Long-chain Fatty acyl-CoA Research Articles

P5-01-10: A Potential New Marker for, and Facilitator of, Hormone Independence.

Abstract Triple negative breast cancers (TNBC) are generally more aggressive and have a worse prognosis than cancers that express estrogen (ER), progesterone (PGR) and/or human epidermal growth factor receptor 2 (HER2). Recent data suggests that another hormone receptor, that for androgen (AR) may be relevant in terms of further stratifying TNBC, with quadruple negative breast cancer (QNBC) having a worse prognosis than TNBC. In addition, TNBC vary with respect to prognosis and sensitivity to specific treatments, and foreknowledge of treatment outcomes has been the goal of numerous studies designed to generate predictive gene signatures. Development of these signatures involves the use of algorithms that can potentially exclude important biomarkers that may appear to be too weakly expressed to make the cut-off. We report here that the lipid metabolic enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4) falls into this category of a heretofore-unrecognized biomarker for aggressive breast cancer. We have previously reported that ACSL4 expression is negatively correlated with the expression of ER and AR in both breast cancer cell lines and tumor samples. We now report a positive correlation between ACSL4 mRNA expression and QNBC status. In two separate studies involving a total of 71 separate breast cancer cell lines, values for ACSL4 expression derived from microarray data indicated increased expression in QNBC cell lines, p=3.75E-08 and p=4.59E-08, when compared with cell lines expressing one or more receptor biomarkers. An analysis of data derived from 178 tumor samples indicated a similar correlation, p=2.2E-03. ACSL4 expression was also correlated with molecular subtype. When breast cancer cell lines were classified by subtype, 24 of 26 luminal cell lines were negative for ACSL4 expression, 8 of 8 claudin-low cell lines were positive for ACSL4 expression, as were 12 of 17 basal-like cell lines. Three basal-like cell lines expressed both ACSL4 and HER2. Upon further examination, these lines were demonstrated to express relatively low levels of other markers that have been suggested to correlate with HER2 sensitivity (ESR1, XBP1, FASN, ERBB3), as well as to express relatively high levels of markers correlated with HER2 resistance (CD44, CAV2). In order to determine the relationship between ACSL4 expression and estrogen independence, ACSL4-negative MCF-7 cells were inducibly transfected with ACSL4 cDNA. Induction of ACSL4 resulted in abrogation of the proliferation response to estrogen, accompanied by a reduction in expression of ER, AR and PGR. In addition, induced cells were less sensitive to treatment with tamoxifen, etoposide and triacsin C. Information derived from RAF-1-transfected MCF-7 microarray data suggest that ACSL4 expression can be regulated by RAF-1. To further explore this notion, QNBC cells, MDA-MB-231, which express high levels of ACSL4, were treated with siRNA for RAF-1 and ACSL4 levels monitored by RT-PCR and immunoblot. The fall in levels of expression of RAF-1 was accompanied by a similar decrease in expression of ACSL4. Reduction of ACSL4, however, had no effect on RAF-1 expression. In summary, these data demonstrate a possible role for ACSL4 as a biomarker for prognosis and treatment outcome, as well as a facilitator of hormone resistance. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-10.

Lipid sensing in the gut, brain and liver

Elevation of lipid levels affects energy and glucose homeostasis. Organs such as the gut, brain and liver detect a rise in lipids and orchestrate a biochemical, molecular, neuronal and physiological network of responses that alters appetite and the rate of hepatic glucose production. The factors involved in these responses are unclear but the formation of esterified lipids (long-chain fatty acyl-CoAs) and subsequent activation of protein kinase Cδ remain a common sensing mechanism in all three organs. In this paper, we discuss the mechanisms underlying lipid sensing within the gut, brain and liver and their physiological impact on the regulation of glucose and energy homeostasis.

Tyrosine phosphatase SHP2 regulates the expression of acyl-CoA synthetase ACSL4

Acyl-CoA synthetase 4 (ACSL4) is implicated in fatty acid metabolism with marked preference for arachidonic acid (AA). ACSL4 plays crucial roles in physiological functions such as steroid synthesis and in pathological processes such as tumorigenesis. However, factors regulating ACSL4 mRNA and/or protein levels are not fully described. Because ACSL4 protein expression requires tyrosine phosphatase activity, in this study we aimed to identify the tyrosine phosphatase involved in ACSL4 expression. NSC87877, a specific inhibitor of the tyrosine phosphatase SHP2, reduced ACSL4 protein levels in ACSL4-rich breast cancer cells and steroidogenic cells. Indeed, overexpression of an active form of SHP2 increased ACSL4 protein levels in MA-10 Leydig steroidogenic cells. SHP2 has to be activated through a cAMP-dependent pathway to exert its effect on ACSL4. The effects could be specifically attributed to SHP2 because knockdown of the phosphatase reduced ACSL4 mRNA and protein levels. Through the action on ACSL4 protein levels, SHP2 affected AA-CoA production and metabolism and, finally, the steroidogenic capacity of MA-10 cells: overexpression (or knockdown) of SHP2 led to increased (or decreased) steroid production. We describe for the first time the involvement of SHP2 activity in the regulation of the expression of the fatty acid-metabolizing enzyme ACSL4.

Alterations in Lipid Metabolism Mediate Inflammation, Fibrosis, and Proliferation in a Mouse Model of Chronic Cholestatic Liver Injury

The liver controls central processes of lipid and bile acid homeostasis. We aimed to investigate whether alterations in lipid metabolism contribute to the pathogenesis of chronic cholestatic liver disease in mice. We used microarray and metabolic profiling analyses to identify alterations in systemic and hepatic lipid metabolism in mice with disruption of the gene ATP-binding cassette sub-family B member 4 (Abcb4(-/-) mice), a model of inflammation-induced cholestatic liver injury, fibrosis, and cancer. Alterations in Abcb4(-/-) mice, compared with wild-type mice, included deregulation of genes that control lipid synthesis, storage, and oxidation; decreased serum levels of cholesterol and phospholipids; and reduced hepatic long-chain fatty acyl-CoAs (LCA-CoA). Feeding Abcb4(-/-) mice the side chain-modified bile acid 24-norursodeoxycholic acid (norUDCA) reversed their liver injury and fibrosis, increased serum levels of lipids, lowered phospholipase and triglyceride hydrolase activities, and restored hepatic LCA-CoA and triglyceride levels. Additional genetic and nutritional studies indicated that lipid metabolism contributed to chronic cholestatic liver injury; crossing peroxisome proliferator-activated receptor (PPAR)-α-deficient mice with Abcb4(-/-) mice (to create double knockouts) or placing Abcb4(-/-) mice on a high-fat diet protected against liver injury, with features similar to those involved in the response to norUDCA. Placing pregnant Abcb4(-/-) mice on high-fat diets prevented liver injury in their offspring. However, fenofibrate, an activator of PPARα, aggravated liver injury in Abcb4(-/-) mice. Alterations in lipid metabolism contribute to the pathogenesis and progression of cholestatic liver disease in mice.

Factors to consider in using [U-C]palmitate for analysis of sphingolipid biosynthesis by tandem mass spectrometry

This study describes the use of a stable-isotope labeled precursor ([U-¹³C]palmitate) to analyze de novo sphingolipid biosynthesis by tandem mass spectrometry. It also describes factors to consider in interpreting the data, including the isotope's location (¹³C appears in three isotopomers and isotopologues: [M + 16] for the sphingoid base or N-acyl fatty acid, and [M + 32] for both); the isotopic enrichment of palmitoyl-CoA; and its elongation, desaturation, and incorporation into N-acyl-sphingolipids. For HEK293 cells incubated with 0.1 mM [U-¹³C]palmitic acid, ∼60% of the total palmitoyl-CoA was ¹³C-labeled by 3 h (which was near isotopic equilibrium); with this correction, the rates of de novo biosynthesis of C16:0-ceramide, C16:0-monohexosylceramide, and C16:0-sphingomyelins were 62 ± 3, 13 ± 2, and 60 ± 11 pmol/h per mg protein, respectively, which are consistent with an estimated rate of appearance of C16:0-ceramide using exponential growth modeling (119 ± 11 pmol/h per mg protein). Including estimates for the very long-chain fatty acyl-CoAs, the overall rate of sphingolipid biosynthesis can be estimated to be at least ∼1.6-fold higher. Thus, consideration of these factors gives a more accurate picture of de novo sphingolipid biosynthesis than has been possible to-date, while acknowledging that there are inherent limitations to such approximations.

Bioinformatic Analysis of Leishmania donovani Long-Chain Fatty Acid-CoA Ligase as a Novel Drug Target.

Fatty acyl-CoA synthetase (fatty acid: CoA ligase, AMP-forming; (EC 6.2.1.3)) catalyzes the formation of fatty acyl-CoA by a two-step process that proceeds through the hydrolysis of pyrophosphate. Fatty acyl-CoA represents bioactive compounds that are involved in protein transport, enzyme activation, protein acylation, cell signaling, and transcriptional control in addition to serving as substrates for beta oxidation and phospholipid biosynthesis. Fatty acyl-CoA synthetase occupies a pivotal role in cellular homeostasis, particularly in lipid metabolism. Our interest in fatty acyl-CoA synthetase stems from the identification of this enzyme, long-chain fatty acyl-CoA ligase (LCFA) by microarray analysis. We found this enzyme to be differentially expressed by Leishmania donovani amastigotes resistant to antimonial treatment. In the present study, we confirm the presence of long-chain fatty acyl-CoA ligase gene in the genome of clinical isolates of Leishmania donovani collected from the disease endemic area in India. We predict a molecular model for this enzyme for in silico docking studies using chemical library available in our institute. On the basis of the data presented in this work, we propose that long-chain fatty acyl-CoA ligase enzyme serves as an important protein and a potential target candidate for development of selective inhibitors against leishmaniasis.

The Vibrio cholerae fatty acid regulatory protein, FadR, represses transcription of plsB, the gene encoding the first enzyme of membrane phospholipid biosynthesis

Glycerol-3-phosphate (sn-glycerol-3-P, G3P) acyltransferase catalyses the first committed step in the biosynthesis of membrane phospholipids, the acylation of G3P to form 1-acyl G3P (lysophosphatidic acid). The paradigm G3P acyltransferase is the Escherichia coli plsB gene product which acylates position-1 of G3P using fatty acids in thioester linkage to either acyl carrier protein (ACP) or CoA as acyl donors. Although the E. coli plsB gene was discovered about 30 years ago, no evidence for transcriptional control of its expression has been reported. However A.E. Kazakov and co-workers (J Bacteriol 2009; 191: 52-64) reported the presence of a putative FadR binding site upstream of the candidate plsB genes of Vibrio cholerae and three other Vibrio species suggesting that plsB might be regulated by FadR, a GntR family transcription factor thus far known only to regulate fatty acid synthesis and degradation. We report that the V. cholerae plsB homologue restored growth of E. coli strain BB26-36 which is a G3P auxotroph due to an altered G3P acyltransferase activity. The plsB promoter was also mapped and the predicted FadR-binding palindrome was found to span positions -19 to -35, upstream of the transcription start site. Gel shift assays confirmed that both V. cholerae FadR and E. coli FadR bound the V. cholerae plsB promoter region and binding was reversed upon addition of long-chain fatty acyl-CoA thioesters. The expression level of the V. cholerae plsB gene was elevated two- to threefold in an E. coli fadR null mutant strain indicating that FadR acts as a repressor of V. cholerae plsB expression. In both E. coli and V. cholerae the β-galactosidase activity of transcriptional fusions of the V. cholerae plsB promoter to lacZ increased two- to threefold upon supplementation of growth media with oleic acid. Therefore, V. cholerae co-ordinates fatty acid metabolism with 1-acyl G3P synthesis.

Acyl-CoA binding proteins interact with the acyl-CoA binding domain of mitochondrial carnitine palmitoyl transferase I

Although the rate limiting step in mitochondrial fatty acid oxidation, catalyzed by carnitine palmitoyl transferase I (CPTI), utilizes long-chain fatty acyl-CoAs (LCFA-CoA) as a substrate, how LCFA-CoA is transferred to CPTI remains elusive. Based on secondary structural predictions and conserved tryptophan residues, the cytoplasmic C-terminal domain was hypothesized to be the LCFA-CoA binding site and important for interaction with cytoplasmic LCFA-CoA binding/transport proteins to provide a potential route for LCFA-CoA transfer. To begin to address this question, the cytoplasmic C-terminal region of liver CPTI (L-CPTI) was recombinantly expressed and purified. Data herein showed for the first time that the L-CPTI C-terminal 89 residues were sufficient for high affinity binding of LCFA-CoA (K (d) = 2-10 nM) and direct interaction with several cytoplasmic LCFA-CoA binding proteins (K (d) < 10 nM), leading to enhanced CPTI activity. Furthermore, alanine substitutions for tryptophan in L-CPTI (W391A and W452A) altered secondary structure, decreased binding affinity for LCFA-CoA, and almost completely abolished L-CPTI activity, suggesting that these amino acids may be important for ligand stabilization necessary for L-CPTI activity. Moreover, while decreased activity of the W452A mutant could be explained by decreased binding of lipid binding proteins, W391 itself seems to be important for activity. These data suggest that both interactions with lipid binding proteins and the peptide itself are important for optimal enzyme activity.

Malonyl-CoA mediates leptin hypothalamic control of feeding independent of inhibition of CPT-1a

Hypothalamic fatty acid metabolism is involved in central nervous system controls of feeding and energy balance. Malonyl-CoA, an intermediate of fatty acid biosynthesis, is emerging as a significant player in these processes. Notably, hypothalamic malonyl-CoA has been implicated in leptin's feeding effect. Leptin treatment increases malonyl-CoA level in the hypothalamic arcuate nucleus (Arc), and this increase is required for leptin-induced decrease in food intake. However, the intracellular downstream mediators of malonyl-CoA's feeding effect have not been identified. A primary biochemical action of malonyl-CoA is the inhibition of the acyltransferase activity of carnitine palmitoyltransferase-1 (CPT-1). In the hypothalamus, the predominant isoform of CPT-1 that possesses the acyltransferase activity is CPT-1 liver type (CPT-1a). To address the role of CPT-1a in malonyl-CoA's anorectic action, we used a recombinant adenovirus expressing a mutant CPT-1a that is insensitive to malonyl-CoA inhibition. We show that Arc overexpression of the mutant CPT-1a blocked the malonyl-CoA-mediated inhibition of CPT-1 activity. However, the overexpression of this mutant did not affect the anorectic actions of leptin or central cerulenin for which an increase in Arc malonyl-CoA level is also required. Thus, CPT-1a does not appear to be involved in the malonyl-CoA's anorectic actions induced by leptin. Furthermore, long-chain fatty acyl-CoAs, substrates of CPT-1a, dissociate from malonyl-CoA's actions in the Arc under different feeding states. Together, our results suggest that Arc intracellular mechanisms of malonyl-CoA's anorectic actions induced by leptin are independent of CPT-1a. The data suggest that target(s), rather than CPT-1a, mediates malonyl-CoA action on feeding.

Long-chain acyl-CoA synthetase 4 modulates prostaglandin E2 release from human arterial smooth muscle cells

Long-chain acyl-CoA synthetases (ACSLs) catalyze the thioesterification of long-chain FAs into their acyl-CoA derivatives. Purified ACSL4 is an arachidonic acid (20:4)-preferring ACSL isoform, and ACSL4 is therefore a probable regulator of lipid mediator production in intact cells. Eicosanoids play important roles in vascular homeostasis and disease, yet the role of ACSL4 in vascular cells is largely unknown. In the present study, the ACSL4 splice variant expressed in human arterial smooth muscle cells (SMCs) was identified as variant 1. To investigate the function of ACSL4 in SMCs, ACSL4 variant 1 was overexpressed, knocked-down by small interfering RNA, or its enzymatic activity acutely inhibited in these cells. Overexpression of ACSL4 resulted in a markedly increased synthesis of arachidonoyl-CoA, increased 20:4 incorporation into phosphatidylethanolamine, phosphatidylinositol, and triacylglycerol, and reduced cellular levels of unesterified 20:4. Accordingly, secretion of prostaglandin E₂ (PGE₂) was blunted in ACSL4-overexpressing SMCs compared with controls. Conversely, acute pharmacological inhibition of ACSL4 activity resulted in increased release of PGE₂. However, long-term downregulation of ACSL4 resulted in markedly reduced PGE₂ secretion. Thus, ACSL4 modulates PGE₂ release from human SMCs. ACSL4 may regulate a number of processes dependent on the release of arachidonic acid-derived lipid mediators in the arterial wall.

Role of Cardiac Steatosis and Lipotoxicity in Obesity Cardiomyopathy

See related article, pp 216–222 The pandemic of obesity is a devastating health problem and contributes to premature morbidity and mortality. Results from clinical and experimental studies have identified a variety of unfavorable consequences of obesity including cardiovascular diseases, pulmonary diseases, cancer, and sleep disorders. An obesity-triggered parallel increase in the prevalence of type 2 diabetes mellitus is also expected to add to the overall cardiovascular burden of obesity. Components of metabolic syndrome such as dyslipidemia, hyperglycemia, insulin resistance, and hypertension are thought to play pivotal roles in obesity-associated sequelae responsible for atherosclerosis, cardiac hypertrophy, and ventricular dysfunction. The presence of 1 or more of these metabolic syndrome components can adversely affect multiple metabolic pathways resulting in alterations in glucose and lipid metabolism, fatty acid (FA) transport/storage/oxidation, oxygen consumption, redox status, and high-energy phosphate metabolism. Although the precise mechanism(s) of action responsible for metabolic derangement-induced cardiac abnormalities in obesity remains poorly understood, 1 theory that has received increasing attention focuses on lipid transport and storage, excessive FA oxidation (FAO), and lipotoxic injury to the heart.1,2 When energy intake exceeds expenditure, fat is stored as triacylglycerol (TG) in adipose tissue. In turn, once fat levels exceed the storage capacity of adipocytes, a variety of neutral lipids are released and accumulated in other cells and tissues including the heart. The presence of lipid inclusions within cardiomyocytes, a condition referred to as cardiac steatosis, has been confirmed in obesity and diabetes.3,4 Although recent evidence indicates that cardiac steatosis, increased availability of FA and excess FAO contribute to cardiac anomalies associated with obesity and type 2 diabetes, it has also been suggested that cardiac steatosis may be a compensatory mechanism used to neutralize FAs and their metabolites through esterification to neutral lipids. Generation of ATP for normal cardiac …

Expression and regulation of GPAT isoforms in cultured human keratinocytes and rodent epidermis

Phospholipids are required for epidermal lamellar body formation. Glycerol 3-phosphate acyltransferases (GPATs) catalyze the initial step in the biosynthesis of glycerolipids. Little is known about the expression and regulation of GPATs in epidermis/keratinocytes. Here, we demonstrate that GPAT 1, 3, and 4 are expressed in epidermis/keratinocytes, whereas GPAT2 is not detected. In mouse epidermis, GPAT 3 and 4 are mainly localized to the upper layers whereas GPAT1 is found in both the upper and lower layers. GPAT1 and 3 mRNA increase during fetal rat epidermal development. No change in GPAT expression was observed in adult mice following acute permeability barrier disruption. Calcium-induced human keratinocyte differentiation increased GPAT3 mRNA whereas both GPAT1 and 4 mRNA levels decreased. In parallel, total GPAT activity increased 2-fold in differentiated keratinocytes attributable to an increase in N-ethylmaleimide (NEM) sensitive GPAT activity localized to microsomes with little change in NEM resistant activity, consistent with an increase in GPAT3. Furthermore, PPARγ or PPARδ activators increased GPAT3 mRNA, microsomal GPAT activity, and glycerol lipid synthesis without affecting the expression of GPAT1 or 4. Finally, both PPARγ and PPARδ activators increased GPAT3 mRNA via increasing its transcription. Thus, multiple isoforms of GPAT are expressed and differentially regulated in epidermis/keratinocytes.

Regulation of C. elegans Fat Uptake and Storage by Acyl-CoA Synthase-3 Is Dependent on NR5A Family Nuclear Hormone Receptor nhr-25

Acyl-CoA synthases are important for lipid synthesis and breakdown, generation of signaling molecules, and lipid modification of proteins, highlighting thechallenge of understanding metabolic pathwayswithin intact organisms. From a C. elegans mutagenesis screen, we found that loss of ACS-3, a long-chain acyl-CoA synthase, causes enhanced intestinal lipid uptake, de novo fat synthesis, and accumulation of enlarged, neutral lipid-rich intestinal depots. Here, we show that ACS-3 functions in seam cells, epidermal cells anatomically distinct from sites of fat uptake and storage, and that acs-3 mutant phenotypes require the nuclear hormone receptor NHR-25, a key regulator of C. elegans molting. Our findings suggest that ACS-3-derived long-chain fattyacyl-CoAs, perhaps incorporated into complexligands such as phosphoinositides, modulate NHR-25 function, which in turn regulates an endocrine program of lipid uptake and synthesis. These results reveal a link between acyl-CoA synthase function and an NR5A family nuclear receptor in C.elegans.

Loss of stearoyl-CoA desaturase 1 rescues cardiac function in obese leptin-deficient mice

The heart of leptin-deficient ob/ob mice is characterized by pathologic left ventricular hypertrophy along with elevated triglyceride (TG) content, increased stearoyl-CoA desaturase (SCD) activity, and increased myocyte apoptosis. In the present study, using an ob/ob;SCD1(-/-) mouse model, we tested the hypothesis that lack of SCD1 could improve steatosis and left ventricle (LV) function in leptin deficiency. We show that disruption of the SCD1 gene improves cardiac function in ob/ob mice by correcting systolic and diastolic dysfunction without affecting levels of plasma TG and FFA. The improvement is associated with reduced expression of genes involved in FA transport and lipid synthesis in the heart, as well as reduction in cardiac FFA, diacylglycerol, TG, and ceramide levels. The rate of FA beta-oxidation is also significantly lower in the heart of ob/ob;SCD1(-/-) mice compared with ob/ob controls. Moreover, SCD1 deficiency reduces cardiac apoptosis in ob/ob mice due to increased expression of antiapoptotic factor Bcl-2 and inhibition of inducible nitric oxide synthase and caspase-3 activities. Reduction in myocardial lipid accumulation and inhibition of apoptosis appear to be one of the main mechanisms responsible for improved LV function in ob/ob mice caused by SCD1 deficiency.

Fatty acids regulate CREBh via transcriptional mechanisms that are dependent on proteasome activity and insulin

Excess fatty acids are closely associated with metabolic dysfunction. The deleterious effects of fatty acids relate, in part, to their ability to up-regulate pro-inflammatory cytokines and propagate a state of systemic inflammation. CREBh is a recently identified transcription factor that appears to be required for hepatic synthesis of C-reactive protein. Recent data suggest that fatty acids can up-regulate CREBh, thus establishing a potential molecular link between fatty acids and inflammation. The aim of this study was to examine the nature and mechanisms of fatty acid-mediated regulation of CREBh. H4IIE liver cells were incubated in the absence or presence of varying concentrations (50-500 μM) of albumin-bound, long-chain saturated (palmitate, stearate) or unsaturated (oleate, linoleate) fatty acids (1-16 h). All fatty acids significantly increased CREBh gene expression via transcriptional mechanisms, at concentrations as low as 50 μM. Palmitate- or oleate-mediated up-regulation of CREBh was not inhibited by triacsin C, an inhibitor of long-chain fatty acyl CoA synthetase, or by the PPARα antagonist, MK886. Inhibition of proteasome activity with MG132 or lactacystin, or inclusion of insulin reduced palmitate- and oleate-mediated increases in CREBh mRNA. Finally, we examined fatty acid regulation of CREBh in vivo. Male Wistar rats were exposed to a 4-h pancreatic clamp combined with infusion of saline or a mixed lipid emulsion. CREBh mRNA and protein were significantly increased in rats exposed to the lipid infusion compared to the saline group. Collectively, these results may have important implications for metabolic diseases characterized by excess fatty acids, insulin resistance, and inflammation.

Adipose Acyl-CoA Synthetase-1 Directs Fatty Acids toward β-Oxidation and Is Required for Cold Thermogenesis

Long-chain acyl-CoA synthetase-1 (ACSL1) contributes 80% of total ACSL activity in adipose tissue and was believed to be essential for the synthesis of triacylglycerol. We predicted that an adipose-specific knockout of ACSL1 (Acsl1(A-/-)) would be lipodystrophic, but compared to controls, Acsl1(A-/-) mice had 30% greater fat mass when fed a low-fat diet and gained weight normally when fed a high-fat diet. Acsl1(A-/-) adipocytes incorporated [(14)C]oleate into glycerolipids normally, but fatty acid (FA) oxidation rates were 50%-90% lower than in control adipocytes and mitochondria. Acsl1(A-/-) mice were markedly cold intolerant, and beta(3)-adrenergic agonists did not increase oxygen consumption, despite normal adrenergic signaling in brown adipose tissue. The reduced adipose FA oxidation and marked cold intolerance of Acsl1(A-/-) mice indicate that normal activation of FA for oxidation in adipose tissue in vivo requires ACSL1. Thus, ACSL1 has a specific function in directing the metabolic partitioning of FAs toward beta-oxidation in adipocytes.

Acyl-CoA binding protein gene ablation induces pre-implantation embryonic lethality in mice.

Unique among the intracellular lipid binding proteins, acyl-CoA binding protein (ACBP) exclusively binds long-chain fatty acyl-CoAs (LCFA-CoAs). To test if ACBP is an essential protein in mammals, the ACBP gene was ablated by homologous recombination in mice. While ACBP heterozygotes appeared phenotypically normal, intercrossing of the heterozygotes did not produce any live homozygous deficient (null) ACBP((-/-)) pups. Heterozygous and wild type embryos were detected at all post-implantation stages, but no homozygous ACBP-null embryos were obtained-suggesting that an embryonic lethality occurred at a pre-implantation stage of development, or that embryos never formed. While ACBP-null embryos were not detected at any blastocyst stage, ACBP-null embryos were detected at the morula (8-cell), cleavage (2-cell), and zygote (1-cell) pre-implantation stages. Two other LCFA-CoA binding proteins, sterol carrier protein-2 (SCP-2) and sterol carrier protein-x (SCP-x) were significantly upregulated at these stages. These findings demonstrate for the first time that ACBP is an essential protein required for embryonic development and its loss of function may be initially compensated by concomitant upregulation of two other LCFA-CoA binding proteins, but only at the earliest pre-implantation stages. The fact that ACBP is the first known intracellular lipid binding protein whose deletion results in embryonic lethality suggests its vital importance in mammals.

Western diet changes cardiac acyl-CoA composition in obese rats: a potential role for hepatic lipogenesis

The "lipotoxic footprint" of cardiac maladaptation in diet-induced obesity is poorly defined. We investigated how manipulation of dietary lipid and carbohydrate influenced potential lipotoxic species in the failing heart. In Wistar rats, contractile dysfunction develops at 48 weeks on a high-fat/high-carbohydrate "Western" diet, but not on low-fat/high-carbohydrate or high-fat diets. Cardiac content of the lipotoxic candidates--diacylglycerol, ceramide, lipid peroxide, and long-chain acyl-CoA species--was measured at different time points by high-performance liquid chromatography and biochemical assays, as was lipogenic capacity in the heart and liver by qRT-PCR and radiometric assays. Changes in membranes fluidity were also monitored using fluorescence polarization. We report that Western feeding induced a 40% decrease in myocardial palmitoleoyl-CoA content and a similar decrease in the unsaturated-to-saturated fatty acid ratio. These changes were associated with impaired cardiac mitochondrial membrane fluidity. At the same time, hepatic lipogenic capacity was increased in animals fed Western diet (+270% fatty acid elongase activity compared with high-fat diet), while fatty acid desaturase activity decreased over time. Our findings suggest that dysregulation of lipogenesis is a significant component of heart failure in diet-induced obesity.

Expression of Long-chain Fatty Acyl-CoA Synthetase 4 in Breast and Prostate Cancers Is Associated with Sex Steroid Hormone Receptor Negativity

Previous studies have shown that key enzymes involved in lipid metabolic pathways are differentially expressed in normal compared with tumor tissues. However, the precise role played by dysregulated expression of lipid metabolic enzymes and altered lipid homeostasis in carcinogenesis remains to be established. Fatty acid synthase is overexpressed in a variety of cancers, including breast and prostate. The purpose of the present study was to examine the expression patterns of additional lipid metabolic enzymes in human breast and prostate cancers. This was accomplished by analysis of published expression databases, with confirmation by immunoblot assays. Our results indicate that the fatty acid-activating enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4), is differentially expressed in human breast cancer as a function of estrogen receptor alpha (ER) status. In 10 separate studies, ACSL4 messenger RNA (mRNA) was overexpressed in ER-negative breast tumors. Of 50 breast cancer cell lines examined, 17 (89%) of 19 ER-positive lines were negative for ACSL4 mRNA expression and 20 (65%) of 31 ER-negative lines expressed ACSL4 mRNA. The inverse relationship between ER expression and ACSL4 expression was also observed for androgen receptor status in both breast and prostate cancers. Furthermore, loss of steroid hormone sensitivity, such as that observed in Raf1-transfected MCF-7 cells and LNCaP-AI cells, was associated with induction of ACSL4 expression. Ablation of ACSL4 expression inMDA-MB-231 breast cancer cells had no effect on cell proliferation; however, sensitivity to the cytotoxic effects of triacsin C was increased three-fold in the cells lacking ACSL4.

ACSVL3 depletion in U87 glioma cells affects both fatty acid and cholesterol homeostasis

Human malignant gliomas overexpress ACSVL3, a member of the very long‐chain fatty acyl‐CoA synthetase family. Depletion of ACSVL3 in cultured human U87 glioma cells by RNA interference decreased both their malignant growth properties in vitro and their ability to produce tumors in mice. To understand how ACSVL3 knockdown (KD) in U87 cells promotes these beneficial effects, we investigated fatty acid and cholesterol homeostasis. Incorporation of [14C]C16:0 into several lipids, including phosphatidylinositol, phosphatidylethanolamine, and non‐hydroxy ceramide, was reduced in KD U87 cells. Although the mass of triacylglycerol (TAG) in U87 cells was barely detectable, [14C]C16:0 incorporation into TAG was robust, and was decreased in KD glioma cells. Surprisingly, the mass of free cholesterol was reduced by ~40% in ACSVL3 KD cells. This was due, in part, to decreased de novo cholesterol synthesis, as measured by [14C]acetate incorporation. Acetate incorporation into free fatty acids was also reduced, indicating that ACSVL3 depletion also affects de novo fatty acid synthesis. We hypothesize that ACSVL3 depletion affects fatty acid and cholesterol homeostasis via a common mechanism. Studies to elucidate this mechanism are in progress. Supported by NIH grant NS062043 and a grant from the National Brain Tumor Society.