Functional Analysis of Long-chain Acyl-CoA Synthetase 1 in 3T3-L1 Adipocytes

ACSL1 (acyl-CoA synthetase 1), the major acyl-CoA synthetase of adipocytes, has been proposed to function in adipocytes as mediating free fatty acid influx, esterification, and storage as triglyceride. To test this hypothesis, ACSL1 was stably silenced (knockdown (kd)) in 3T3-L1 cells, differentiated into adipocytes, and evaluated for changes in lipid metabolism. Surprisingly, ACSL1-silenced adipocytes exhibited no significant changes in basal or insulin-stimulated long-chain fatty acid uptake, lipid droplet size, or tri-, di-, or monoacylglycerol levels when compared with a control adipocyte line. However, ACSL1 kd adipocytes displayed a 7-fold increase in basal and a ∼15% increase in forskolin-stimulated fatty acid efflux without any change in glycerol release, indicating a role for the protein in fatty acid reesterification following lipolysis. Consistent with this proposition, ACSL1 kd cells exhibited a decrease in activation and phosphorylation of AMP-activated protein kinase and its primary substrate acetyl-CoA carboxylase. Moreover, ACSL1 kd adipocytes displayed an increase in phosphorylated protein kinase Cθ and phosphorylated JNK, attenuated insulin signaling, and a decrease in insulin-stimulated glucose uptake. These findings identify a primary role of ACSL1 in adipocytes not in control of lipid influx, as previously considered, but in lipid efflux and fatty acid-induced insulin resistance.Fatty acid influx and efflux mechanisms and their regulation affect lipid storage and metabolism in adipocytes. Imbalances in adipose lipid metabolism have been shown to significantly contribute to the development of obesity and associated metabolic diseases, such as type 2 diabetes, hypertension, and cardiovascular disease (1–3). Although the molecular mechanisms involved in fatty acid efflux are still undefined, several proteins implicated in fatty acid influx have been proposed: CD36 (fatty acid translocase), acyl-CoA synthetases (fatty acid transport protein (FATP)² and acyl-CoA synthetase (ACSL) family members), plasma membrane fatty acid-binding protein, and caveolin-1 (4–9).FATPs and long-chain ACSLs are membrane-bound enzymes that catalyze the ATP-dependent esterification of long chain (ACSL) and very long-chain (FATP) fatty acids to their acyl-CoA derivatives (10, 11). Both types of CoA synthetases have common ATP/AMP binding and fatty acid binding signature motifs. In mammals, six different isoforms of FATP (FATP1–FATP6) and five different isoforms of ACSL (ACSL1, -3, -4, -5, and -6) have been identified with tissue-specific expression patterns (12). White adipose tissue predominantly express FATP1, FATP4, and ACSL1, whereas brown adipose tissue in addition expresses ACSL5. Our recent results have confirmed a major role of FATP1 and CD36, but not FATP4, in insulin-stimulated LCFA uptake in 3T3-L1 adipocytes (6).ACSL1 is a ∼78-kDa intrinsic membrane protein localized to multiple sites in a variety of different cells. In liver, ACSL1 has been shown to be localized to the endoplasmic reticulum and mitochondria-associated membranes, whereas in adipocytes, ACSL1 was also found associated with the plasma membrane, the lipid droplet surface (13), and glucose transporter 4-containing vesicles (14, 15). Recent studies have postulated a cooperative role of FATP1 and ACSL1 in the movement of LCFAs across the plasma membrane via a process termed vectoral acylation (16), in which the CoA- and ATP-dependent esterification of internalized fatty acid provides the thermodynamic force necessary for net lipid influx. Evidence supporting this hypothesis came from a functional cloning strategy that identified mouse ACSL1 along with FATP1 as proteins involved in LCFA transport (17). In contrast to the role of ACSL1 in LCFA uptake and triglyceride synthesis in adipocytes, overexpression of ACSL1 in rat primary hepatocytes channeled fatty acids toward diacylglycerol and phospholipids synthesis and increased reacylation of hydrolyzed fatty acids into triglyceride (18).Since lipid flux is defined by the location and activity of its regulatory enzymes and proteins, overexpression strategies can result in changes in metabolism potentially distinct from the endogenous function. To that end, our laboratory has recently undertaken a gene silencing approach to the evaluation of proteins implicated in adipocyte fatty acid influx and efflux, and prior studies have focused on FATP1, FATP4, and CD36 (6). In this report, we evaluated the adipose-specific role(s) of ACSL1 using stable gene-silencing strategies in 3T3-L1 adipocytes using lentiviral delivery of shRNA. We report herein that, contrary to previous reports, in 3T3-L1 adipocytes, ACSL1 does not facilitate the basal or insulin-stimulated component of LCFA uptake. ACSL1 is, however, involved in the reesterification of hydrolyzed fatty acids released during basal and forskolin-stimulated lipolysis, thereby regulating their availability and efflux from the cell. Additionally, fatty acid reesterification by ACSL1 during lipolysis plays a major role in regulating the AMP-activated protein kinase (AMPK) as well as the PKCθ and JNK pathways leading to insulin resistance. Such findings bring to light a new interpretation of the role of ACSL1 and other acyl-CoA synthetases in the control of intermediary metabolism and lipid-mediated signal transduction.     

Mapping of the hormone-sensitive lipase binding site on the adipocyte fatty acid-binding protein (AFABP). Identification of the charge quartet on the AFABP/aP2 helix-turn-helix domain

The hormone-sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP/aP2) form a physical complex that affects basal and hormone-stimulated adipocyte fatty acid efflux. Previous work has established that AFABP/aP2-HSL complex formation requires that HSL be in its activated, phosphorylated form and AFABP/aP2 have a bound fatty acid. To identify the HSL binding site of AFABP/aP2 a combination of alanine-scanning mutagenesis and fluorescence resonance energy transfer was used. Mutation of Asp17, Asp18, Lys21, or Arg30 (but not other amino acids in the helix-turn-helix region) to alanine inhibited interaction with HSL without affecting fatty acid binding. The cluster of residues on the helical domain of AFABP/aP2 form two ion pairs (Asp17-Arg30 and Asp18-Lys21) and identifies the region we have termed the charge quartet as the HSL interaction site. To demonstrate direct association, the non-interacting AFABP/aP2-D18K mutant was rescued by complementary mutation of HSL (K196E). The charge quartet is conserved on other FABPs that interact with HSL such as the heart and epithelial FABPs but not on non-interacting proteins from the liver or intestine and may be a general protein interaction domain utilized by fatty acid-binding proteins in regulatory control of lipid metabolism.     

Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm

Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty acids are activated to CoA esters, which bind with high affinity to the acyl-CoA-binding protein (ACBP). Thus, the availability of known and potential PPAR ligands may be regulated by lipid-binding proteins. In this report we show by transient transfection of CV-1 cells that coexpression of ACBP and adipocyte lipid-binding protein (ALBP) exerts a ligand- and PPAR subtype-specific attenuation of PPAR-mediated trans-activation, suggesting that lipid-binding proteins, when expressed at high levels, may function as negative regulators of PPAR activation by certain ligands. Expression of ACBP, ALBP, and keratinocyte lipid-binding protein (KLBP) is induced during adipocyte differentiation, a process during which PPARgamma plays a prominent role. We present evidence that endogenous ACBP, ALBP, and KLBP not only localize to the cytoplasm but also exhibit a prominent nuclear localization in 3T3-L1 adipocytes. In addition, forced expression of ACBP, ALBP, and KLBP in CV-1 cells resulted in a substantial accumulation of all three proteins in the nucleus. These results suggest that lipid-binding proteins, contrary to the general assumption, may exert their action in the nucleus as well as in the cytoplasm.     

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.     

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.     

Purification of murine adipocyte lipid-binding protein. Characterization as a fatty acid- and retinoic acid-binding protein

An adipose-specific protein has been purified from murine 3T3-L1 adipocytes to greater than 98% homogeneity. A purification procedure was developed utilizing a combination of gel filtration, cation exchange chromatography, and covalent chromatography on activated-thiol Sepharose 4B. The protein exists as a single polypeptide with a molecular weight of about 15,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein contains 2 mol of reduced sulfhydryl groups per mol of protein and an amino terminus blocked to sequencing. Automated Edman degradation of trypsin and CNBr-derived peptides has verified that the purified protein is that predicted by the mRNA (Bernlohr, D. A., Angus, C. W., Lane, M. D., Bolanowski, M. A., and Kelly, T. J. Jr. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5468-5472). Based on sequence analysis, the 15-kDa adipocyte protein is considered to be a member of a family of tissue-specific, cytosolic lipid-binding proteins. Utilizing a liposome assay, the purified protein binds both oleic acid and retinoic acid saturably with approximately 1 mol of ligand bound per mol of protein. Dissociation constants determined from Scatchard analysis were 3 and 50 microM, respectively. This report represents the first demonstration of a member of this family of structurally related proteins that is capable of binding both fatty acid and retinoic acid. Hence, we propose the name adipocyte lipid-binding protein, or ALBP.     

Purification, properties, and regulation of glutamic dehydrogenase of Bacillus licheniformis

Cell-free extracts of Bacillus licheniformis and B. cereus were found to contain high specific activities of nicotinamide adenine dinucleotide phosphate (NADP)-dependent-l-glutamate dehydrogenase [EC 1.4.1.4; l-glutamate: NADP oxidoreductase (deaminating)]. Maximum specific activities were found in extracts of cells during the late exponential phase of growth when ammonium ion served as the sole source of nitrogen. Extremely low specific activities were detected throughout the growth cycle when l-glutamate or Casamino Acids served as the source of carbon and nitrogen. The enzyme was purified 55-fold from crude extracts of B. licheniformis, and apparent kinetic constants were determined. Sigmoidal saturation kinetics were not observed, and various adenylates had no effect on the enzyme. Repression of enzyme synthesis during growth on l-glutamate or Casamino Acids was partially overcome by additions of glucose or pyruvate, and this apparent derepression was totally abolished by inhibitors of ribonucleic acid and protein synthesis. Similarly, additions of l-glutamate or Casamino Acids to cells growing on glucose-ammonium ion resulted in strong repression of enzyme synthesis. It is suggested that the enzyme serves an anabolic role in metabolism. Nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase activity was not detected in five species of Bacillus, irrespective of nutritional conditions or of the physiological age of cells.     

Properties of the Bacillus licheniformis A5 glutamine synthetase purified from cells grown in the presence of ammonia or nitrate.

The glutamine synthetase from Bacillus licheniformis A5 was purified by using a combination of polyethylene glycol precipitation and chromatography on Bio-Gel A 1.5m. The resulting preparation was judged to be homogeneous by the criteria of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, equilibrium analytical ultracentrifugation, and electron microscopic analysis. The enzyme is a dodecamer with a molecular weight of approximately 616,000, and its subunit molecular weight is 51,000. Under optimal assay conditions (pH 6.6, 37 degrees C) apparent Km values for glutamate, ammonia, and manganese.adenosine 5'-triphosphate (1:1 ratio) were 3.6, 0.4, and 0.9 mM, respectively. Glutamine synthetase activity was inhibited approximately 50% by the addition of 5 mM glutamine, alanine, glycine, serine, alpha-ketoglutarate, carbamyl phosphate, adenosine 5'-diphosphate, or inosine 5'-triphosphate to the standard glutamine synthetase assay system, whereas 5 mM adenosine 5'-monophosphate or pyrophosphate caused approximately 90% inhibition of enzyme activity. Phosphorylribosyl pyrophosphate at 5 mM enhanced activity approximately 60%. We were unable to detect any physical or kinetic differences in the properties of the enzyme when it was purified from cells grown in the presence of ammonia or nitrate as sole nitrogen source. The data indicate that B. licheniformis A5 contains one species of glutamine synthetase whose catalytic activity is not regulated by a covalent modification system.     

Purification and regulation of glucose-6-phosphate dehydrogenase from Bacillus licheniformis

d-Glucose-6-phosphate nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase (EC 1.1.1.49) from Bacillus licheniformis has been purified approximately 600-fold. The enzyme appears to be constitutive and exhibits activity with either oxidized NAD (NAD(+)) or oxidized NADP (NADP(+)) as electron acceptor. The enzyme has a pH optimum of 9.0 and has an absolute requirement for cations, either monovalent or divalent. The enzyme exhibits a K(m) of approximately 5 muM for NADP(+), 3 mM for NAD(+), and 0.2 mM for glucose-6-phosphate. Reduced NADP (NADPH) is a competitive inhibitor with respect to NADP(+) (K(m) = 10 muM). Phosphoenolpyruvate (K(m) = 1.6 mM), adenosine 5'-triphosphate (K(m) = 0.5 mM), adenosine diphosphate (K(m) = 1.5 mM), and adenosine 5'-monophosphate (K(m) = 3.0 mM) are competitive inhibitors with respect to NAD(+). The molecular weight as estimated from sucrose density centrifugation and molecular sieve chromatography is 1.1 x 10(5). Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is composed of two similar subunits of approximately 6 x 10(4) molecular weight. The intracellular levels of glucose-6-phosphate, NAD(+), and NADP(+) were measured and found to be approximately 1 mM, 0.9 mM, and 0.2 mM, respectively, during logarithmic growth. From a consideration of the substrate pool sizes and types of inhibitors, we conclude that this single constitutive enzyme may function in two roles in the cell-NADH production for energetics and NADPH production for reductive biosynthesis.     

Isotopic Study of Control of the Lysine Biosynthetic Pathway During Sporulation of Bacillus cereus

The extent of incorporation of aspartate into dipicolinic acid and into various amino compounds was determined in Bacillus cereus at various times before, during, and near the end of synthesis of dipicolinic acid. The purpose of this study was to gain further information on the in vivo control of the biosynthesis of amino acids derived from aspartate. Control of the lysine biosynthetic pathway was of particular interest with regard to sporulation, owing to the important role of diaminopimelate and dipicolinate in the structure of the spore. As synthesis of dipicolinate was initiated, incorporation of carbon derived from aspartate was funneled preferentially into this compound as compared with others of the aspartate group. Incorporation into lysine essentially stopped just before the synthesis of dipicolinate began. This is consistent with the previously observed disappearance at this time of diaminopimelic acid decarboxylase in cell-free extracts. Synthesis of diaminopimelate continued during the time of synthesis of dipicolinate. The previous suggestion that diaminopimelate might exert negative control on one of the enzymes between dihydrodipicolinate and diaminopimelate is thus considered unlikely. The possibility is discussed that synthesis of dipicolinate is favored by an increase in the rate of synthesis of dihydrodipicolinate rather than by a block in its rate of utilization.