In mammals, a family of five acyl-CoA synthetases (ACSLs), each the product of a separate gene, activates long chain fatty acids to form acyl-CoAs. Because the ACSL isoforms have overlapping preferences for fatty acid chain length and saturation and are expressed in many of the same tissues, the individual function of each isoform has remained uncertain. Thus, we constructed a mouse model with a liver-specific knock-out of ACSL1, a major ACSL isoform in liver. Eliminating ACSL1 in liver resulted in a 50% decrease in total hepatic ACSL activity and a 25-35% decrease in long chain acyl-CoA content. Although the content of triacylglycerol was unchanged in Acsl1 L؊/؊ liver after mice were fed either low or high fat diets, in isolated primary hepatocytes the absence of ACSL1 diminished the incorporation of [ 14 C]oleate into triacylglycerol. Further, small but consistent increases were observed in the percentage of 16:0 in phosphatidylcholine and phosphatidylethanolamine and of 18:1 in phosphatidylethanolamine and lysophosphatidylcholine, whereas concomitant decreases were seen in 18:0 in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lysophosphatidylcholine. In addition, decreases in long chain acylcarnitine content and diminished production of acid-soluble metabolites from [ 14 C]oleate suggested that hepatic ACSL1 is important for mitochondrial ␤-oxidation of long chain fatty acids. Because the Acsl1 L؊/؊ mice were not protected from developing either high fat diet-induced hepatic steatosis or insulin resistance, our study suggests that lowering the content of hepatic acyl-CoA without a concomitant decrease in triacylglycerol and other lipid intermediates is insufficient to protect against hepatic insulin resistance. Acyl-CoA synthetase (ACSL)3 activates long chain fatty acid (FA) to acyl-CoA, thereby enhancing vectorial FA transport across the plasma membrane (1) and providing substrates for most downstream pathways that metabolize FA. ACSL1 is one of five ACSL isoforms, each encoded by a separate gene. Its mRNA expression is highest in adipose tissue, liver, and heart (2); and because Acsl1 mRNA and total ACSL1 activity increase 160-fold (3) and 100-fold (4), respectively, in differentiating 3T3-L1 adipocytes, ACSL1 has been thought to be important in activating FA destined for triacylglycerol (TAG) synthesis. In support of this idea, overexpressing ACSL1 in mouse heart increases cardiac myocyte TAG accumulation 12-fold and induces apoptotic pathways, cardiac hypertrophy, left ventricular dysfunction, and heart failure (5). However, Acsl1 mRNA expression is up-regulated in liver and adipose tissue by activators of peroxisome proliferator-activated factor ␣ (PPAR␣) (6, 7) via a PPAR response element in the promoter region of Acsl1 (8), suggesting a possible function related to the ␤-oxidation of fatty acids. Moreover, overexpression of ACSL1 in rat primary hepatocytes increases oleate incorporation into diacylglycerol (DAG) but does not increase TAG mass (9). Thus, the exact role of ACSL1 ...

Microsomal and mitochondrial isoforms of glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyze the committed step in glycerolipid synthesis. The mitochondrial isoform, mtGPAT, was believed to control the positioning of saturated fatty acids at the sn-1 position of phospholipids, and nutritional, hormonal, and overexpression studies suggested that mtGPAT activity is important for the synthesis of triacylglycerol. To determine whether these purported functions were true, we constructed mice deficient in mtGPAT. mtGPAT ؊/؊ mice weighed less than controls and had reduced gonadal fat pad weights and lower hepatic triacylglycerol content, plasma triacylglycerol, and very low density lipoprotein triacylglycerol secretion. As predicted, in mtGPAT ؊/؊ liver, the palmitate content was lower in triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. Positional analysis revealed that mtGPAT ؊/؊ liver phosphatidylethanolamine and phosphatidylcholine had about 21% less palmitate in the sn-1 position and 36 and 40%, respectively, more arachidonate in the sn-2 position. These data confirm the important role of mtGPAT in the synthesis of triacylglycerol, in the fatty acid content of triacylglycerol and cholesterol esters, and in the positioning of specific fatty acids, particularly palmitate and arachidonate, in phospholipids. The increase in arachidonate may be functionally significant in terms of eicosanoid production.

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 2/2 ) 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 [ 14 C]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 2/2 mice" are actually deficient in a novel NEMsensitive 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.-Nagle, C. A., L. Vergnes, H. DeJong, S. Wang, T. M. Lewin, K. Reue, and R. A. Coleman. Identification of a novel sn-glycerol-3-phosphate acyltransferase isoform, GPAT4, as the enzyme deficient in Agpat6 2/2 mice. J. Lipid Res. 2008. 49: 823-831.

Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes phosphatidylcholine synthesis. PEMT knockout mice have fatty livers, and it is possible that, in humans, nonalcoholic fatty liver disease (NAFLD) might be associated with PEMT gene polymorphisms. DNA samples from 59 humans without fatty liver and from 28 humans with NAFLD were genotyped for a single nucleotide polymorphism in exon 8 of PEMT which leads to a V175M substitution. V175M is a loss of function mutation, as determined by transiently transfecting McArdle-RH7777 cells with constructs of wildtype PEMT open reading frame or the V175M mutant. Met/Met at residue 175 (loss of function SNP) occurred in 67.9% of the NAFLD subjects and in only 40.7% of control subjects (p< 0.03). For the first time we report that a polymorphism of the human PEMT gene (V175M) is associated with diminished activity and may confer susceptibility to NAFLD.

The metabolism of long-chain fatty acids in brain and their incorporation into signaling molecules such as diacylglycerol and LPA and into structural components of membranes, including myelin, requires activation by long-chain acyl-CoA synthetase (ACSL). Because ACSL3 and ACSL6 are the predominant ACSL isoforms in brain, we cloned and characterized these isoforms from rat brain and identified a novel ACSL6 clone (ACSL6_v2). ACSL6_v2 and the previously reported ACSL6_v1 represent splice variants that include exon 13 or 14, respectively. Homologue sequences of both of these variants are present in the human and mouse databases. ACSL3, ACSL6_v1, and ACSL6_v2 with Flag-epitopes at the C-termini were expressed in Escherichia coli and purified on Flag-affinity columns. The three recombinant proteins were characterized. Compared to ACSL4, another brain isoform, ACSL3, ACSL6_v1, and ACSL6_v2 showed similarities in kinetic values for CoA, palmitate, and arachidonate, but their apparent Km values for oleate were 4- to 6-fold lower than for ACSL4. In a direct competition assay with palmitate, all the polyunsaturated fatty acids tested were strong competitors only for ACSL4 with IC50 values of 0.5 to 5 microM. DHA was also strongly preferred by ACSL6_v2. The apparent Km value for ATP of ACSL6_v1 was 8-fold higher than that of ACSL6_v2. ACSL3 and the two variants of ACSL6 were more resistant than ACSL4 to heat inactivation. Despite the high amino acid identity between ACSL3 and ACSL4, rosiglitazone inhibited only ACSL4. Triacsin C, an inhibitor of ACSL1 and ACSL4, also inhibited ACSL3, but did not inhibit the ACSL6 variants. These data further document important differences in the closely related ACSL isoforms and show that amino acid changes near the consensus nucleotide binding site alter function in the two splice variants of ACSL6.

OBJECTIVE -Polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PCDFs) are important and persistent organic pollutants (POPs) in humans. Recent cross-sectional studies have detected increased concentrations of serum POPs in diabetic patients. We aimed to examine the association between previous high exposures to PCBs and PCDFs and the cumulative incidence of type 2 diabetes and hypertension.RESEARCH DESIGN AND METHODS -During the late 1970s, the consumption of rice-bran oil laced with PCBs poisoned thousands of Taiwanese. Between 1993 and 2003, we examined 1,054 Yucheng ("oil disease") victims against neighborhood reference subjects using a protocol blinded for POP exposure. Here, we report the results derived from 378 Yucheng subjects and 370 matched references.RESULTS -The diabetes risk to members of the Yucheng cohort relative to their reference subjects was significantly increased for women (odds ratio [OR] 2.1 [95% CI 1.1-4.5]) but not for men after considering age, BMI, cigarette smoking, and alcohol intake. Yucheng women diagnosed with chloracne had adjusted ORs of 5.5 (95% CI 2.3-13.4) for diabetes and 3.5 (1.7-7.2) for hypertension compared with those who were chloracne free.CONCLUSIONS -Yucheng women, who had endured previous exposure to PCBs and PCDFs, suffered from increased incidences of diabetes, particularly those who had retained significant levels of pollutant as evident from chloracne. When planning treatments against diabetes, the body burden of PCBs and dioxins should be carefully considered, especially for women.

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