regular intervals. Liver and muscle tissues appeared to express several FABPs possibly linked to different metabolic functions. FABPs mRNA expression did not change with dietary inclusion of 75% rapeseed oil, whereas FABP3 protein expression seemed to be affected by dietary rapeseed oil inclusion. Significant changes in red muscle FABP3 mRNA expression correlate to significant changes in total beta-oxidation capacity during the energy consuming process of smoltification.

Amino Acid Sequence↗

PubMed2005

Lipid metabolism and adipokine levels in fatty acid-binding protein null and transgenic mice.

Fatty acid-binding proteins (FABPs) facilitate the diffusion of fatty acids within cellular cytoplasm. Compared with C57Bl/6J mice maintained on a high-fat diet, adipose-FABP (A-FABP) null mice exhibit increased fat mass, decreased lipolysis, increased muscle glucose oxidation, and attenuated insulin resistance, whereas overexpression of epithelial-FABP (E-FABP) in adipose tissue results in decreased fat mass, increased lipolysis, and potentiated insulin resistance. To identify the mechanisms that underlie these processes, real-time PCR analyses indicate that the expression of hormone-sensitive lipase is reduced, while perilipin A is increased in A-FABP/aP2 null mice relative to E-FABP overexpressing mice. In contrast, de novo lipogenesis and expression of genes encoding lipoprotein lipase, CD36, long-chain acyl-CoA synthetase 5, and diacylglycerol acyltransferase are increased in A-FABP/aP2 null mice relative to E-FABP transgenic animals. Consistent with an increase in de novo lipogenesis, there was an increase in adipose C16:0 and C16:1 acyl-CoA pools. There were no changes in serum free fatty acids between genotypes. Serum levels of resistin were decreased in the E-FABP transgenic mice, whereas serum and tissue adiponectin were increased in A-FABP/aP2 null mice and decreased in E-FABP transgenic animals; leptin expression was unaffected. These results suggest that the balance between lipolysis and lipogenesis in adipocytes is remodeled in the FABP null and transgenic mice and is accompanied by the reprogramming of adipokine expression in fat cells and overall changes in plasma adipokines.

Adipocytes↗

PubMed2005

Perilipin targets a novel pool of lipid droplets for lipolytic attack by hormone-sensitive lipase.

Adipocytes serve as the principal energy reservoir of the body; however, the subcellular organization of the machinery regulating lipid trafficking and metabolism is poorly understood. Mobilization of stored triglyceride is thought be controlled by interactions among intracellular lipases and proteins that coat lipid storage droplets. A major limitation of previous studies of hormone-mediated lipolysis, however, is the use of cultured model adipocytes whose three-dimensional architectures do not resemble those in real adipose tissue. To address this limitation, we investigated the intracellular targeting of perilipin, a major lipid coat protein, and hormone-sensitive lipase in three preparations that exhibit more appropriate morphologies: 3T3-L1 adipocytes grown in three-dimensional matrix, dissociated mature adipocytes from mouse adipose tissue, and adipocytes within intact fat pads. High resolution imaging of native and fluorescently tagged proteins indicate that: 1) perilipin preferentially targets a special class of peripheral lipid storage droplets, but not the major or central lipid storage droplets, 2) the peripheral droplets are the sites of attack by hormone-sensitive lipase, and 3) perilipin and hormone-sensitive lipase are continuously colocalized following lipolytic activation. These results indicate that in white adipose tissue, lipolysis takes place in a specialized subcellular domain that is distinct from the major lipid storage site and is defined by perilipin.

3T3-L1 Cells↗

PubMed2005

Enzymatic properties of purified murine fatty acid transport protein 4 and analysis of acyl-CoA synthetase activities in tissues from FATP4 null mice.

Fatty acid transport protein 4 (FATP4) is an integral membrane protein expressed in the plasma and internal membranes of the small intestine and adipocyte as well as in the brain, kidney, liver, skin, and heart. FATP4 has been hypothesized to be bifunctional, exhibiting both fatty acid transport and acyl-CoA synthetase activities that work in concert to mediate fatty acid influx across biological membranes. To determine whether FATP4 is an acyl-CoA synthetase, the murine protein was engineered to contain a C-terminal FLAG epitope tag, expressed in COS1 cells via adenovirus-mediated infection and purified to near homogeneity using alpha-FLAG affinity chromatography. Kinetic analysis of the enzyme was carried out for long chain (palmitic acid, C16:0) and very long chain (lignoceric acid, C24:0) fatty acids as well as for ATP and CoA. FATP4 exhibited substrate specificity for C16:0 and C24:0 fatty acids with a V(max)/K(m) (C16:0)/V(max)/K(m) (C24:0) of 1.5. Like purified FATP1, FATP4 was insensitive to inhibition by triacsin C but was sensitive to feedback inhibition by acyl-CoA. Although purified FATP4 exhibited high levels of palmitoyl-CoA and lignoceroyl-CoA synthetase activity, extracts from the skin and intestine of FATP4 null mice exhibited reduced esterification for C24:0, but not C16:0 or C18:1, suggesting that in vivo, defects in very long chain fatty acid uptake may underlie the skin disorder phenotype of null mice.

Animals↗

PubMed2004

Physical association between the adipocyte fatty acid-binding protein and hormone-sensitive lipase: a fluorescence resonance energy transfer analysis.

Previous in vitro studies have established that hormone sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP) form a physical complex that presumably positions the FABP to accept a product fatty acid generated during catalysis. To assess AFABP-HSL interaction within a cellular context, we have used lipocytes derived from 293 cells (C8PA cells) and examined physical association using fluorescence resonance energy transfer. Transfection of C8PA cells with cyan fluorescent protein (CFP)-HSL, yellow fluorescent protein (YFP)-adipocyte FABP, or YFP-liver FABP revealed that under basal conditions each protein was cytoplasmic. In the presence of 20 microm forskolin, CFP-HSL translocated to the triacylglycerol droplet, coincident with BODIPY-FA labeled depots. Fluorescence resonance energy transfer analysis demonstrated that CFP-HSL associated with YFP-adipocyte FABP in both basal and forskolin-treated cells. In contrast, little if any fluorescence resonance energy transfer could be detected between CFP-HSL and YFP-liver FABP. These results suggest that a pre-lipolysis complex containing at least AFABP and HSL exists and that the complex translocates to the surface of the lipid droplet.

Adipocytes↗

PubMed2004

Investigation of in vivo fatty acid metabolism in AFABP/aP2(-/-) mice.

The metabolic impact of the murine adipocyte fatty acid-binding protein (AFABP/aP2) on lipid metabolism was investigated in the AFABP/aP2(-/-) mouse and compared with wild-type C57BL/6J littermates. Mice were weaned on a high-fat diet (59% of energy from fat) and acclimated to meal feeding. Stable isotopes were administered, and indirect calorimetry was performed to quantitate fatty acid flux, dietary fatty acid utilization, and substrate oxidation. Consistent with previous in situ and in vitro studies, fasting serum nonesterified fatty acid (NEFA) release was significantly reduced in AFABP/aP2(-/-) (17.1 +/- 9.0 vs. 51.9 +/- 22.9 mg.kg(-1).min(-1)). AFABP/aP2(-/-) exhibited higher serum NEFA (1.4 +/- 0.6 vs. 0.8 +/- 0.4 mmol/l, AFABP/aP2(-/-) vs. C57BL/6J, respectively) and triacylglycerol (TAG; 0.23 +/- 0.09 vs. 0.13 +/- 0.10 mmol/l) and accumulated more TAG in liver tissue (2.9 +/- 2.3 vs. 1.1 +/- 0.8% wet wt) in the fasted state. For the liver-TAG pool, 16.4 +/- 7.3% of TAG-fatty acids were derived from serum NEFA in AFABP/aP2(-/-). In contrast, a significantly greater portion of C57BL/6J liver-TAG was derived from serum NEFA (42.3 +/- 25.5%) during tracer infusion. For adipose-TAG stores, only 0.29 +/- 0.04% was derived from serum NEFA in AFABP/aP2(-/-), and, in C57BL/6J, 1.85 +/- 0.97% of adipose-TAG was derived from NEFA. In addition, AFABP/aP2(-/-) preferentially oxidized glucose relative to fatty acids in the fed state. These data demonstrate that in vivo disruption of AFABP/aP2(-/-) leads to changes in the following two major metabolic processes: 1) decreased adipose NEFA efflux and 2) preferential utilization of glucose relative to fatty acids.

Adipose Tissue↗

PubMed2004

Fatty acid binding proteins stabilize leukotriene A4: competition with arachidonic acid but not other lipoxygenase products.

Leukotriene A(4) (LTA(4)) is a chemically reactive conjugated triene epoxide product derived from 5-lipoxygenase oxygenation of arachidonic acid. At physiological pH, this reactive compound has a half-life of less than 3 s at 37 degrees C and approximately 40 s at 4 degrees C. Regardless of this aqueous instability, LTA(4) is an intermediate in the formation of biologically active leukotrienes, which can be formed through either intracellular or transcellular biosynthesis. Previously, epithelial fatty acid binding protein (E-FABP) present in RBL-1 cells was shown to increase the half-life of LTA(4) to approximately 20 min at 4 degrees C. Five FABPs (adipocyte FABP, intestinal FABP, E-FABP, heart/muscle FABP, and liver FABP) have now been examined and also found to increase the half-life of LTA(4) at 4 degrees C to approximately 20 min with protein present. Stabilization of LTA(4) was examined when arachidonic acid was present to compete with LTA(4) for the binding site on E-FABP. Arachidonate has an apparent higher affinity for E-FABP than LTA(4) and was able to completely block stabilization of the latter. When E-FABP is not saturated with arachidonate, FABP can still stabilize LTA(4). Several lipoxygenase products, including 5-hydroxyeicosatetraenoic acid, 5,6-dihydroxyeicosatetraenoic acid, and leukotriene B(4), were found to have no effect on the stability of LTA(4) induced by E-FABP even when present at concentrations 3-fold higher than LTA(4).

Animals↗

PubMed2004

Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family.

By consensus, the acyl-CoA synthetase (ACS) community, with the advice of the human and mouse genome nomenclature committees, has revised the nomenclature for the mammalian long-chain acyl-CoA synthetases. ACS is the family root name, and the human and mouse genes for the long-chain ACSs are termed ACSL1,3-6 and Acsl1,3-6, respectively. Splice variants of ACSL3, -4, -5, and -6 are cataloged. Suggestions for naming other family members and for the nonmammalian acyl-CoA synthetases are made.

Acyl Coenzyme A↗

PubMed2003

Stabilization of leukotriene A4 by epithelial fatty acid-binding protein in the rat basophilic leukemia cell.

Leukotriene A(4) (LTA(4)) is a chemically unstable triene epoxide product of 5-lipoxygenase metabolism of arachidonic acid. Despite this chemical reactivity and its synthesis at the perinuclear membrane, LTA(4) is enzymatically converted into the cysteinyl leukotrienes and leukotriene B(4). Furthermore, LTA(4) participates in transcellular biosynthesis and is thus transferred between cells as an intact molecule. A cytosolic fatty acid-binding protein present in the rat basophilic leukemia cells was identified using mass spectrometry. This protein was determined to be the stabilizing factor present in the cell cytosol responsible for increasing the effective chemical half-life of LTA(4). Rat epithelial fatty acid-binding protein (E-FABP) was isolated using partial protein purification and immunoprecipitation. In-gel digestion with trypsin followed by peptide fingerprint analysis using matrix-assisted laser desorption ionization mass spectrometry and sequencing the major tryptic peptide obtained from liquid chromatography/mass spectrometry/mass spectrometry analysis identified E-FABP in the active fraction. Semi-quantitative Western blot analysis indicated that E-FABP in the cytosolic fraction of RBL-1 cells was present at approximately 1-3 pmol/10(6) cells. E-FABP (9 microm) was tested for its ability to stabilize LTA(4), and at 37 degrees C E-FABP was able to increase the half-life of LTA(4) from the previously reported half-life less than 3 s to a half-life of approximately 7 min. These results present a novel function for the well studied fatty acid-binding protein as a participant in leukotriene biosynthesis that permits LTA(4) to be available for further enzymatic processing in various cellular regions.

Animals↗

PubMed2003

Fatty acid-binding protein-hormone-sensitive lipase interaction. Fatty acid dependence on binding.

Adipose lipolysis is mediated, in part, via interaction of fatty acid-binding protein (FABP) with hormone-sensitive lipase (HSL). Mice with reduced FABP content in fat (adipocyte FABP null) exhibit diminished fat cell lipolysis, whereas transgenic mice with increased FABP content in fat (epithelial FABP transgenic) exhibit enhanced lipolysis. To examine the relationship between the binding of FABP to HSL and activation of catalytic activity, isothermal titration microcalorimetry as well as kinetic analysis using a variety of FABP isoforms have been employed. In the absence of fatty acids, no FABP-HSL association could be demonstrated for any FABP form. However, in the presence of 10 microm oleate, A-FABP and E-FABP each bound to HSL with high affinity (Kd of 0.5 and 3 nM, respectively) in a approximately 1:1 molar stoichiometry, whereas liver FABP and intestinal FABP did not exhibit any association. To compare binding to catalysis, each FABP isoform was incubated with HSL in vitro, and enzymatic activity was assessed. Importantly, each FABP form stimulated HSL activity approximately 2-fold using cholesteryl oleate as substrate but exhibited no activation using p-nitrophenyl butyrate. The activation by A-FABP was dependent upon its fatty acid binding properties because a non-fatty acid binding mutant, R126Q, failed to activate HSL. These results suggest that binding and activation of HSL by FABPs are separate and distinct functions and that HSL contains a site for fatty acid binding that allows for FABP association.

Anilino Naphthalenesulfonates↗

PubMed2003

Characterization of the Acyl-CoA synthetase activity of purified murine fatty acid transport protein 1.

Fatty acid transport protein 1 (FATP1) is an approximately 63-kDa plasma membrane protein that facilitates the influx of fatty acids into adipocytes as well as skeletal and cardiac myocytes. Previous studies with FATP1 expressed in COS1 cell extracts suggested that FATP1 exhibits very long chain acyl-CoA synthetase (ACS) activity and that such activity may be linked to fatty acid transport. To address the enzymatic activity of the isolated protein, murine FATP1 and ACS1 were engineered to contain a C-terminal Myc-His tag expressed in COS1 cells via adenoviral-mediated infection and purified to homogeneity using nickel affinity chromatography. Kinetic analysis of the purified enzymes was carried out for long chain palmitic acid (C16:0) and very long chain lignoceric acid (C24:0) as well as for ATP and CoA. FATP1 exhibited similar substrate specificity for fatty acids 16-24 carbons in length, whereas ACS1 was 10-fold more active on long chain fatty acids relative to very long chain fatty acids. The very long chain acyl-CoA synthetase activity of the two enzymes was comparable as were the Km values for both ATP and coenzyme A. Interestingly, FATP1 was insensitive to inhibition by triacsin C, whereas ACS1 was inhibited by micromolar concentrations of the compound. These data represent the first characterization of purified FATP1 and indicate that the enzyme is a broad substrate specificity acyl-CoA synthetase. These findings are consistent with the hypothesis that that fatty acid uptake into cells is linked to their esterification with coenzyme A.

Adenoviridae↗

PubMed2003

Role of the fatty acid binding protein mal1 in obesity and insulin resistance.

The metabolic syndrome is a cluster of metabolic and inflammatory abnormalities including obesity, insulin resistance, type 2 diabetes, hypertension, dyslipidemia, and atherosclerosis. The fatty acid binding proteins aP2 (fatty acid binding protein [FABP]-4) and mal1 (FABP5) are closely related and both are expressed in adipocytes. Previous studies in aP2-deficient mice have indicated a significant role for aP2 in obesity-related insulin resistance, type 2 diabetes, and atherosclerosis. However, the biological functions of mal1 are not known. Here, we report the generation of mice with targeted null mutations in the mal1 gene as well as transgenic mice overexpressing mal1 from the aP2 promoter/enhancer to address the role of this FABP in metabolic regulation in the presence or absence of obesity. To address the role of the second adipocyte FABP in metabolic regulation in the presence and deficiency of obesity, absence of mal1 resulted in increased systemic insulin sensitivity in two models of obesity and insulin resistance. Adipocytes isolated from mal1-deficient mice also exhibited enhanced insulin-stimulated glucose transport capacity. In contrast, mice expressing high levels of mal1 in adipose tissue display reduced systemic insulin sensitivity. Hence, our results demonstrate that mal1 modulates adipose tissue function and contributes to systemic glucose metabolism and constitutes a potential therapeutic target in insulin resistance.

Animals↗

PubMed2002

Covalent modification of epithelial fatty acid-binding protein by 4-hydroxynonenal in vitro and in vivo. Evidence for a role in antioxidant biology.

4-Hydroxynonenal (4-HNE) is a cytotoxic alpha,beta-unsaturated acyl aldehyde that is naturally produced from lipid peroxidation and cleavage in response to oxidative stress and aging. Such reactive lipids covalently modify cellular target proteins, thereby affecting biological structure and function. Herein we report the identification of the epithelial fatty acid-binding protein (E-FABP) as a molecular target for 4-HNE modification both in vitro and in vivo. 4-HNE covalently modified (t(12) < 60 s) E-FABP in vitro, as revealed by a combination of matrix-assisted laser desorption ionization-time of flight mass spectrometry and immunochemical reactivity using antibodies directed to 4-HNE-protein conjugates. Identification of Cys-120 as the major site of modification was determined through tandem mass spectral sequencing of tryptic peptides, as well as analysis of E-FABP mutants C120A, C127A, and C120A/C127A. The in vitro modification of Cys-120 by 4-HNE was relatively insensitive to pH (6.4-8.4), and temperature (4-37 degrees C) but was markedly potentiated by noncovalently bound fatty acids. 4-HNE-modified E-FABP was more stable than unmodified E-FABP to chemical denaturation by guanidine hydrochloride, as assessed by changes in intrinsic tryptophan fluorescence. Analysis of soluble protein extracts from rat retina with antibodies directed to 4-HNE-protein conjugates revealed immunoreactivity with a 15-kDa protein that was identified by electrospray ionization and matrix-assisted laser desorption ionization-time of flight mass spectrometry as E-FABP. Evaluation of retinal pigment epithelial cell extracts derived from E-FABP null mice by two-dimensional gel electrophoresis using anti-4-HNE antibodies revealed increased modification in the null cells relative to those from wild type cells. These results indicate that E-FABP is a molecular target for 4-HNE modification and the hypothesis that E-FABP functions as an antioxidant protein by scavenging reactive lipids through covalent modification of Cys-120.

Aldehydes↗

PubMed2002

Testing of the portal hypothesis: analysis of a V32G, F57G, K58G mutant of the fatty acid binding protein of the murine adipocyte.

The portal region of fatty acid binding proteins is hypothesized to function as a dynamic aperture, controlling accessibility of external ligands to the internal fatty acid binding cavity. To test this hypothesis, a triple mutant of the murine FABP4 has been developed (V32G, F57G, K58G, referred to as the portal mutant) that is predicted to constitutively enlarge the opening due to a reduction in the molecular dimensions of the side chains of key portal amino acids. The portal mutant was purified from expressing Escherichia coli, its stability was evaluated, and the thermodynamics and kinetics of ligand binding were compared to that of wild-type protein. Introduction of the three amino acid substitutions caused no significant change in the stability of the protein with a free energy of unfolding of 13.7 kJ/mol as compared to 14.0 kJ/mol for the wild-type protein. The portal mutant exhibited a modest decrease (4-fold) in ligand binding affinity using the fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) as a surrogate ligand. 1,8-ANS displacement assays revealed that the binding affinity for oleate increased from a K0.5 of 196 +/- 15 nM for the wild-type protein to 165 +/- 8 for the portal mutant, while that for arachidonate decreased from the wild type of 186 +/- 11 nM to 418 +/- 26 nM for the portal mutant. To evaluate cavity accessibility, rate of 1,8-ANS binding was assessed between the portal and wild-type protein. Using equimolar amounts of ligand and protein at 4 degrees, 1,8-ANS bound within the cavity to 95% saturation (t0.95) in 750 ms, while the mutant protein was fully modified in less than 1.4 ms. To independently evaluate cavity accessibility, modification of the sole protein cysteine residue, C117 residing within the cavity near C2-C4 of the bound ligand, was monitored using 5,5'-dithio-bis(2-nitrobenzoic acid) modification. The half time for modification (t0.5) for the wild-type protein was approximately 20 s, while that for V32G F57G K58G occurred in less than a second. As such, enlargement of the portal region of FABP4 markedly increased the accessibility of ligands to the cavity while having only modest effects on ligand affinity. Taken together, these data provide support for the portal region hypothesis and suggest dynamic fluctuations in this region regulate cavity access, but not ligand affinity or selectivity.

Adipocytes↗

PubMed2002

FATP1 channels exogenous FA into 1,2,3-triacyl-sn-glycerol and down-regulates sphingomyelin and cholesterol metabolism in growing 293 cells.

Biosynthesis of lipids was investigated in growing 293 cells stably expressing fatty acid (FA) transport protein 1 (FATP1), a bifunctional polypeptide with FA transport as well as fatty acyl-CoA synthetase activity. In short-term (30 s) incubations, FA uptake was increased in FATP1 expressing cells (C8 cells) compared with the vector (as determined by BODIPY 3823 staining and radioactive FA uptake). In long-term (4 h) incubations, incorporation of [(14)C]acetate, [3H]oleic acid, or [(14)C]lignoceric acid into 1,2,3-triacyl-sn-glycerol (TG) was elevated in C8 cells compared with vector, whereas incorporation of radiolabel into glycerophospholipids was unaltered. The increase in TG biosynthesis correlated with an increase in 1,2-diacyl-sn-glycerol acyltransferase activity in C8 cells compared with vector. In contrast, incorporation of [(14)C]acetate into sphingomyelin (SM) and cholesterol, and [3H]oleic acid or [(14)C]lignoceric acid into SM was reduced due to a reduction in de novo biosynthesis of these lipids in C8 cells compared with vector. The results indicate that exogenously supplied FAs, and their subsequently produced acyl-CoAs, are preferentially channeled by an FATP1 linked mechanism into the TG biosynthetic pathway and that such internalized lipids down-regulate de novo SM and cholesterol metabolism in actively growing 293 cells.

Biological Transport↗