Identification of a novel sn-glycerol-3-phosphate acyltransferase isoform, GPAT4, as the enzyme deficient in Agpat6−/− mice

The regulation of triacylglycerol (TAG) synthesis and metabolism plays an important role in whole body energy homeostasis in mammals, and dysregulation of TAG synthesis and oxidation pathways has been implicated in the pathogenesis of obesity, lipodystrophy, cardiovascular disease, insulin resistance, and type 2 diabetes (1). De novo TAG synthesis begins with the formation of lysophosphatidic acid (LPA) through the acylation of the sn-1 position of glycerol-3-phosphate by sn-glycerol-3-phosphate acyltransferase (GPAT) (2). The second enzyme in de novo TAG synthesis, sn-1-acylglycerol-3-phosphate O-acyltransferase (AGPAT), acylates LPA to form phosphatidic acid (PA). Phosphatidic acid phosphohydrolase (also called lipin) converts PA to diacylglycerol (DAG) (3, 4), and diacylglycerol acyltransferase acylates DAG to form TAG. PA and DAG are also precursors for the glycerophospholipids. Three mammalian GPAT isoforms have been cloned (5–7). GPAT1 and GPAT2 are located in the outer mitochondrial membrane, whereas GPAT3 is located in the endoplasmic reticulum. GPAT1 is resistant to inactivation by sulfhydryl agents like N-ethylmaleimide (NEM), prefers palmitoyl-CoA as a substrate compared with oleoyl-CoA, and plays a regulatory role in liver TAG synthesis (2). GPAT2 is NEM-sensitive, has no preference for palmitoyl-CoA, and is highly expressed in rodent testis (8, 9). Recently, the gene product previously called AGPAT8 in the National Center for Biotechnology Information database was shown to have NEM-sensitive GPAT activity and to lack AGPAT activity; this gene product was renamed GPAT3 (7). Because small interfering RNA knockdown of Gpat3 in 3T3-L1 adipocytes reduces total GPAT activity by ∼60% (7, 10), it is likely that GPAT3 is important for TAG synthesis in white adipose tissue (WAT). However, GPAT3 is unlikely to play a major role in TAG synthesis in liver, where its mRNA expression is very low (7, 10). In liver, GPAT1 accounts for 30–50% of the total GPAT activity (2). The remaining hepatic GPAT activity is NEM-sensitive and microsomal, suggesting that one or more additional microsomal NEM-sensitive GPAT isoforms must exist. Eight genes with predicted amino acid sequence similarities to GPAT1 have been named AGPAT isoforms 1–8. AGPAT1 and -2 are well characterized and have high AGPAT activity (11, 12). Mutations in AGPAT2 cause generalized human congenital lipodystrophy, with absent abdominal and subcutaneous adipose tissue, systemic insulin resistance, and hypertriglyceridemia (13). Compared with AGPAT1 and -2, the AGPAT activities reported for AGPAT3 to -5 are extremely low (14); no enzyme activity has been reported for AGPAT6 or -7. We previously characterized mice with a targeted deletion in Agpat6 (Agpat6−/− mice; {"type":"entrez-protein","attrs":{"text":"NP_848934","term_id":"30520329","term_text":"NP_848934"}}NP_848934) (10, 15). These mice have a subdermal lipodystrophy, resistance to high-fat diet-induced obesity, and a 50% reduction of TAG content in liver, adipose tissue, and mammary epithelium, suggesting an important role for AGPAT6 in TAG synthesis in these tissues. AGPAT6 shares amino acid sequence homology with other glycerolipid acyltransferases at conserved motifs I–V (Table 1) and is closely related to GPAT1 and -2 in motifs I–IV, which were identified as important for acyltransferase catalysis and glycerol-3-phosphate binding domains (16). AGPAT6 shares 66% amino acid homology with “AGPAT8,” recently identified as GPAT3 (7), and confocal microscopy shows that AGPAT6 localizes to the endoplasmic reticulum (15). These observations raised the possibility that AGPAT6, like AGPAT8, is in fact a GPAT enzyme. Here, we demonstrate that expression of this enzyme in Cos-7 cells increases GPAT, but not AGPAT, activity. Furthermore, tissues from Agpat6−/− mice show reduced GPAT activity but normal AGPAT activity. Our studies thus establish that Agpat6 encodes the primary microsomal NEM-sensitive GPAT activity in liver, GPAT4. TABLE 1 Acyltransferase motifs in Escherichia coli and mouse AGPAT and GPAT isoforms