Expression and identification of p90 as the murine mitochondrial glycerol-3-phosphate acyltransferase

Yet, Shaw Fang; Lee, Sun-Joo; Hahm, Young Tae; Sul, Hei Sook

doi:10.1021/bi00087a029

Cited by 83 publications

(69 citation statements)

References 28 publications

(34 reference statements)

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“…The mitochondria were not contaminated with endoplasmic reticulum (Table I), and no immunoreactive protein was detected in the microsome fraction (Fig. 3A), consistent with previous reports that the microsomal and mitochondrial GPAT isoforms are distinct (1,2,11) and that their amino acid sequences are very different. A two-dimensional Western blot analysis was performed in order to distinguish the anti-GPAT immunoreactive proteins in wild type and mtGPAT Ϫ/Ϫ liver mitochondria.…”

Section: Resultssupporting

confidence: 90%

“…Because only the mitochondrial GPAT isoform has been cloned (11,12), changes in activity of the two isoenzymes can be compared, but not changes in mRNA abundance or protein expression. In general, mtGPAT mRNA, protein, and activity in liver and adipose tissue increase with carbohydrate feeding and with insulin stimulation, whereas microsomal GPAT activity does not change (11,(13)(14)(15). In addition, mtGPAT mRNA is upregulated by sterol regulatory element-binding protein-1c, a potent activator of lipogenesis (16).…”

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confidence: 99%

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Identification of a New Glycerol-3-phosphate Acyltransferase Isoenzyme, mtGPAT2, in Mitochondria

Lewin

Schwerbrock

Lee

et al. 2004

Journal of Biological Chemistry

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Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step of glycerolipid synthesis. Two distinct GPAT isoenzymes had been identified in mammalian tissues, an N-ethylmaleimide (NEM)-sensitive isoform in the endoplasmic reticulum membrane (microsomal GPAT) and an NEM-resistant form in the outer mitochondrial membrane (mtGPAT). Although only mtGPAT has been cloned, the microsomal and mitochondrial GPAT isoforms can be distinguished, because they differ in acyl-CoA substrate preference, sensitivity to inhibition by dihydroxyacetone phosphate and polymixin B, temperature sensitivity, and ability to be activated by acetone. The preponderance of evidence supports a role for mtGPAT in synthesizing the precursors for triacylglycerol synthesis. In mtGPAT ؊/؊ mice, PCR genotyping and Northern analysis showed successful knockout of mtGPAT; however, we detected a novel NEM-sensitive GPAT activity in mitochondrial fractions and an anti-mtGPAT immunoreactive protein in liver mitochondria, but not in microsomes. Rigorous analysis using two-dimensional gel electrophoresis revealed that the anti-mtGPAT immunoreactive proteins in wild type and mtGPAT ؊/؊ liver mitochondria have different isoelectric points. These results suggested the presence of a second GPAT in liver mitochondria from mtGPAT ؊/؊ mice. Characterization of this GPAT activity in liver from mtGPAT null mice showed that, unlike the mtG-PAT activity in wild type samples, activity in mtGPAT knockout mitochondria did not prefer palmitoyl-CoA, was sensitive to inactivation by NEM, was inhibited by dihydroxyacetone phosphate and polymixin B, was temperature-sensitive, and was not activated by acetone. We conclude that a novel GPAT (mtGPAT2) with antigenic epitopes similar to those of mtGPAT is detectable in mitochondria from the livers of mtGPAT ؊/؊ mice.The initial and rate-limiting step of glycerolipid synthesis is the acylation of glycerol 3-phosphate with long-chain fatty acylCoA to form 1-acyl-glycerol 3-phosphate (LPA). 1 This reaction is catalyzed by two glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) isoenzymes that are encoded by different genes (1). One isoform is present in the endoplasmic reticulum membrane (microsomal GPAT), and the other is located in the outer mitochondrial membrane (mtGPAT). Although the microsomal GPAT has not been cloned or purified, its activity is easily distinguished, because, unlike mtGPAT, the microsomal isoform is inhibited by sulfhydryl reagents (2). In most tissues, the microsomal GPAT activity is 10 times higher than that found in the mitochondrial fraction, but in liver, mtGPAT contributes 30 -50% of the total activity (1).Microsomal GPAT and mtGPAT also differ in their acyl-CoA substrate preference. The microsomal isoform esterifies both saturated and unsaturated long-chain acyl-CoAs equally well, but mtGPAT prefers C16:0-CoA (2). In rat liver, kidney, and heart, mtGPAT activity is 3-10-fold higher with C16:0-CoA than with other long-chain saturated or unsaturated acyl-CoA substrates (3...

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Section: Resultssupporting

confidence: 90%

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confidence: 99%

Identification of a New Glycerol-3-phosphate Acyltransferase Isoenzyme, mtGPAT2, in Mitochondria

Lewin

Schwerbrock

Lee

et al. 2004

Journal of Biological Chemistry

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“…Our laboratory originally cloned mtGPAT, which was the first mammalian enzyme in TAG and phospholipid biosynthesis to be cloned [15][16][17]. While mammalian cells are known to have two GPATs, one in the ER (microsomal GPAT) and another in the mitochondria (mtGPAT), microsomal GPAT has not been cloned [13,18].…”

Section: Biosynthesis Of Triacylglycerolmentioning

confidence: 99%

“…Most enzymes in TAG synthesis are intrinsic membrane proteins and their substrates and products are hydrophobic, making purification and kinetic analysis difficult. Cloning and characterization of the enzymes mentioned previously has only been achieved during the last decade following advances in molecular biological techniques.Our laboratory originally cloned mtGPAT, which was the first mammalian enzyme in TAG and phospholipid biosynthesis to be cloned [15][16][17]. While mammalian cells are known to have two GPATs, one in the ER (microsomal GPAT) and another in the mitochondria (mtGPAT), microsomal GPAT has not been cloned [13,18].…”

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Triacylglycerol Metabolism In Adipose Tissue

et al. 2007

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Triacylglycerol (TAG) in adipose tissue serves as the major energy storage form in higher eukaryotes. Obesity, resulting from excess white adipose tissue, has increased dramatically in recent years resulting in a serious public health problem. Understanding of adipocyte-specific TAG synthesis and hydrolysis is critical to the development of strategies to treat and prevent obesity and its closely associated diseases, for example, Type 2 diabetes, hypertension and atherosclerosis. In this review, we present an overview of the major enzymes in TAG synthesis and lipolysis, including the recent discovery of a novel adipocyte TAG hydrolase. Keywords1-acylglycerol-3-phosphate acyltransferase; desnutrin/adipose triglyceride lipase; diacylglycerol acyltransferase; glycerol-3-phosphate acyltransferase; hormone-sensitive lipase; lipolysis; phosphatidic acid phosphatase; triacylglycerol White adipose tissue (WAT) is the major energy reserve in higher eukaryotes. The primary purposes of WAT are synthesis and storage of triacylglycerol (TAG) in periods of energy excess, and hydrolysis of TAG to generate fatty acids for use by other organs during periods of energy deprivation [1]. Adipose tissue also secretes adipokines that regulate energy intake and metabolism.The prevalence of obesity resulting from excess WAT has increased dramatically in recent years resulting in a serious public health problem, since obesity is closely associated with a number of disorders including Type 2 diabetes, hypertension and atherosclerosis [2]. While † Author for correspondence, University of California, Department of Nutritional Sciences & Toxicology, Berkeley, CA 94720, USA, adipocyte number has been shown to increase (hyperplasia) in morbid obesity, obesity is primarily attributed to adipocyte hypertrophy that occurs when TAG synthesis (esterification) exceeds TAG breakdown (lipolysis), resulting in elevated TAG storage [1]. Although it has been postulated that large adipocyte size may contribute to insulin resistance, the molecular mechanism is not clear.Paradoxically, the metabolic abnormalities typically found in obesity are also associated with lipodystrophies that are characterized by selective loss of adipose tissue from particular regions of the body [3][4][5][6]. Although the underlying molecular mechanisms are not clear, metabolic complications may result from ectopic storage of TAG in tissues such as the liver and muscle [7][8][9]. A proper capacity for TAG storage in adipocytes is clearly important for normal metabolic regulation. In view of the wide range of health problems associated with improper fat storage and release, it is critical to understand adipocyte-specific regulation of TAG synthesis and hydrolysis. Biosynthesis of triacylglycerolThe synthesis of phosphatidic acid and its subsequent conversion to TAG was described more than 40 years ago by Kennedy and coworkers [10], and is summarized in Figure 1. The initial committal step in this pathway is the formation of lysophosphatidic acid (1-acylglycerol-3-phosphate)...

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“…The gene encoding the mammalian mitochondrial NEM-resistant GPAT1 (mtGPAT1, GPAM) was identified a decade ago and was shown to play a key role in liver TAG synthesis (6)(7)(8). Although initial reports suggested that mtGPAT1-deficient mice have decreased adipose tissue mass (6), more recent studies found little effect of mtGPAT1 deficiency on the development of obesity (7).…”

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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.

203

236

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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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Expression and identification of p90 as the murine mitochondrial glycerol-3-phosphate acyltransferase

Cited by 83 publications

References 28 publications

Identification of a New Glycerol-3-phosphate Acyltransferase Isoenzyme, mtGPAT2, in Mitochondria

Identification of a New Glycerol-3-phosphate Acyltransferase Isoenzyme, mtGPAT2, in Mitochondria

Triacylglycerol Metabolism In Adipose Tissue

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

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