SummaryFour 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 longchain acyl-CoA. This step initiates the synthesis of all the glycerolipids and evidence from gain-offunction 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. KeywordsGlycerolipid; phospholipid; membrane; lipid droplet; lysophosphatidic acid; diacylglycerol Glycerol-3-phosphate acyltransferases are members of the pfam 01553 family of acyltransferasesAfter Eugene Kennedy and his colleagues showed that the esterification of glycerol-3-phosphate with a long-chain acyl-CoA was the initial step in the synthesis of phospholipids [1] and triacylglycerol (TAG) [2], Pullman's group reported that this sn-glycerol-3-phosphate acyltransferase activity (GPAT; EC 2.3.1.15) was comprised of what appeared to be two isoforms, one located in the mitochondrial outer membrane and the other in the endoplasmic reticulum [3]. The endoplasmic reticulum (microsomal) activity was inhibited by sulfhydryl reagents such as N-ethylmaleimide (NEM) and exhibited no preference for particular acyl-CoA species, whereas the mitochondrial activity was resistant to NEM inactivation and preferred to use saturated acyl-CoAs like 16:0-CoA and 18:0-CoA [4]. With the identification of four genes encoding separate GPAT isoenzymes [5-11], we now know that GPAT mediated regulation of glycerolipid synthesis is more complex than anyone had previously thought; investigators are currently struggling with the question as to why four separate isoforms are required for glycerolipid biosynthesis.Gpat1, the first mammalian GPAT isoform cloned [5,6], resides in the outer mitochondrial membrane, is resistant NEM inactivation and prefers to use saturated acyl-CoAs [4]. A second mitochondrial GPAT, GPAT2, also resides in the outer mitochondrial membrane, but its activity is inhibited by NEM and it has no long-chain acyl-CoA preference [12]. The NEM-
Cancer cachexia is a syndrome of unintentional weight loss that is characterized by wasting of both skeletal muscle and adipose tissue. Glucose intolerance and insulin resistance have been associated with cancer cachexia. However, it is unknown whether resistance to insulin has a role in the development of cachexia. In the present study, male CD2F1 mice with colon-26 adenocarcinoma tumors underwent an insulin tolerance test before the onset of weight loss. Compared to mice without tumors, mice with tumors had a profoundly blunted blood glucose response to insulin. Corroborating these findings, mice with tumors had decreased phosphorylation of Akt in quadriceps muscle and epididymal adipose tissue at the end of the study. Expression of Akt-regulated genes Atrogin-1, MuRF-1, and Bnip3 was increased in muscle, suggesting a role for decreased insulin signaling in the induction of both proteasomal proteolysis and autophagy in cachectic muscle. Rosiglitazone treatment increased serum adiponectin, insulin sensitivity, and body weight, and decreased Atrogin-1 and MuRF-1 expression in the skeletal muscle of tumor-bearing mice. In conclusion, insulin resistance is an early event in mice with cachexia induced by colon-26 tumors. Rosiglitazone improves insulin sensitivity and decreases early markers of cachexia. These data provide evidence that insulin resistance is not only present in cachexia, but also has a role in cachexia pathogenesis. Correction of insulin resistance may be a novel therapeutic target for the treatment of cancer cachexia.
Increased flux through the glycerolipid synthesis pathway impairs the ability of insulin to inhibit hepatic gluconeogenesis, but the exact mechanism remains unknown. To determine the mechanism by which glycerolipids impair insulin signaling, we overexpressed glycerol-3-phosphate acyltransferase-1 (GPAT1) in primary mouse hepatocytes. GPAT1 overexpression impaired insulin-stimulated phosphorylation of Akt-S473 and -T308, diminished insulin-suppression of glucose production, significantly inhibited mTOR complex 2 (mTORC2) activity and decreased the association of mTOR and rictor. Conversely, in hepatocytes from Gpat1 −/− mice, mTORrictor association and mTORC2 activity were enhanced. However, this increase in mTORC2 activity in Gpat1 −/− hepatocytes was ablated when rictor was knocked down. To determine which lipid intermediate was responsible for inactivating mTORC2, we overexpressed GPAT1, AGPAT, or lipin to increase the cellular content of lysophosphatidic acid (LPA), phosphatidic acid (PA), or diacylglycerol (DAG), respectively. The inhibition of mTOR/rictor binding and mTORC2 activity coincided with the levels of PA and DAG species that contained 16:0, the preferred substrate of GPAT1. Furthermore, di-16:0-PA strongly inhibited mTORC2 activity and disassociated mTOR/rictor in vitro. Taken together, these data reveal a signaling pathway by which phosphatidic acid synthesized via the glycerol-3-phosphate pathway inhibits mTORC2 activity by decreasing the association of rictor and mTOR, thereby down-regulating insulin action. These data demonstrate a critical link between nutrient excess, TAG synthesis, and hepatic insulin resistance.triacylglycerol | palmitate | hepatic steatosis
Four glycerol-3-phosphate acyltransferase (GPAT) isoforms, each encoded by a separate gene, catalyze the initial step in glycerolipid synthesis; in liver, the major isoforms are GPAT1 and GPAT4. To determine whether each of these hepatic isoforms performs a unique function in the metabolism of fatty acid, we measured the incorporation of de novo synthesized fatty acid or exogenous fatty acid into complex lipids in primary mouse hepatocytes from control, Gpat1−/−, and Gpat4−/− mice. Although hepatocytes from each genotype incorporated a similar amount of exogenous fatty acid into triacylglycerol (TAG), only control and Gpat4−/− hepatocytes were able to incorporate de novo synthesized fatty acid into TAG. When compared with controls, Gpat1−/− hepatocytes oxidized twice as much exogenous fatty acid. To confirm these findings and to assess hepatic β-oxidation metabolites, we measured acylcarnitines in liver from mice after a 24-h fast and after a 24-h fast followed by 48 h of refeeding with a high sucrose diet to promote lipogenesis. Confirming the in vitro findings, the hepatic content of long-chain acylcarnitine in fasted Gpat1−/− mice was 3-fold higher than in controls. When compared with control and Gpat4−/− mice, after the fasting-refeeding protocol, Gpat1−/− hepatic TAG was depleted, and long-chain acylcarnitine content was 3.5-fold higher. Taken together, these data demonstrate that GPAT1, but not GPAT4, is required to incorporate de novo synthesized fatty acids into TAG and to divert them away from oxidation.
OBJECTIVEHepatic 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.RESEARCH DESIGN AND METHODSHepatic lipids, insulin sensitivity, and hepatic insulin signaling were compared in lean (Lep+/?), lean-Gpat1−/−, ob/ob (Lepob/ob), and ob/ob-Gpat1−/− mice.RESULTSCompared 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.CONCLUSIONSThese 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.
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