Mutations in 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) cause congenital generalized lipodystrophy. To understand the molecular mechanisms underlying the metabolic complications associated with AGPAT2 deficiency, Agpat2 null mice were generated. Agpat2−/− mice develop severe lipodystrophy affecting both white and brown adipose tissue, severe insulin resistance, diabetes, and hepatic steatosis. The expression of lipogenic genes and rates of de novo fatty acid biosynthesis were increased ~4-fold in Agpat2−/− mouse livers. The mRNA and protein levels of monoacylglycerol acyltransferase isoform 1 were markedly increased in the livers of Agpat2−/− mice suggesting that the alternative monoacylglycerol pathway for triglyceride biosynthesis is activated in the absence of AGPAT2. Feeding a fat-free diet reduced liver triglycerides by ~50% in Agpat2−/− mice. These observations suggest that both dietary fat and hepatic triglyceride biosynthesis via a novel monoacylglycerol pathway may contribute to hepatic steatosis in Agpat2−/− mice.
AGPAT; LPAAT; MGAT; phosphatidic acid phosphatases; acyltransferase; phospholipids; lipodystrophy; hepatic steatosis
Triglyceride synthesis in most mammalian tissues involves the sequential addition of fatty acids to a glycerol backbone, with unique enzymes required to catalyze each acylation step. Acylation at the sn-2 position requires 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) activity. To date, seven Agpat genes have been identified based on activity and/or sequence similarity, but their physiological functions have not been well established. We have generated a mouse model deficient in AGPAT6, which is normally expressed at high levels in brown adipose tissue (BAT), white adipose tissue (WAT), and liver. Agpat6-deficient mice exhibit a 25% reduction in body weight and resistance to both diet-induced and genetically induced obesity. The reduced body weight is associated with increased energy expenditure, reduced triglyceride accumulation in BAT and WAT, reduced white adipocyte size, and lack of adipose tissue in the subdermal region. In addition, the fatty acid composition of triacylglycerol, diacylglycerol, and phospholipid is altered, with proportionally greater polyunsaturated fatty acids at the expense of monounsaturated fatty acids. Thus, Agpat6 plays a unique role in determining triglyceride content and composition in adipose tissue and liver that cannot be compensated by other members of the Agpat family.
acyltransferase; gene-trap; adipose tissue; energy expenditure; 1-acylglycerol-3-phosphate O-acyltransferase
De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. In this work we show that mitochondrial GPAT2 overexpression in CHO-K1 cells increased TAG content and both GPAT and AGPAT activities 2-fold with arachidonoyl-CoA as a substrate, indicating specificity for this fatty acid.
Methods and Results
Incubation of GPAT2-transfected CHO-K1 cells with [1-14C]arachidonate for 3 h increased incorporation of [14C]arachidonate into TAG by 40%. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2's role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein.
These results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.
Triglycerides and phospholipids play an important role in epidermal permability barrier formation and function. They are synthesized de novo in the epidermis via the glycerol-3-phosphate pathway, catalyzed sequentially by a group of enzymes that have multiple isoforms including glycerol-3-phosphate acyltransferase (GPAT), 1-acylglycerol-3-phosphate acyltransferase (AGPAT), Lipin and diacylglycerol acyltransferase (DGAT). Here we review the current knowledge of GPAT, AGPAT, Lipin and DGAT enzymes in keratinocytes/epidermis focusing on the expression levels of the various isoforms and their localization in mouse epidermis. Additionally, the factors regulating their gene expression, including calcium induced differentiation, PPAR and LXR activators, and the effect of acute permeability barrier disruption will be discussed.
glycerol-3-phosphate acyltransferase; 1-acylglycerol-3-phosphate acyltransferase; lipin; diacylglycerol acyltransferase; human keratinocytes; epidermis
Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step in glycerolipid synthesis. Several mammalian GPAT activities have been recognized, including N-ethylmaleimide (NEM)-sensitive isoforms in microsomes and mitochondria and an NEM-resistant form in mitochondrial outer membrane (GPAT1). We have now cloned a second mitochondrial isoform, GPAT2 from mouse testis. The open reading frame encodes a protein of 798 amino acids with a calculated mass of 88.8 kDa and 27% amino acid identity to GPAT1. Testis mRNA expression was 50-fold higher than in liver or brown adipose tissue, but the specific activity of NEM-sensitive GPAT in testis mitochondria was similar to that in liver. When Cos-7 cells were transiently transfected with GPAT2, NEM-sensitive GPAT activity increased 30%. Confocal microscopy confirmed a mitochondrial location. Incubation of GPAT2-transfected Cos-7 cells with trace (3 μM; 0.25μCi) [1-14C]oleate for 6 h increased incorporation of [14C]oleate into TAG 84%. In contrast, incorporation into phospholipid species was lower than in control cells. Although a polyclonal antibody raised against full-length GPAT1 detected an ∼89 kDa band in liver and testis from GPAT1 null mice and both 89 and 80 kDa bands in BAT from the knockout animals, the GPAT2 protein expressed in Cos-7 cells was only 80 kDa. In vitro translation showed a single product of 89 kDa. Unlike GPAT1, GPAT2 mRNA abundance in liver was not altered by fasting or refeeding. GPAT2 is likely to have a specialized function in testis.
AGPAT isoforms catalyze the acylation of lysophosphatidic acid (LPA) to form phosphatidic acid (PA). AGPAT2 mutations are associated with defective adipogenesis. Muscle and adipose tissue share common precursor cells. We investigated the role of AGPAT isoforms in skeletal muscle development. We demonstrate that small interference RNA-mediated knockdown of AGPAT1 expression prevents the induction of myogenin, a key transcriptional activator of the myogenic program, and inhibits the expression of myosin heavy chain. This effect is rescued by transfection with AGPAT1 but not AGPAT2. Knockdown of AGPAT2 has no effect. The regulation of myogenesis by AGPAT1 is associated with alterations on actin cytoskeleton. The role of AGPAT1 on actin cytoskeleton is further supported by colocalization of AGPAT1 to areas of active actin polymerization. AGPAT1 overexpression was not associated with an increase in PA levels. Our observations strongly implicate AGPAT1 in the development of skeletal muscle, specifically to terminal differentiation. These findings are linked to the regulation of actin cytoskeleton.
Cytoskeleton; Phosphatidic acid; AGPAT2; C2C12; Skeletal muscle; Actin
Integral membrane lysophospholipid acyltransferases (AT) are involved in many reactions that produce phospholipids and triglycerides. Enzymes that utilize lysophosphatidic acid (LPA) as an acceptor substrate have been termed LPAATs, and several are members of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) gene family. Amino acid sequence comparisons with other acyltransferases reveal that AGPATs contain four conserved motifs (I–IV), whose invariant residues appear to be important for catalysis and/or substrate recognition. Although the enzymatic activities of many AGPATs are known, for many members their structural organization within membranes and their exact biological functions are unclear. Recently, a new function for AGPATs was discovered when it was determined that human AGPAT3/LPAAT3 is involved in the structure and function of the Golgi complex. Here we have determined the topological orientation of human AGPAT3/LPAAT3. AGPAT3/LPAAT3 possesses two transmembrane domains, one of which separates motifs I and II, which are thought to form a functional unit that is critical for enzymatic activity. This is a surprising result but similar to a recent study on the topology of human LPAAT 1. The data is consistent with a structural arrangement in which motif I is located in the cytoplasm and motif II is in the endoplasmic reticulum and Golgi lumen, suggesting a different model for AGPAT3/LPAAT3’s enzymatic mechanism.
Golgi complex; 1-acylglycerol-3-phosphate O-acyltransferase; AGPAT3; lysophosphatidic acid acyltransferase; LPAAT3; membrane topology
In analyzing the sequence tags for mutant mouse embryonic stem (ES) cell lines in BayGenomics (a mouse gene-trapping resource), we identified a novel gene, Agpat6, with sequence similarities to previously characterized glycerolipid acyltransferases. Agpat6’s closest family member is another novel gene that we have provisionally designated Agpat8. Both Agpat6 and Agpat8 are conserved from plants, nematodes, and flies to mammals. AGPAT6, which is predicted to contain multiple membrane-spanning helices, is found exclusively within the endoplasmic reticulum in mammalian cells. To gain insights into the in vivo importance of Agpat6, we used the Agpat6 ES cell line from BayGenomics to create Agpat6-deficient (Agpat6−/−) mice. Agpat6−/− mice lacked full-length Agpat6 transcripts, as judged by northern blots. One of the most striking phenotypes of Agpat6−/− mice was a defect in lactation. Pups nursed by Agpat6−/− mothers die perinatally. Normally, Agpat6 is expressed at high levels in the mammary epithelium of breast tissue, but not in the surrounding adipose tissue. Histological studies revealed that the aveoli and ducts of Agpat6−/− lactating mammary glands were underdeveloped, and there was a dramatic decrease in size and number of lipid droplets within mammary epithelial cells and ducts. Also, the milk from Agpat6−/− mice was markedly depleted in diacylglycerols and triacylglycerols. Thus, we identified a novel glycerolipid acyltransferase of the endoplasmic reticulum, AGPAT6, which is crucial for the production of milk fat by the mammary gland.
LPAAT; acyltransferase; transacylase; milk fat
Loss-of-function mutations in AGPAT2, encoding 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2), produce congenital generalised lipodystrophy (CGL). We screened the AGPAT2 gene in two siblings who presented with pseudoacromegaly, diabetes and severe dyslipidaemia and identified a novel mutation in AGPAT2 causing a single amino acid substitution, p.Cys48Arg. We subsequently investigated the molecular pathogenic mechanism linking both this mutation and the previously reported p.Leu228Pro mutation to clinical disease. Wild-type and mutant AGPAT2 were expressed in control and AGPAT2-deficient preadipocyte cell lines. mRNA and protein expression was determined, and the ability of each AGPAT2 species to rescue adipocyte differentiation in AGPAT2-deficient cells was assessed. Protein levels of both p.Cys48Arg and p.Leu228Pro AGPAT2 were significantly reduced compared with that of wild-type AGPAT2 despite equivalent mRNA levels. Stable expression of wild-type AGPAT2 partially rescued adipogenesis in AGPAT2 deficient preadipocytes, whereas stable expression of p.Cys48Arg or p.Leu228Pro AGPAT2 did not. In conclusion, unusually severe dyslipidaemia and pseudoacromegaloid overgrowth in patients with diabetes should alert physicians to the possibility of lipodystrophy. Both the previously unreported pathogenic p.Cys48Arg mutation in AGPAT2, and the known p.Leu228Pro mutation result in decreased AGPAT2 protein expression in developing adipocytes. It is most likely that the CGL seen in homozygous carriers of these mutations is largely accounted for by loss of protein expression.
Glycerol-3-phosphate acyltransferase-1 (GPAT1), which is located on the outer mitochondrial membrane comprises up to 30% of total GPAT activity in the heart. It is one of at least four mammalian GPAT isoforms known to catalyze the initial, committed, and rate limiting step of glycerolipid synthesis. Because excess triacylglycerol (TAG) accumulates in cardiomyocytes in obesity and type 2 diabetes, we determined whether lack of GPAT1 would alter the synthesis of heart TAG and phospholipids after a 2-week high sucrose diet or a 3-month high fat diet. Even in the absence of hypertriglyceridemia, TAG increased 2-fold with both diets in hearts from wildtype mice. In contrast, hearts from Gpat1−/− mice contained 20–80% less TAG than the wildtype controls. In addition, hearts from Gpat1−/− mice fed the high-sucrose diet incorporate 60% less [14C]palmitate into heart TAG as compared to wildtype mice. Because GPAT1 prefers 16:0-CoA to other long chain acyl-CoA substrates, we determined the fatty acid composition of heart phospholipids. Compared to wildtype littermate controls, hearts from Gpat1−/− mice contained a lower amount of 16:0 in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine/phosphatidylinositol and significantly more C20:4n6. Phosphatidylcholine and phosphatidylethanolamine from Gpat1−/− hearts also contained higher amounts of 18:0 and 18:1. Although at least three other GPAT isoforms are expressed in the heart, our data suggest that GPAT1 contributes significantly to cardiomyocyte TAG synthesis during lipogenic or high fat diets and influences the incorporation of 20:4n6 into heart phospholipids.
obesity; type 2 diabetes; lipotoxicity; diabetic cardiomyopathy; arachidonic acid
Microsomal and mitochondrial isoforms of glycerol-3-phosphate acyltransferase (GPAT; E.C. 220.127.116.11) catalyze the committed step in glycerolipid synthesis. The mitochondrial isoform, mtGPAT, was believed to control the positioning of saturated fatty acids at the sn-1 position of phospholipids, and nutritional, hormonal, and overexpression studies suggested that mtGPAT activity is important for the synthesis of triacylglycerol. To determine whether these purported functions were true, we constructed mice deficient in mtGPAT. mtGPAT−/− mice weighed less than controls and had reduced gonadal fat pad weights and lower hepatic triacylglycerol content, plasma triacylglycerol, and very low density lipoprotein triacylglycerol secretion. As predicted, in mtGPAT−/− liver, the palmitate content was lower in triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. Positional analysis revealed that mtGPAT−/− liver phosphatidylethanolamine and phosphatidylcholine had about 21% less palmitate in the sn-1 position and 36 and 40%, respectively, more arachidonate in the sn-2 position. These data confirm the important role of mtGPAT in the synthesis of triacylglycerol, in the fatty acid content of triacylglycerol and cholesterol esters, and in the positioning of specific fatty acids, particularly palmitate and arachidonate, in phospholipids. The increase in arachidonate may be functionally significant in terms of eicosanoid production.
Mammals express four isoforms of glycerol-3-phosphate acyltransferase (GPAT). The mitochondrial isoform GPAT1 may have been the acyltransferase that appeared first in evolution. The hepatopancreas of the crustacean Macrobrachium borellii has a high capacity for triacylglycerol (TAG) biosynthesis and storage. In order to understand the mechanism of glycerolipid biosynthesis in M. borellii, we investigated its hepatopancreas GPAT activity. In hepatopancreas mitochondria, we identified a GPAT activity with characteristics similar to those of mammalian GPAT1. The activity was resistant to inactivation by SH-reactive N-ethylmaleimide, it was activated by polymyxin-B, and its preferred substrate was palmitoyl-CoA. The reaction products were similar to those of mammalian GPAT1. A 70-kDa protein band immunoreacted with an anti-rat liver GPAT1 antibody. Surprisingly, we did not detect high GPAT specific activity in hepato-pancreas microsomes. GPAT activity in microsomes was consistent with mitochondrial contamination, and its properties were similar to those of the mitochondrial activity. In microsomes, TAG synthesis was not dependent on the presence of glycerol-3 phosphate as a substrate, and the addition of monoacylglycerol as a substrate increased TAG synthesis 2-fold. We conclude that in M. borellii the de novo triacylglycerol biosynthetic pathway can be completed in the mitochondria. In contrast, TAG synthesis in the ER may function via the monoacylglycerol pathway.
Triacylglycerol; Crustacean; Glycerolipid synthesis
Congenital generalized lipodystrophy (CGL) is a rare auto-somal recessive disorder characterized by extreme paucity of adipose tissue from birth, and early onset of metabolic complications related to insulin resistance. Mutations in three genes, 1-acylglycerol 3-phosphate-O-acyltransferase 2 (AGPAT2), Berardinelli Seip Congenital Lipodystrophy 2 (BSCL2), and Caveolin-1 (CAV1) are associated with the three subtypes of this disorder, CGL1, CGL2 and CGL3, respectively. We report two siblings of Hispanic origin who displayed characteristic features of CGL such as generalized loss of subcutaneous fat from birth, acanthosis nigricans, acromegaloid habitus, umbilical prominence, hepatosplenomegaly, hypoleptinemia, dyslipidemia, and insulin resistance. However, no disease causing variants were detected in the DNA sequence of AGPAT2, BSCL2 or CAV1 genes. Further, whole body magnetic resonance imaging (MRI) in the two siblings revealed marked loss of subcutaneous, intraabdominal and intrathoracic fat like in other patients with CGL, but preservation of bone marrow fat which is invariably lost in all patients with CGL1 and CGL2, but not in the patient reported with CGL3. They also had generalized muscle weakness during infancy and early childhood associated with a nearly fivefold increase in serum creatine kinase (CK) levels, but with normal muscle biopsy and electrophysiologic studies. Both patients were also found to have atlantoaxial dislocation requiring surgical intervention. Thus, this pedigree represents a novel subtype of CGL characterized by generalized loss of body fat but with preservation of bone marrow fat, congenital muscular weakness and cervical spine instability. The genetic basis of this novel subtype remains to be determined.
congenital generalized lipodystrophy; adipose tissue; insulin resistance; congenital myopathy; cervical spine instability
Glycerol 3-phosphate acyltransferase-1 (GPAT1), catalyzes the committed step in phospholipid and triacylglycerol synthesis. Because both GPAT1 and carnitine-palmitoyltransferase 1 are located on the outer mitochondrial membrane (OMM) it has been suggested that their reciprocal regulation controls acyl-CoA metabolism at the OMM. To determine whether GPAT1, like carnitine-palmitoyltransferase 1, is enriched in both mitochondrial contact sites and OMM, and to correlate protein location and enzymatic function, we used Percoll and sucrose gradient fractionation of rat liver to obtain submitochondrial fractions. Most GPAT1 protein was present in a vesicular membrane fraction associated with mitochondria (MAV) but GPAT specific activity in this fraction was low. In contrast, highest GPAT1 specific activity was present in purified mitochondria. Contact sites from crude mitochondria, which contained markers for both endoplasmic reticulum (ER) and mitochondria, also showed high expression of GPAT1 protein but low specific activity, whereas contact sites isolated from purified mitochondria lacked ER markers and expressed highly active GPAT1. To determine how GPAT1 is targeted to mitochondria, recombinant protein was synthesized in vitro and its incorporation into crude and purified mitochondria was assayed. GPAT1 was rapidly incorporated into mitochondria, but not into microsomes. Incorporation was ATP-driven, and lack of GPAT1 removal by alkali and a chaotropic agent showed that GPAT1 had become an integral membrane protein after incorporation. These results demonstrate that two pools of GPAT1 are present in rat liver mitochondria: an active one, located in OMM and a less active one, located in membranes (ER-contact sites and mitochondrial associated vesicles) associated with both mitochondria and ER.
Triacylglycerol synthesis; Protein targeting; Mitochondria-endoplasmic reticulum interaction
GPAT1, one of four known glycerol-3-phosphate acyltransferase isoforms, is located on the mitochondrial outer membrane, allowing reciprocal regulation with carnitine palmitoyltransferase-1. GPAT1 is upregulated transcriptionally by insulin and SREBP-1c and downregulated acutely by AMP-activated protein kinase, consistent with a role in triacylglycerol synthesis. Knockout and overexpression studies suggest that GPAT1 is critical for the development of hepatic steatosis and that steatosis initiated by overexpression of GPAT1 causes hepatic, and perhaps also peripheral, insulin resistance. Future questions include the function of GPAT1 in relation to the other GPAT isoforms and whether the lipid intermediates synthesized by GPAT and downstream enzymes in the pathway of glycerolipid biosynthesis participate in intracellular signaling pathways.
insulin resistance; diacylglycerol; lysophosphatidate; sterol regulatory element binding protein; hepatic steatosis
Congenital generalized lipodystrophy (CGL) is an autosomal recessive disease characterized by the generalized scant of adipose tissue. CGL type 1 is caused by mutations in gene encoding 1-acylglycerol-3-phosphate O-acyltransferase-2 (AGPAT2). A clinical and molecular genetic investigation was performed in affected and unaffected members of two families with CGL type 1. The AGPAT2 coding region was sequenced in index cases of the two families. The presence of the identified mutations in relevant parents was tested. We identified a novel nonsense mutation (c.685G>T, p.Glu229*) and a missense substitution (c.514G>A, p.Glu172Lys). The unaffected parents in both families were heterozygous carrier of the relevant mutation. The results expand genotype–phenotype spectrum in CGL1 and will have applications in prenatal and early diagnosis of the disease. This is the first report of Persian families identified with AGPAT2 mutations.
► First diagnosis of congenital generalized lipodystrophy type 1 in Persian population. ► Molecular analysis identified a novel nonsense mutation and a missense substitution in the AGPAT2. ► The patients did not have diabetes mellitus or hyperinsulinemia. ► The mutations found are candidates for CGL screening. ► The results expand the knowledge about the genotype–phenotype correlations in CGL.
Congenital generalized lipodystrophy; CGL; Berardinelli-Seip syndrome; AGPAT2
Hepatic 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 METHODS
Hepatic lipids, insulin sensitivity, and hepatic insulin signaling were compared in lean (Lep+/?), lean-Gpat1−/−, ob/ob (Lepob/ob), and ob/ob-Gpat1−/− mice.
Compared 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.
These 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.
The adipocytes synthesize and store triglycerides as lipid droplets surrounded by various proteins and phospholipids at its surface. Recently, the molecular basis of some of the genetic syndromes of lipodystrophies has been elucidated and some of these genetic loci have been found to contribute to lipid droplet formation in adipocytes. The two main types of genetic lipodystrophies are congenital generalized lipodystrophy (CGL) and familial partial lipodystrophy (FPL). So far, three CGL loci: 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2), Berardinelli-Seip Congenital Lipodystrophy 2 (BSCL2) and caveolin 1 (CAV1) and four FPL loci: lamin A/C (LMNA), peroxisome proliferator-activated receptor γ (PPARG), v-AKT murine thymoma oncogene homolog 2 (AKT2) and zinc metalloprotease (ZMPSTE24), have been identified. AGPAT2 plays a critical role in the synthesis of glycerophospholipids and triglycerides required for lipid droplet formation. Another protein, seipin (encoded by BSCL2 gene), has been found to induce lipid droplet fusion. CAV1 is an integral component of caveolae and might contribute towards lipid droplet formation. PPARγ and AKT2 play important role in adipogenesis and lipid synthesis. In this review, we discuss and speculate about the contribution of various lipodystrophy genes and their products in the lipid droplet formation.
Lipodystrophy; AGPAT2; BSCL2; CAV1; LMNA; PPARG; AKT2; ZMPSTE24; Lipid droplet; Acyltransferases
Four 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 long-chain acyl-CoA. This step initiates the synthesis of all the glycerolipids and evidence from gain-of-function 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.
Glycerolipid; phospholipid; membrane; lipid droplet; lysophosphatidic acid; diacylglycerol
Glucocorticoids play important roles in the regulation of distinct aspects of adipocyte biology. Excess glucocorticoids in adipocytes are associated with metabolic disorders, including central obesity, insulin resistance and dyslipidemia. To understand the mechanisms underlying the glucocorticoid action in adipocytes, we used chromatin immunoprecipitation sequencing to isolate genome-wide glucocorticoid receptor (GR) binding regions (GBRs) in 3T3-L1 adipocytes. Furthermore, gene expression analyses were used to identify genes that were regulated by glucocorticoids. Overall, 274 glucocorticoid-regulated genes contain or locate nearby GBR. We found that many GBRs were located in or nearby genes involved in triglyceride (TG) synthesis (Scd-1, 2, 3, GPAT3, GPAT4, Agpat2, Lpin1), lipolysis (Lipe, Mgll), lipid transport (Cd36, Lrp-1, Vldlr, Slc27a2) and storage (S3-12). Gene expression analysis showed that except for Scd-3, the other 13 genes were induced in mouse inguinal fat upon 4-day glucocorticoid treatment. Reporter gene assays showed that except Agpat2, the other 12 glucocorticoid-regulated genes contain at least one GBR that can mediate hormone response. In agreement with the fact that glucocorticoids activated genes in both TG biosynthetic and lipolytic pathways, we confirmed that 4-day glucocorticoid treatment increased TG synthesis and lipolysis concomitantly in inguinal fat. Notably, we found that 9 of these 12 genes were induced in transgenic mice that have constant elevated plasma glucocorticoid levels. These results suggested that a similar mechanism was used to regulate TG homeostasis during chronic glucocorticoid treatment. In summary, our studies have identified molecular components in a glucocorticoid-controlled gene network involved in the regulation of TG homeostasis in adipocytes. Understanding the regulation of this gene network should provide important insight for future therapeutic developments for metabolic diseases.
Glycerol 3-phosphate acyltransferase (GPAT) isozymes are central control points for fat synthesis in mammals. Development of inhibitors of these membrane-bound enzymes could lead to an effective treatment for obesity, but is thwarted by an absence of direct structural information. Based on a highly successful study involving conformationally constrained glycerol 3-phosphate analogs functioning as potent glycerol 3-phosphate dehydrogenase inhibitors, several series of cyclic bisubstrate and transition state analogues were designed, synthesized, and tested as GPAT inhibitors. The weaker in vitro inhibitory activity of these compounds compared to a previously described benzoic acid series was then examined in docking experiments with the soluble squash chloroplast GPAT crystal structure. These in silico experiments indicate that cyclopentyl and cyclohexyl scaffolds prepared in this study may be occluded from the enzyme active site by two protein loops that sterically guard the phosphate binding region. In view of these findings, future GPAT inhibitor design will be driven toward compounds based on planar frameworks able to slide between these loops and enter the active site, resulting in improved inhibitory activity.
Glycerol 3-Phosphate Acyltransferase; GPAT; obesity; triacylglycerol
Fatty liver is commonly associated with insulin resistance and type 2 diabetes, but it is unclear whether triacylglycerol accumulation or an excess flux of lipid intermediates in the pathway of triacyglycerol synthesis are sufficient to cause insulin resistance in the absence of genetic or diet-induced obesity. To determine whether increased glycerolipid flux can, by itself, cause hepatic insulin resistance, we used an adenoviral construct to overexpress glycerol-sn-3-phosphate acyltransferase-1 (Ad-GPAT1), the committed step in de novo triacylglycerol synthesis. After 5–7 days, food intake, body weight, and fat pad weight did not differ between Ad-GPAT1 and Ad-enhanced green fluorescent protein control rats, but the chow-fed Ad-GPAT1 rats developed fatty liver, hyperlipidemia, and insulin resistance. Liver was the predominant site of insulin resistance; Ad-GPAT1 rats had 2.5-fold higher hepatic glucose output than controls during a hyperinsulinemic-euglycemic clamp. Hepatic diacylglycerol and lysophosphatidate were elevated in Ad-GPAT1 rats, suggesting a role for these lipid metabolites in the development of hepatic insulin resistance, and hepatic protein kinase Cε was activated, providing a potential mechanism for insulin resistance. Ad-GPAT1-treated rats had 50% lower hepatic NF-κB activity and no difference in expression of tumor necrosis factor-α and interleukin-β, consistent with hepatic insulin resistance in the absence of increased hepatic inflammation. Glycogen synthesis and uptake of 2-deoxyglucose were reduced in skeletal muscle, suggesting mild peripheral insulin resistance associated with a higher content of skeletal muscle triacylglycerol. These results indicate that increased flux through the pathway of hepatic de novo triacylglycerol synthesis can cause hepatic and systemic insulin resistance in the absence of obesity or a lipogenic diet.
The incidence of obesity and other diseases associated with an increased triacylglycerol mass is growing rapidly, particularly in the United States. Glycerol 3-phosphate acyltransferase (GPAT) catalyzes the rate-limiting step of glycerolipid biosynthesis, the acylation of glycerol 3-phosphate with saturated long chain acyl-CoAs. In an effort to produce small molecule inhibitors of this enzyme, a series of benzoic and phosphonic acids was designed and synthesized. In vitro testing of this series has led to the identification of several compounds, in particular 2-(nonylsulfonamido)benzoic acid (15g), possessing moderate GPAT inhibitory activity in an intact mitochondrial assay.
Lipid droplets (LDs) store metabolic energy and membrane lipid precursors. With excess metabolic energy, cells synthesize triacylglycerol (TG) and form LDs that grow dramatically. It is unclear how TG synthesis relates to LD formation and growth. Here, we identify two LD subpopulations: smaller LDs of relatively constant size, and LDs that grow larger. The latter population contains isoenzymes for each step of TG synthesis. Glycerol-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the first and rate-limiting step, relocalizes from the endoplasmic reticulum (ER) to a subset of forming LDs, where it becomes stably associated. ER-to-LD targeting of GPAT4 and other LD-localized TG synthesis isozymes is required for LD growth. Key features of GPAT4 ER-to-LD targeting and function in LD growth are conserved between Drosophila and mammalian cells. Our results explain how TG synthesis is coupled with LD growth and identify two distinct LD subpopulations based on their capacity for localized TG synthesis.
The presence of the acyl dihydroxyacetone phosphate (acyl DHAP) pathway in yeasts was investigated by examining three key enzyme activities of this pathway in Saccharomyces cerevisiae. In the total membrane fraction of S. cerevisiae, we confirmed the presence of both DHAP acyltransferase (DHAPAT; Km = 1.27 mM; Vmax = 5.9 nmol/min/mg of protein) and sn-glycerol 3-phosphate acyltransferase (GPAT; Km = 0.28 mM; Vmax = 12.6 nmol/min/mg of protein). The properties of these two acyltransferases are similar with respect to thermal stability and optimum temperature of activity but differ with respect to pH optimum (6.5 for GPAT and 7.4 for DHAPAT) and sensitivity toward the sulfhydryl blocking agent N-ethylmaleimide. Total membrane fraction of S. cerevisiae also exhibited acyl/alkyl DHAP reductase (EC 18.104.22.168) activity, which has not been reported previously. The reductase has a Vmax of 3.8 nmol/min/mg of protein for the reduction of hexadecyl DHAP (Km = 15 microM) by NADPH (Km = 20 microM). Both acyl DHAP and alkyl DHAP acted as substrates. NADPH was the specific cofactor. Divalent cations and N-ethylmaleimide inhibited the enzymatic reaction. Reductase activity in the total membrane fraction from aerobically grown yeast cells was twice that from anaerobically grown cells. Similarly, DHAPAT and GPAT activities were also greater in aerobically grown yeast cells. The presence of these enzymes, together with the absence of both ether glycerolipids and the ether lipid-synthesizing enzyme (alkyl DHAP synthase) in S. cerevisiae, indicates that non-ether glycerolipids are synthesized in this organism via the acyl DHAP pathway.