Agpat6—a novel lipid biosynthetic gene required for triacylglycerol production in mammary epithelium

Beigneux, Anne P.; Vergnes, Laurent; Qiao, Xin; Quatela, Steven; Davis, Ryan M.; Watkins, Steven M.; Coleman, Rosalind A.; Walzem, Rosemary L.; Philips, Mark R.; Reue, Karen; Young, Stephen G.

doi:10.1194/jlr.m500556-jlr200

Cited by 114 publications

(135 citation statements)

References 33 publications

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“…Western blots of HEK293 cells transfected with a tagged AGPAT6 expressed a 48-kDa AGPAT protein (Fig. 1A), consistent with the size of mouse AGPAT6 (20). Aside from the 48-kDa protein, a smaller protein was visible in transfected cells, presumably a breakdown product.…”

Section: Resultsmentioning

confidence: 63%

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AGPAT6 Is a Novel Microsomal Glycerol-3-phosphate Acyltransferase

Chen

Kuo

et al. 2008

Journal of Biological Chemistry

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AGPAT6 is a member of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family that appears to be important in triglyceride biosynthesis in several tissues, but the precise biochemical function of the enzyme is unknown. In the current study, we show that AGPAT6 is a microsomal glycerol-3-phosphate acyltransferase (GPAT). Membranes from HEK293 cells overexpressing human AGPAT6 had higher levels of GPAT activity. Substrate specificity studies suggested that AGPAT6 was active against both saturated and unsaturated long-chain fatty acyl-CoAs. Both glycerol 3-phosphate and fatty acyl-CoA increased the GPAT activity, and the activity was sensitive to N-ethylmaleimide, a sulfhydryl-modifying reagent. Purified AGPAT6 protein possessed GPAT activity but not AGPAT activity. Using [ 13 C 7 ]oleic acid labeling and mass spectrometry, we found that overexpression of AGPAT6 increased both lysophosphatidic acid and phosphatidic acid levels in cells. In these studies, total triglyceride and phosphatidylcholine levels were not significantly altered, although there were significant changes in the abundance of specific phosphatidylcholine species. Human AGPAT6 is localized to endoplasmic reticulum and is broadly distributed in tissues. Membranes of mammary epithelial cells from Agpat6-deficient mice exhibited markedly reduced GPAT activity compared with membranes from wildtype mice. Reducing AGPAT6 expression in HEK293 cells through small interfering RNA knockdown suggested that AGPAT6 significantly contributed to HEK293 cellular GPAT activity. Our data indicate that AGPAT6 is a microsomal GPAT, and we propose renaming this enzyme GPAT4.

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Section: Resultsmentioning

confidence: 63%

“…A deficiency of AGPAT6 in mice results in resistance to dietinduced and genetic forms of obesity. Brown fat mass was significantly reduced, and DG and TG levels in milk and subdermal fat were reduced by nearly 90% (20,21). These studies suggest that AGPAT6 has a significant role in lipid biosynthesis.…”

mentioning

confidence: 60%

AGPAT6 Is a Novel Microsomal Glycerol-3-phosphate Acyltransferase

Chen

Kuo

et al. 2008

Journal of Biological Chemistry

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“…However, systematic annotation of any plsC-containing protein homologous to AGPAT as an additional member of this family has led to some confusion, because not all appear to use lysoPA as substrate. No acyltransferase activity could be detected for AGPAT6 (zeta isoform) with lysoPA or any other lysophospholipid (11). We report that member 7 (eta form) acylates lysoPC rather than lysoPA and therefore belongs to the LPCAT family.…”

Section: Discussionmentioning

confidence: 87%

“…Previous attempts for its isolation suggested that protein(s) of an apparent molecular mass of 50-60 kDa could be labeled with a photoreactive azido-lysoPC derivative in a partially purified microsomal fraction containing a LPCAT activity (10). To identify candidate LPCATs in RBCs, we searched databases for proteins carrying the E. coli plsC domain, which is characteristic of all known 1-acyl-sn-glycerol-3-phosphate acyltransferases (11)(12)(13), and excluded all known enzymes that have been described as lysophosphatidic acid acyltransferases (LPAATs). We focused particularly on two gene products of unknown function with a predicted molecular mass of 60 kDa, annotated in human as AYTL1 and AYTL2 and as Aytl1 and Aytl2 in mouse (Fig.…”

Section: Resultsmentioning

confidence: 99%

Mammalian acyl-CoA:lysophosphatidylcholine acyltransferase enzymes

Soupène¹,

Fyrst²,

Kuypers³

2008

Proc. Natl. Acad. Sci. U.S.A.

103

107

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The mammalian RBC lacks de novo lipid synthesis but maintains its membrane composition by rapid turnover of acyl moieties at the sn-2 position of phospholipids. Plasma-derived fatty acids are esterified to acyl-CoA by acyl-CoA synthetases and transferred to lysophospholipids by acyl-CoA:lysophospholipid acyltransferases. We report the characterization of three lysophosphatidylcholine (lysoPC) acyltransferases (LPCATs), products of the AYTL1, -2, and -3 genes. These proteins are three members of a LPCAT family, of which all three genes are expressed in an erythroleukemic cell line. Aytl2 mRNA was detected in mouse reticulocytes, and the presence of the product of the human ortholog was confirmed in adult human RBCs. The three murine Aytl proteins generated phosphatidylcholine from long-chain acyl-CoA and lysoPC when expressed in Escherichia coli membranes. Spliced variants of Aytl1, affecting a conserved catalytic motif, were identified. Calcium and magnesium modulated LPCAT activity of both Aytl1 and -2 proteins that exhibit EF-hand motifs at the C terminus. Characterization of the product of the Aytl2 gene as the phosphatidylcholine reacylating enzyme in RBCs represents the identification of a plasma membrane lysophospholipid acyltransferase and establishes the function of a LPCAT protein.phosphatidylcholine ͉ RBC ͉ AYTL2

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“…GPAT3 is closely related (Ϸ80% identity within the acyltransferase domain) to a previously identified gene of unknown enzymatic function, AGPAT6. During the preparation of this manuscript, two publications reported on tissue distribution and in vivo functions of AGPAT6 (35,36), indicating an important role of AGPAT6 in TAG biosynthesis. The gene we named GPAT3 was provisionally designated as AGPAT8 in these two manuscripts; however, no enzymatic activity data were provided for both AGPAT6 and GPAT3/AGPAT8.…”

Section: Methodsmentioning

confidence: 99%

Molecular identification of microsomal acyl-CoA:glycerol-3-phosphate acyltransferase, a key enzyme in de novo triacylglycerol synthesis

Cao

Li²,

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

197

235

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Acyl-CoA:glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step during de novo synthesis of triacylglycerol. It has been well recognized that mammals possess multiple enzymatically distinct proteins with GPAT activity. Although the mitochondrialassociated GPAT has been cloned and extensively characterized, the molecular identity of the endoplasmic reticulum (ER)-associated GPAT, which accounts for the majority of total GPAT activity in most tissues, has remained elusive. Here we report the identification of genes encoding human and mouse ER-associated GPAT (termed GPAT3). GPAT3 is a member of the acyltransferase family predominantly expressed in tissues characterized by active lipid metabolism, such as adipose tissue, small intestine, kidney, and heart. Ectopic expression of GPAT3 leads to a significant increase in N-ethylmaleimide-sensitive GPAT activity, whereas acyltransferase activity toward a variety of other lysophospholipids, as well as neutral lipid substrates, is not altered. Overexpression of GPAT3 in mammalian cells results in increased triacylglycerol, but not phospholipid, formation. GPAT3 is localized to the ER when overexpressed in COS-7 cells. GPAT3 mRNA is dramatically up-regulated during adipocyte differentiation, is reciprocally regulated in adipose tissue and liver of ob/ob mice, and is up-regulated in mice treated with a peroxisome proliferator-activated receptor ␥ (PPAR␥) agonist. A substantial loss of GPAT activity in 3T3-L1 adipocytes was achieved by reducing GPAT3 mRNA levels through GPAT3-specific siRNA knockdown. These findings identify GPAT3 as a previously uncharacterized triacylglycerol biosynthetic enzyme. Similar to other lipogenic enzymes, GPAT3 may be useful as a target for the treatment of obesity.bioinformatics ͉ endoplasmic reticulum ͉ lysophosphatidic acid ͉ regulation

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Agpat6—a novel lipid biosynthetic gene required for triacylglycerol production in mammary epithelium

Abstract: In analyzing the sequence tags for mutant mouse embryonic stem (ES) cell lines in BayGenomics (a mouse gene-trapping resource

Cited by 114 publications

References 33 publications

AGPAT6 Is a Novel Microsomal Glycerol-3-phosphate Acyltransferase

AGPAT6 Is a Novel Microsomal Glycerol-3-phosphate Acyltransferase

Mammalian acyl-CoA:lysophosphatidylcholine acyltransferase enzymes

Molecular identification of microsomal acyl-CoA:glycerol-3-phosphate acyltransferase, a key enzyme in de novo triacylglycerol synthesis

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