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1.  Lipid signals and insulin resistance 
Clinical lipidology  2013;8(6):659-667.
The metabolic syndrome, a cluster of metabolic derangements that include obesity, glucose intolerance, dyslipidemia and hypertension, is a major risk factor for cardiovascular disease. Insulin resistance has been proposed to be the common feature that links obesity to the metabolic syndrome, but the mechanism remains obscure. Although the excess content of triacylglycerol in muscle and liver is highly associated with insulin resistance in these tissues, triacylglycerol itself is not causal but merely a marker. Thus, attention has turned to the accumulation of cellular lipids known to have signaling roles. This review will discuss recent progress in understanding how glycerolipids and related lipid intermediates may impair insulin signaling.
PMCID: PMC3921899  PMID: 24533033
acyl-CoA; ceramide; diacylglycerol; lysophosphatidic acid; phosphatidic acid
2.  Glycerol-3-Phosphate Acyltranferase-2 Behaves as a Cancer Testis Gene and Promotes Growth and Tumorigenicity of the Breast Cancer MDA-MB-231 Cell Line 
PLoS ONE  2014;9(6):e100896.
The de novo synthesis of glycerolipids in mammalian cells begins with the acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferase (GPAT). GPAT2 is a mitochondrial isoform primarily expressed in testis under physiological conditions. Because it is aberrantly expressed in multiple myeloma, it has been proposed as a novel cancer testis gene. Using a bioinformatics approach, we found that GPAT2 is highly expressed in melanoma, lung, prostate and breast cancer, and we validated GPAT2 expression at the protein level in breast cancer by immunohistochemistry. In this case GPAT2 expression correlated with a higher histological grade. 5-Aza-2′ deoxycytidine treatment of human cells lines induced GPAT2 expression suggesting epigenetic regulation of gene expression. In order to evaluate the contribution of GPAT2 to the tumor phenotype, we silenced its expression in MDA-MB-231 cells. GPAT2 knockdown diminished cell proliferation, anchorage independent growth, migration and tumorigenicity, and increased staurosporine-induced apoptosis. In contrast, GPAT2 over-expression increased cell proliferation rate and resistance to staurosporine-induced apoptosis. To understand the functional role of GPAT2, we performed a co-expression analysis in mouse and human testis and found a significant association with semantic terms involved in cell cycle, DNA integrity maintenance, piRNA biogenesis and epigenetic regulation. Overall, these results indicate the GPAT2 would be directly associated with the control of cell proliferation. In conclusion, we confirm GPAT2 as a cancer testis gene and that its expression contributes to the tumor phenotype of MDA-MB-231 cells.
PMCID: PMC4072688  PMID: 24967918
3.  Expanding roles for lipid droplets 
PMCID: PMC4040132  PMID: 21531573
4.  Acyl-coenzyme A synthetases in metabolic control 
Current opinion in lipidology  2010;21(3):212-217.
Purpose of review
The 11 long-chain (ACSL) and very long chain acyl-coenzyme A (acyl-CoA) synthetases [(ACSVL)/fatty acid transport protein] are receiving considerable attention because it has become apparent that their individual functions are not redundant.
Recent findings
Recent studies have focused on the structure of the acyl-CoA synthetases, their post-translational modification, their ability to activate fatty acids of varying chain lengths, and their role in directing fatty acids into different metabolic pathways. An unsettled controversy focuses on the ACSVL isoforms and whether these have both enzymatic and transport functions. Another issue is whether conversion of a fatty acid to an acyl-CoA produces an increase in the AMP/ATP ratio that is sufficient to activate AMP-activated kinase.
FuturestudiesarerequiredtodeterminethesubcellularlocationofeachACSLandACSVL isoform and the functional importance of phosphorylation and acetylation. Purification and crystallization of mammalian ACSL and ACSVL isoforms is needed to confirm the mechanism of action and discover how these enzymesdiffer in their affinity for fatty acids of differentchainlengths.Functionally,itwillbeimportanttolearnhowtheACSLisoformscan direct their acyl-CoA products toward independent downstream pathways.
PMCID: PMC4040134  PMID: 20480548
β-oxidation; acyl-CoA synthetase; AMP-activated kinase; fatty acid; fatty acid transport protein; glycerolipid synthesis
5.  Propylisopropylacetic acid (PIA), a constitutional isomer of valproic acid, uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: a potential drug for bipolar disorder 
Biochimica et biophysica acta  2013;1831(4):880-886.
Mood stabilizers used for treating bipolar disorder (BD) selectively downregulate arachidonic acid (AA) turnover (deacylation-reacylation) in brain phospholipids, when given chronically to rats. In vitro studies suggest that one of these, valproic acid (VPA), which is teratogenic, reduces AA turnover by inhibiting the brain acyl-CoA synthetase (Acsl)-4 mediated acylation of AA to AA-CoA. We tested whether non-teratogenic VPA analogues might also inhibit Acsl-4 catalyzed acylation, and thus have potential anti-BD action.
Rat Acsl4-flag protein was expressed in E. coli, and the ability of three VPA analogues, propylisopropylacetic acid (PIA), propylisopropylacetamide (PID) and N-methyl-2,2,3,3-tetramethylcyclopropanecarboxamide (MTMCD), and of sodium butyrate, to inhibit conversion of AA to AA-CoA by Acsl4 was quantified using Michaelis-Menten kinetics.
Acsl4-mediated conversion of AA to AA-CoA in vitro was inhibited uncompetitively by PIA, with a Ki of 11.4 mM compared to a published Ki of 25 mM for VPA, while PID, MTMCD and sodium butyrate had no inhibitory effect.
PIA's ability to inhibit conversion of AA to AA-CoA by Acsl4 in vitro suggests that, like VPA, PIA may reduce AA turnover in brain phospholipids in unanesthetized rats, and if so, may be effective as a non-teratogenic mood stabilizer in BD patients.
PMCID: PMC3593989  PMID: 23354024
bipolar disorder; valproate; arachidonic acid; acyl-CoA synthetase 4; mood stabilizer; Acsl4; brain; MTMCD; N-methyl-2,2,3,3-tetramethylcyclopropanecarboxamide; PIA; propylisopropylacetic; PID; propylisopropylacetamide; rat; butyrate; inhibition; uncompetitive; enzyme; anticonvulsant
6.  Endothelial Acyl-CoA Synthetase 1 is not Required for Inflammatory and Apoptotic Effects of a Saturated Fatty Acid-Rich Environment 
Saturated fatty acids, such as palmitic and stearic acid, cause detrimental effects in endothelial cells (ECs) and have been suggested to contribute to macrophage accumulation in adipose tissue and the vascular wall in states of obesity and insulin resistance. Long-chain fatty acids are believed to require conversion into acyl-CoA derivatives to exert most of their detrimental effects, a reaction catalyzed by acyl-CoA synthetases (ACSL). The objective of this study was to investigate the role of ACSL1, an ACSL isoform previously shown to mediate inflammatory effects in myeloid cells, in regulating EC responses to a saturated fatty acid-rich environment in vitro and in vivo.
Methods and Results
Saturated fatty acids caused increased inflammatory activation, ER stress, and apoptosis in mouse microvascular ECs. Forced ACSL1 overexpression exacerbated the effects of saturated fatty acids on apoptosis and ER stress. However, endothelial ACSL1-deficiency did not protect against the effects of saturated fatty acids in vitro, nor did it protect insulin resistant mice fed a saturated fatty acid-rich diet from macrophage adipose tissue accumulation or increased aortic adhesion molecule expression.
Endothelial ACSL1 is not required for inflammatory and apoptotic effects of a saturated fatty acid-rich environment.
PMCID: PMC3553857  PMID: 23241406
Acyl-CoA synthetase; Apoptosis; Endothelium; Inflammation
7.  Contribution of novel ATGL missense mutations to the clinical phenotype of NLSD-M: a strikingly low amount of lipase activity may preserve cardiac function 
Human Molecular Genetics  2012;21(24):5318-5328.
The lack of adipose triglyceride lipase (ATGL), a patatin-like phospholipase domain-containing enzyme that hydrolyzes fatty acids from triacylglycerol (TAG) stored in multiple tissues, causes the autosomal recessive disorder neutral lipid storage disease with myopathy (NLSD-M). In two families of Lebanese and Italian origin presenting with NLSD-M, we identified two new missense mutations in highly conserved regions of ATGL (p.Arg221Pro and p.Asn172Lys) and a novel nonsense mutation (p.Trp8X). The Lebanese patients harbor homozygous p.Arg221Pro, whereas the Italian patients are heterozygotes for p.Asn172Lys and the p.Trp8X mutation. The p.Trp8X mutation results in a complete absence of ATGL protein, while the p.Arg221Pro and p.Asn172Lys mutations result in proteins with minimal lipolytic activity. Although these mutations did not affect putative catalytic residues or the lipid droplet (LD)-binding domain of ATGL, cytosolic LDs accumulated in cultured skin fibroblasts from the patients. The missense mutations might destabilize a random coil (p.Asn172Lys) or a helix (p.Arg221Pro) structure within or proximal to the patatin domain of the lipase, thereby interfering with the enzyme activity, while leaving intact the residues required to localize the protein to LDs. Overexpressing wild-type ATGL in one patient's fibroblasts corrected the metabolic defect and effectively reduced the number and area of cellular LDs. Despite the poor lipase activity in vitro, the Lebanese siblings have a mild myopathy and not clinically evident myocardial dysfunction. The patients of Italian origin show a late-onset and slowly progressive skeletal myopathy. These findings suggest that a small amount of correctly localized lipase activity preserves cardiac function in NLSD-M.
PMCID: PMC3510752  PMID: 22990388
8.  Phosphorylation and Acetylation of Acyl-CoA Synthetase- I 
Long chain acyl-CoA synthetase 1 (ACSL1) contributes 50 to 90% of total ACSL activity in liver, adipose tissue, and heart and appears to direct the use of long chain fatty acids for energy. Although the functional importance of ACSL1 is becoming clear, little is understood about its post-translational regulation. In order to investigate the post-translational modifications of ACSL1 under different physiological conditions, we overexpressed ACSL1 in hepatocytes, brown adipocytes, and 3T3-L1 differentiated adipocytes, treated these cells with different hormones, and analyzed the resulting phosphorylated and acetylated amino acids by mass spectrometry. We then compared these results to the post-translational modifications observed in vivo in liver and brown adipose tissue after mice were fasted or exposed to a cold environment. We identified universal N-terminal acetylation, 15 acetylated lysines, and 25 phosphorylation sites on ACSL1. Several unique acetylation and phosphorylation sites occurred under conditions in which fatty acid β-oxidation is normally enhanced. Thirteen of the acetylated lysines had not previously been identified, and none of the phosphorylation sites had been previously identified. Site-directed mutagenesis was used to introduce mutations at three potential acetylation and phosphorylation sites believed to be important for ACSL1 function. At the ATP/AMP binding site and at a highly conserved site near the C terminus, modifications of Ser278 or Lys676, respectively, totally inhibited ACSL1 activity. In contrast, mutations of Lys285 that mimicked acetylation (Lys285Ala and Lys285Gln) reduced ACSL activity, whereas full activity was retained by Lys285Arg, suggesting that acetylation of Lys285 would be likely to decrease ACSL1 activity. These results indicate that ACSL1 is highly modified post-translationally. Several of these modifications would be expected to alter enzymatic function, but others may affect protein stability or protein-protein interactions.
PMCID: PMC3772793  PMID: 24039348
Fatty acid metabolism; Beta-oxidation; Post-translational modification; Triacylglycerol synthesis; phosphorylation; Acetylation
9.  Triacylglycerol Synthesis Enzymes Mediate Lipid Droplet Growth by Relocalizing from the ER to Lipid Droplets 
Developmental cell  2013;24(4):384-399.
Lipid droplets (LDs) store metabolic energy and membrane lipid precursors. With excess metabolic energy, cells synthesize triacylglycerol (TG) and form LDs that grow dramatically. It is unclear how TG synthesis relates to LD formation and growth. Here, we identify two LD subpopulations: smaller LDs of relatively constant size, and LDs that grow larger. The latter population contains isoenzymes for each step of TG synthesis. Glycerol-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the first and rate-limiting step, relocalizes from the endoplasmic reticulum (ER) to a subset of forming LDs, where it becomes stably associated. ER-to-LD targeting of GPAT4 and other LD-localized TG synthesis isozymes is required for LD growth. Key features of GPAT4 ER-to-LD targeting and function in LD growth are conserved between Drosophila and mammalian cells. Our results explain how TG synthesis is coupled with LD growth and identify two distinct LD subpopulations based on their capacity for localized TG synthesis.
PMCID: PMC3727400  PMID: 23415954
10.  Mammalian Triacylglycerol Metabolism: Synthesis, Lipolysis and Signaling 
Chemical reviews  2011;111(10):6359-6386.
PMCID: PMC3181269  PMID: 21627334
11.  Glycerol-3-Phosphate Acyltransferase-2 Is Expressed in Spermatic Germ Cells and Incorporates Arachidonic Acid into Triacylglycerols 
PLoS ONE  2012;7(8):e42986.
De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. In this work we show that mitochondrial GPAT2 overexpression in CHO-K1 cells increased TAG content and both GPAT and AGPAT activities 2-fold with arachidonoyl-CoA as a substrate, indicating specificity for this fatty acid.
Methods and Results
Incubation of GPAT2-transfected CHO-K1 cells with [1-14C]arachidonate for 3 h increased incorporation of [14C]arachidonate into TAG by 40%. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2's role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein.
These results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.
PMCID: PMC3414494  PMID: 22905194
12.  Valproate uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: Relevance to valproate’s efficacy against bipolar disorder 
Biochimica et biophysica acta  2010;1811(3):163-169.
The ability of chronic valproate (VPA) to reduce arachidonic acid (AA) turnover in brain phospholipids of unanesthetized rats has been ascribed to its inhibition of acyl-CoA synthetase (Acsl)-mediated activation of AA to AA-CoA. Our aim was to identify a rat Acsl isoenzyme that could be inhibited by VPA in vitro.
Rat Acsl3-, Acsl6v1- and Acsl6v2-, and Acsl4-flag proteins were expressed in E. coli, and the ability of VPA to inhibit their activation of long-chain fatty acids to acyl-CoA was estimated using Michaelis-Menten kinetics.
VPA uncompetitively inhibited Acsl4-mediated conversion of AA and of docosahexaenoic (DHA) but not of palmitic acid to acyl-CoA, but did not affect AA conversion by Acsl3, Acsl6v1 or Acsl6v2. Acsl4-mediated conversion of AA to AA-CoA showed substrate inhibition and had a 10-times higher catalytic efficiency than did conversion of DHA to DHA-CoA. Butyrate, octanoate, or lithium did not inhibit AA activation by Acsl4.
VPA’s ability to inhibit Acsl4 activation of AA and of DHA to their respective acyl-CoAs, related to the higher catalytic efficiency of AA than DHA conversion, may account for VPA’s selective reduction of AA turnover in rat brain phospholipids, and contribute to VPA’s efficacy against bipolar disorder.
PMCID: PMC3037030  PMID: 21184843
bipolar disorder; valproate; arachidonic acid; acyl-CoA synthetase; mood stabilizer; Acsl4; brain; rat
13.  Mice deficient in mitochondrial glycerol-3-phosphate acyltransferase-1 have diminished myocardial triacylglycerol accumulation during lipogenic diet and altered phospholipid fatty acid composition 
Biochimica et Biophysica Acta  2008;1781(6-7):352-358.
Glycerol-3-phosphate acyltransferase-1 (GPAT1), which is located on the outer mitochondrial membrane comprises up to 30% of total GPAT activity in the heart. It is one of at least four mammalian GPAT isoforms known to catalyze the initial, committed, and rate limiting step of glycerolipid synthesis. Because excess triacylglycerol (TAG) accumulates in cardiomyocytes in obesity and type 2 diabetes, we determined whether lack of GPAT1 would alter the synthesis of heart TAG and phospholipids after a 2-week high sucrose diet or a 3-month high fat diet. Even in the absence of hypertriglyceridemia, TAG increased 2-fold with both diets in hearts from wildtype mice. In contrast, hearts from Gpat1−/− mice contained 20–80% less TAG than the wildtype controls. In addition, hearts from Gpat1−/− mice fed the high-sucrose diet incorporate 60% less [14C]palmitate into heart TAG as compared to wildtype mice. Because GPAT1 prefers 16:0-CoA to other long chain acyl-CoA substrates, we determined the fatty acid composition of heart phospholipids. Compared to wildtype littermate controls, hearts from Gpat1−/− mice contained a lower amount of 16:0 in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine/phosphatidylinositol and significantly more C20:4n6. Phosphatidylcholine and phosphatidylethanolamine from Gpat1−/− hearts also contained higher amounts of 18:0 and 18:1. Although at least three other GPAT isoforms are expressed in the heart, our data suggest that GPAT1 contributes significantly to cardiomyocyte TAG synthesis during lipogenic or high fat diets and influences the incorporation of 20:4n6 into heart phospholipids.
PMCID: PMC3285559  PMID: 18522808
obesity; type 2 diabetes; lipotoxicity; diabetic cardiomyopathy; arachidonic acid
14.  The role of lipid droplets in metabolic disease in rodents and humans 
The Journal of Clinical Investigation  2011;121(6):2102-2110.
Lipid droplets (LDs) are intracellular organelles that store neutral lipids within cells. Over the last two decades there has been a dramatic growth in our understanding of LD biology and, in parallel, our understanding of the role of LDs in health and disease. In its simplest form, the LD regulates the storage and hydrolysis of neutral lipids, including triacylglycerol and/or cholesterol esters. It is becoming increasingly evident that alterations in the regulation of LD physiology and metabolism influence the risk of developing metabolic diseases such as diabetes. In this review we provide an update on the role of LD-associated proteins and LDs in metabolic disease.
PMCID: PMC3104768  PMID: 21633178
15.  Agpat6—a Novel Lipid Biosynthetic Gene Required for Triacylglycerol Production in Mammary Epithelium 
Journal of lipid research  2006;47(4):734-744.
In analyzing the sequence tags for mutant mouse embryonic stem (ES) cell lines in BayGenomics (a mouse gene-trapping resource), we identified a novel gene, Agpat6, with sequence similarities to previously characterized glycerolipid acyltransferases. Agpat6’s closest family member is another novel gene that we have provisionally designated Agpat8. Both Agpat6 and Agpat8 are conserved from plants, nematodes, and flies to mammals. AGPAT6, which is predicted to contain multiple membrane-spanning helices, is found exclusively within the endoplasmic reticulum in mammalian cells. To gain insights into the in vivo importance of Agpat6, we used the Agpat6 ES cell line from BayGenomics to create Agpat6-deficient (Agpat6−/−) mice. Agpat6−/− mice lacked full-length Agpat6 transcripts, as judged by northern blots. One of the most striking phenotypes of Agpat6−/− mice was a defect in lactation. Pups nursed by Agpat6−/− mothers die perinatally. Normally, Agpat6 is expressed at high levels in the mammary epithelium of breast tissue, but not in the surrounding adipose tissue. Histological studies revealed that the aveoli and ducts of Agpat6−/− lactating mammary glands were underdeveloped, and there was a dramatic decrease in size and number of lipid droplets within mammary epithelial cells and ducts. Also, the milk from Agpat6−/− mice was markedly depleted in diacylglycerols and triacylglycerols. Thus, we identified a novel glycerolipid acyltransferase of the endoplasmic reticulum, AGPAT6, which is crucial for the production of milk fat by the mammary gland.
PMCID: PMC3196597  PMID: 16449762
LPAAT; acyltransferase; transacylase; milk fat
16.  Mouse Cardiac Acyl Coenzyme A Synthetase 1 Deficiency Impairs Fatty Acid Oxidation and Induces Cardiac Hypertrophy▿ †  
Molecular and Cellular Biology  2011;31(6):1252-1262.
Long-chain acyl coenzyme A (acyl-CoA) synthetase isoform 1 (ACSL1) catalyzes the synthesis of acyl-CoA from long-chain fatty acids and contributes the majority of cardiac long-chain acyl-CoA synthetase activity. To understand its functional role in the heart, we studied mice lacking ACSL1 globally (Acsl1T−/−) and mice lacking ACSL1 in heart ventricles (Acsl1H−/−) at different times. Compared to littermate controls, heart ventricular ACSL activity in Acsl1T−/− mice was reduced more than 90%, acyl-CoA content was 65% lower, and long-chain acyl-carnitine content was 80 to 90% lower. The rate of [14C]palmitate oxidation in both heart homogenate and mitochondria was 90% lower than in the controls, and the maximal rates of [14C]pyruvate and [14C]glucose oxidation were each 20% higher. The mitochondrial area was 54% greater than in the controls with twice as much mitochondrial DNA, and the mRNA abundance of Pgc1α and Errα increased by 100% and 41%, respectively. Compared to the controls, Acsl1T−/− and Acsl1H−/− hearts were hypertrophied, and the phosphorylation of S6 kinase, a target of mammalian target of rapamycin (mTOR) kinase, increased 5-fold. Our data suggest that ACSL1 is required to synthesize the acyl-CoAs that are oxidized by the heart, and that without ACSL1, diminished fatty acid (FA) oxidation and compensatory catabolism of glucose and amino acids lead to mTOR activation and cardiac hypertrophy without lipid accumulation or immediate cardiac dysfunction.
PMCID: PMC3067914  PMID: 21245374
17.  Adipose acyl-CoA synthetase-1 (ACSL1) directs fatty acids towards β-oxidation and is required for cold thermogenesis 
Cell metabolism  2010;12(1):53-64.
Acyl-CoA synthetase-1 (ACSL) 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 (Acsl1A−/−) would be lipodystrophic, but, compared to controls, Acsl1A−/− mice had 30% greater fat mass when fed a low fat diet, and gained weight normally when fed a high fat diet. Acsl1A−/− adipocytes incorporated [14C]oleate into glycerolipids normally, but fatty acid oxidation rates were 50–90% lower than in control adipocytes and mitochondria. Acsl1A−/− mice were markedly cold intolerant, and β3-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 Acsl1A−/− 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 fatty acids towards β-oxidation.
PMCID: PMC2910420  PMID: 20620995
fatty acid oxidation; cold thermogenesis; brown adipose; triacylglycerol
18.  Glycerol-3-Phosphate Acyltransferase 1 Deficiency in ob/ob Mice Diminishes Hepatic Steatosis but Does Not Protect Against Insulin Resistance or Obesity 
Diabetes  2010;59(6):1321-1329.
Hepatic steatosis is strongly associated with insulin resistance, but a causal role has not been established. In ob/ob mice, sterol regulatory element binding protein 1 (SREBP1) mediates the induction of steatosis by upregulating target genes, including glycerol-3-phosphate acyltransferase-1 (Gpat1), which catalyzes the first and committed step in the pathway of glycerolipid synthesis. We asked whether ob/ob mice lacking Gpat1 would have reduced hepatic steatosis and improved insulin sensitivity.
Hepatic lipids, insulin sensitivity, and hepatic insulin signaling were compared in lean (Lep+/?), lean-Gpat1−/−, ob/ob (Lepob/ob), and ob/ob-Gpat1−/− mice.
Compared with ob/ob mice, the lack of Gpat1 in ob/ob mice reduced hepatic triacylglycerol (TAG) and diacylglycerol (DAG) content 59 and 74%, respectively, but increased acyl-CoA levels. Despite the reduction in hepatic lipids, fasting glucose and insulin concentrations did not improve, and insulin tolerance remained impaired. In both ob/ob and ob/ob-Gpat1−/− mice, insulin resistance was accompanied by elevated hepatic protein kinase C-ε activation and blunted insulin-stimulated Akt activation.
These results suggest that decreasing hepatic steatosis alone does not improve insulin resistance, and that factors other than increased hepatic DAG and TAG contribute to hepatic insulin resistance in this genetically obese model. They also show that the SREBP1-mediated induction of hepatic steatosis in ob/ob mice requires Gpat1.
PMCID: PMC2874692  PMID: 20200319
19.  Lysophosphatidic Acid Activates Peroxisome Proliferator Activated Receptor-γ in CHO Cells That Over-Express Glycerol 3-Phosphate Acyltransferase-1 
PLoS ONE  2011;6(4):e18932.
Lysophosphatidic acid (LPA) is an agonist for peroxisome proliferator activated receptor-γ (PPARγ). Although glycerol-3-phosphate acyltransferase-1 (GPAT1) esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO) cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA) or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression.
PMCID: PMC3080373  PMID: 21533082
20.  Acyl-CoA synthesis, lipid metabolism and lipotoxicity 
Biochimica et biophysica acta  2009;1801(3):246-251.
Although the underlying causes of insulin resistance have not been completely delineated, in most analyses, a recurring theme is dysfunctional metabolism of fatty acids. Because the conversion of fatty acids to activated acyl-CoAs is the first and essential step in the metabolism of long-chain fatty acid metabolism, interest has grown in the synthesis of acyl-CoAs, their contribution to the formation of signaling molecules like ceramide and diacylglycerol, and their direct effects on cell function. In this review, we cover the evidence for the involvement of acyl-CoAs in what has been termed lipotoxicity, the regulation of the acyl-CoA synthetases, and the emerging functional roles of acyl-CoAs in the major tissues that contribute to insulin resistance and lipotoxicity, adipose, liver, heart and pancreas.
PMCID: PMC2824076  PMID: 19818872
22.  Clinical and genetic characterization of chanarin-dorfman syndrome patients: first report of large deletions in the ABHD5 gene 
Chanarin-Dorfman syndrome (CDS) is a rare autosomal recessive disorder characterized by nonbullous congenital ichthyosiform erythroderma (NCIE) and an intracellular accumulation of triacylglycerol (TG) droplets in most tissues. The clinical phenotype involves multiple organs and systems, including liver, eyes, ears, skeletal muscle and central nervous system (CNS). Mutations in ABHD5/CGI58 gene are associated with CDS.
Eight CDS patients belonging to six different families from Mediterranean countries were enrolled for genetic study. Molecular analysis of the ABHD5 gene included the sequencing of the 7 coding exons and of the putative 5' regulatory regions, as well as reverse transcript-polymerase chain reaction analysis and sequencing of normal and aberrant ABHD5 cDNAs.
Five different mutations were identified, four of which were novel, including two splice-site mutations (c.47+1G>A and c.960+5G>A) and two large deletions (c.898_*320del and c.662-1330_773+46del). All the reported mutations are predicted to be pathogenic because they lead to an early stop codon or a frameshift producing a premature termination of translation. While nonsense, missense, frameshift and splice-site mutations have been identified in CDS patients, large genomic deletions have not previously been described.
These results emphasize the need for an efficient approach for genomic deletion screening to ensure an accurate molecular diagnosis of CDS. Moreover, in spite of intensive molecular screening, no mutations were identified in one patient with a confirmed clinical diagnosis of CDS, appointing to genetic heterogeneity of the syndrome.
PMCID: PMC3019207  PMID: 21122093
23.  Glycerol-3-phosphate acyltransferases: Rate limiting enzymes of triacylglycerol biosynthesis 
Biochimica et biophysica acta  2008;1791(6):501-506.
Four homologous isoforms of glycerol-3-phosphate acyltransferase (GPAT), each the product of a separate gene, catalyze the synthesis of lysophosphatidic acid from glycerol-3-phosphate and long-chain acyl-CoA. This step initiates the synthesis of all the glycerolipids and evidence from gain-of-function and loss-of-function studies in mice and in cell culture strongly suggests that each isoform contributes to the synthesis of triacylglycerol. Much work remains to fully delineate the regulation of each GPAT isoform and its individual role in triacylglycerol synthesis.
PMCID: PMC2737689  PMID: 19038363
Glycerolipid; phospholipid; membrane; lipid droplet; lysophosphatidic acid; diacylglycerol
24.  Long-chain acyl-CoA synthetases and fatty acid channeling 
Future lipidology  2007;2(4):465-476.
Thirteen homologous proteins comprise the long-chain acyl-CoA synthetase (ACSL), fatty acid transport protein (FATP), and bubblegum (ACSBG) subfamilies that activate long-chain and very-long-chain fatty acids to form acyl-CoAs. Gain- and loss-of-function studies show marked differences in the ability of these enzymes to channel fatty acids into different pathways of complex lipid synthesis. Further, the ability of the ACSLs and FATPs to enhance cellular FA uptake does not always require these proteins to be present on the plasma membrane; instead, FA uptake can be increased by enhancing its conversion to acyl-CoA and its metabolism in downstream pathways. Since altered fatty acid metabolism is a hallmark of numerous metabolic diseases and pathological conditions, the ACSL, FATP and ACSBG isoforms are likely to play important roles in disease etiology.
PMCID: PMC2846691  PMID: 20354580
25.  Identification of a novel sn-glycerol-3-phosphate acyltransferase isoform, GPAT4, as the enzyme deficient in Agpat6−/− mice 
Journal of lipid research  2008;49(4):823.
Elucidation of the metabolic pathways of triacylglycerol (TAG) synthesis is critical to the understanding of chronic metabolic disorders such as obesity, cardiovascular disease, and diabetes. sn-Glycerol-3-phosphate acyltransferase (GPAT) and sn-1-acylglycerol-3-phosphate acyltransferase (AGPAT) catalyze the first and second steps in de novo TAG synthesis. AGPAT6 is one of eight AGPAT isoforms identified through sequence homology, but the enzyme activity for AGPAT6 has not been confirmed. We found that in liver and brown adipose tissue from Agpat6-deficient (Agpat6−/−) mice, N-ethylmaleimide (NEM)-sensitive GPAT specific activity was 65% lower than in tissues from wild-type mice, but AGPAT specific activity was similar. Overexpression of Agpat6 in Cos-7 cells increased an NEM-sensitive GPAT specific activity, but AGPAT specific activity was not increased. Agpat6 and Gpat1 overexpression in Cos-7 cells increased the incorporation of [14C]oleate into diacylglycerol (DAG) or into DAG and TAG, respectively, suggesting that the lysophosphatidic acid, phosphatidic acid, and DAG intermediates initiated by each of these isoforms lie in different cellular pools. Together, these data show that “Agpat6−/− mice” are actually deficient in a novel NEM-sensitive GPAT, GPAT4, and indicate that the alterations in lipid metabolism in adipose tissue, liver, and mammary epithelium of these mice are attributable to the absence of GPAT4
PMCID: PMC2819352  PMID: 18192653
triacylglycerol; phospholipid; lipodystrophy; acyl-coenzyme A; steatosis; sn-l-acylglycerol-3-phosphate O-acyltransferase-deficient mice

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