Overexpression of Human Diacylglycerol Acyltransferase 1, Acyl-CoA:Cholesterol Acyltransferase 1, or Acyl-CoA:Cholesterol Acyltransferase 2 Stimulates Secretion of Apolipoprotein B-containing Lipoproteins in McA-RH7777 Cells

Liang, John J.; Oelkers, Peter; Cui-ying, Guo; Chu, Pi-Chun; Dixon, John R.; Ginsberg, Henry N.; Sturley, Stephen L.

doi:10.1074/jbc.m408507200

Cited by 85 publications

(72 citation statements)

References 66 publications

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“…GOAT shares structural similarities with members of the MBOAT family of acyl transferases (16)(17)(18). When we coexpressed ghrelin with other members of the MBOAT family, including MBOAT1, MBOAT2, MBOAT3, MBOAT5, human-BB1, porcupine, and FKSG89, we were able to achieve ghrelin octanoylation only with GOAT.…”

Section: Discussionmentioning

confidence: 83%

Ghrelin octanoylation mediated by an orphan lipid transferase

Gutiérrez

Solenberg

Perkins

et al. 2008

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

750

703

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The peptide hormone ghrelin is the only known protein modified with an O-linked octanoyl side group, which occurs on its third serine residue. This modification is crucial for ghrelin's physiological effects including regulation of feeding, adiposity, and insulin secretion. Despite the crucial role for octanoylation in the physiology of ghrelin, the lipid transferase that mediates this novel modification has remained unknown. Here we report the identification and characterization of human GOAT, the ghrelin O-acyl transferase. GOAT is a conserved orphan membrane-bound O-acyl transferase (MBOAT) that specifically octanoylates serine-3 of the ghrelin peptide. Transcripts for both GOAT and ghrelin occur predominantly in stomach and pancreas. GOAT is conserved across vertebrates, and genetic disruption of the GOAT gene in mice leads to complete absence of acylated ghrelin in circulation. The occurrence of ghrelin and GOAT in stomach and pancreas tissues demonstrates the relevance of GOAT in the acylation of ghrelin and further implicates acylated ghrelin in pancreatic function.G hrelin is a 28-aa peptide hormone produced principally by stomach tissue with an unusual acyl modification on its critical serine-3 residue. Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor 1a (GHSR1a), and its acyl modification is critical for the activation of the GHSR1a (1). In addition to stimulating growth hormone release from pituitary, ghrelin also promotes food intake, carbohydrate utilization, and adiposity (2-5). Accordingly, ghrelin levels are modulated by changes in nutritional status such as feeding and fasting or exposure to high-fat diets (5, 6). Importantly, ghrelin is the only peptide hormone of peripheral tissue origin that increases food intake (2).More recent studies have identified roles for acylated ghrelin in regulating insulin secretion and blood glucose. Acylated ghrelin occurs in pancreas tissues, and GHSR1a receptor blockade with specific antagonists or treatments with antiserum against acylated ghrelin enhance glucose-induced increases in insulin release whereas acylated ghrelin decreases insulin release (5, 7-10). These observations have further implicated acylated ghrelin in the regulation of metabolism.In stomach tissue and in circulation, acylated forms of ghrelin are modified via an ester linkage with n-octanoic acid and to a lesser extent with decanoyl and decenoyl fatty acids (1, 3). Importantly, the acyl modification in ghrelin is essential for function, with octanoyl and decanoyl fatty acids being optimal (11). Ghrelin is highly conserved in vertebrates, and the third serine residue, which is uniquely modified by the ester-linked acyl group, occurs in all mammal, avian, and fish species (3).The enzyme(s) responsible for acylation of ghrelin has remained unknown. Work by Takada et al. (12) described that porcupine, an enzyme with structural similarities to membrane-bound O-acyl transferases (MBOAT), is required for serine-209 acylation with palmitoleic acid and for transport of...

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Section: Discussionmentioning

confidence: 83%

Ghrelin octanoylation mediated by an orphan lipid transferase

Gutiérrez

Solenberg

Perkins

et al. 2008

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

750

703

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“…Like DGATs, two genes that encode ACAT1 and ACAT2 have been identified with distinct roles (35)(36)(37). Although the membrane topology of ACAT2 is still controversial (38,39), it secretes more VLDL cholesterol compared with ACAT1 when overexpressed in RH7777 cells (27). These data suggest that DGAT1 and ACAT2 might coordinately participate in VLDL formation within the ER lumen.…”

Section: Discussionmentioning

confidence: 83%

“…Overexpression of human DGAT1 in McArdle rat hepatoma cells (RH7777) results in increased TG-rich VLDL secretion (27). When DGAT1 and DGAT2 are overexpressed in RH7777 cells, small lipid droplets around the cell periphery are observed in DGAT1-expressing cells, whereas numerous large cytosolic lipid droplets are observed in DGAT2-expressing cells (Fig.…”

Section: Discussionmentioning

confidence: 99%

Increased Very Low Density Lipoprotein Secretion and Gonadal Fat Mass in Mice Overexpressing Liver DGAT1

Yamazaki

Sasaki

Kakinuma

et al. 2005

Journal of Biological Chemistry

132

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Acyl-CoA:diacylglycerol acyltransferases (DGATs) catalyze the last step in triglyceride (TG) synthesis. The genes for two DGAT enzymes, DGAT1 and DGAT2, have been identified. To examine the roles of liver DGAT1 and DGAT2 in TG synthesis and very low density lipoprotein (VLDL) secretion, liver DGAT1-and DGAT2-overexpressing mice were created by adenovirus-mediated gene transfection. DGAT1-overexpressing mice had markedly increased DGAT activity in the presence of the permeabilizing agent alamethicin. This suggests that DGAT1 possesses latent DGAT activity on the lumen of the endoplasmic reticulum. DGAT1-overexpressing mice showed increased VLDL secretion, resulting in increased gonadal (epididymal or parametrial) fat mass but not subcutaneous fat mass. The VLDL-mediated increase in gonadal fat mass might be due to the 4-fold greater expression of the VLDL receptor protein in gonadal fat than in subcutaneous fat. DGAT2-overexpressing mice had increased liver TG content, but VLDL secretion was not affected. These results indicate that DGAT1 but not DGAT2 has a role in VLDL synthesis and that increased plasma VLDL concentrations may promote obesity, whereas increased DGAT2 activity has a role in steatosis.Triglyceride (TG) 1 is the major energy storage form and is synthesized primarily in three tissues: liver, adipose, and small intestine. In the liver, synthesized TG is either stored in cytoplasmic droplets or secreted as very low density lipoprotein (VLDL) particles. Acyl-CoA:diacylglycerol acyltransferase (DGAT) is a membrane-bound enzyme that catalyzes the last step in the synthesis of TG. Classified by detergent sensitivity, two types of DGATs in microsomes have been proposed: the overt type (on the cytosol) catalyzes the synthesis of TG destined for cytoplasmic droplets, and latent type (on the lumen of the endoplasmic reticulum) catalyzes the synthesis of TG for VLDL formation (1). Because it has been well established that cytosolic droplet TG cannot be incorporated en bloc into VLDL (2), the relative activities of these two functions of DGAT may have a significant impact on the level of triglyceridemia as well as on the development of steatosis.Regarding the molecular aspects of DGAT, the cDNAs of DGATs, viz. DGAT1 and DGAT2, have been recently cloned and sequenced (3, 4). DGAT1 and DGAT2 are unrelated proteins that exhibit DGAT activity. DGAT1 is expressed ubiquitously, with the highest expression levels in the small intestine (3), and the phenotypes of DGAT1-null mice have been extensively examined (5-9). DGAT1-null mice are viable, can still synthesize TG, and have normal 4-h fasted plasma TG levels (5). These mice have reduced adiposity and are resistant to diet-induced obesity through a mechanism that involves increased energy expenditure (5). The increased energy expenditure is due partly to the increased peripheral leptin sensitivity in DGAT1-deficient mice (6). DGAT2 is expressed ubiquitously, with high expression levels in the liver and white adipose tissue (WAT) (4). In contrast to DGAT1-deficient ...

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“…In a separate study mice overexpressing DGAT1, but not DGAT2, had increased latent DGAT activity with a corresponding increase in VLDL secretion (49). Overexpression of DGAT1 was also able to stimulate secretion of TG-rich VLDL from rat hepatoma (McArdle RH7777) cells (50). Taken together, DGAT1 appears to have a role in lipoprotein formation, whereas DGAT2 promotes bulk TG synthesis that accumulates in large cytosolic lipid droplets (17).…”

Section: Discussionmentioning

confidence: 90%

Topological Orientation of Acyl-CoA:Diacylglycerol Acyltransferase-1 (DGAT1) and Identification of a Putative Active Site Histidine and the Role of the N Terminus in Dimer/Tetramer Formation

McFie

Stone

Banman

et al. 2010

Journal of Biological Chemistry

133

125

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Acyl CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerols. Two DGAT enzymes have been identified (DGAT1 and DGAT2) with unique roles in lipid metabolism. DGAT1 is a multifunctional acyltransferase capable of synthesizing diacylglycerol, retinyl, and wax esters in addition to triacylglycerol. Here, we report the membrane topology for murine DGAT1 using protease protections assays and indirect immunofluorescence in conjunction with selective permeabilization of cellular membranes. Topology models based on prediction algorithms suggested that DGAT1 had eight transmembrane domains. In contrast, our data indicate that DGAT1 has three transmembrane domains with the N terminus oriented toward the cytosol. The C-terminal region of DGAT1, which accounts for ϳ50% of the protein, is present in the endoplasmic reticulum lumen and contains a highly conserved histidine residue (His-426) that may be part of the active site. Mutagenesis of His-426 to alanine impaired the ability of DGAT1 to synthesize triacylglycerols as well as retinyl and wax esters in an in vitro acyltransferase assay. Finally, we show that the N-terminal domain of DGAT1 is not required for the catalytic activity of DGAT1 but, instead, may be involved in regulating enzyme activity and dimer/tetramer formation. Acyl coenzyme A:1,2-diacylglycerol acyltransferase (DGAT)2 is a membrane-bound enzyme that catalyzes the biosynthesis of triacylglycerols (TGs) (1). TGs are a class of neutral lipid that represents the major form of stored energy in eukaryotic organisms (2). However, excessive accumulation of TG in tissues can lead to obesity and insulin resistance, whereas increased TG in the blood is a risk factor for atherosclerosis (3)(4)(5).DGAT catalyzes the formation of an ester bond between a long chain fatty acid (fatty acyl coenzyme A (CoA)) and the free hydroxyl group of diacylglycerol (DG), generating TG. Two DGAT genes have now been identified, Dgat1 and Dgat2, which share no sequence homology. DGAT1 belongs to a large family of membrane-bound O-acyltransferases (MBOAT) that includes acyl-CoA:cholesterol acyltransferase-1 and -2 (ACAT-1 and -2), which catalyze cholesterol ester biosynthesis (6 -10). DGAT2 belongs to the DGAT2/acyl-CoA:monoacylglycerol acyltransferase gene family including monoacylglycerol acylCoA acyltransferases 1-3 and wax synthase (9, 11-16). Biochemical analyses have indicated that DGAT1 and DGAT2 have distinct roles in TG metabolism. When overexpressed in cells, DGAT2 yielded a much larger increase in intracellular triacylglycerol than DGAT1 (17). In in vitro assays, DGAT1, but not DGAT2, was capable of using a broad array of acyl acceptors to synthesize diacylglycerol, retinyl, and wax esters in addition to triacylglycerol (18).In vivo experiments in mice have also provided strong evidence that DGAT1 and DGAT2 do not serve redundant roles in lipid metabolism. Dgat1-deficient (Dgat1 Ϫ/Ϫ ) mice were viable with modest reductions in TG con...

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Overexpression of Human Diacylglycerol Acyltransferase 1, Acyl-CoA:Cholesterol Acyltransferase 1, or Acyl-CoA:Cholesterol Acyltransferase 2 Stimulates Secretion of Apolipoprotein B-containing Lipoproteins in McA-RH7777 Cells

Cited by 85 publications

References 66 publications

Ghrelin octanoylation mediated by an orphan lipid transferase

Ghrelin octanoylation mediated by an orphan lipid transferase

Increased Very Low Density Lipoprotein Secretion and Gonadal Fat Mass in Mice Overexpressing Liver DGAT1

Topological Orientation of Acyl-CoA:Diacylglycerol Acyltransferase-1 (DGAT1) and Identification of a Putative Active Site Histidine and the Role of the N Terminus in Dimer/Tetramer Formation

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