Subcellular distribution and some properties of N-ethylmaleimide-sensitive and-insensitive forms of glycerol phosphate acyltransferase in rat adipocytes.
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ABSTRACT:
SUBMITTER: Saggerson ED
PROVIDER: S-EPMC1162077 | biostudies-other | 1980 Jul
REPOSITORIES: biostudies-other
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Project description: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 88.8kDa 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 microM; 0.25 microCi) [1-(14)C]oleate for 6h increased incorporation of [(14)C]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 approximately 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.
| S-EPMC2133398 | biostudies-literature
Project description:Incubation of rat adipocytes with 1 microM-noradrenaline caused a decrease in both the N-ethylmaleimide-sensitive (microsomal) and N-ethylmaleimide-insensitive (mitochondrial) glycerol phosphate acyltransferase activities measured in homogenates from freeze-stopped cells. The effects of noradrenaline on glycerol phosphate acyltransferase activity were apparent over a wide range of concentrations of glycerol phosphate and palmitoyl-CoA. The effect of noradrenaline was reversed within cells by the subsequent addition of insulin or propranolol. Inclusion of albumin in homogenization buffers abolished the effect of noradrenaline on the N-ethylmaleimide-sensitive activity. The effect of noradrenaline on the N-ethylmaleimide-insensitive (mitochondrial) activity was, however, not abolished by inclusion of albumin in buffers for preparation of homogenates from freeze-stopped cells. Inclusion of fluoride in homogenization buffers did not alter the observed effect of noradrenaline. The inactivating effect of noradrenaline persisted through the subcellular fractionation procedures used to isolate adipocyte microsomes (microsomal fractions). The effect of noradrenaline on mitochondrial glycerol phosphate acyltransferase did not persist through subcellular fractionation. Noradrenaline treatment of cells significantly decreased the Vmax. of glycerol phosphate acyltransferase in isolated microsomes without changing the activity of NADPH-cytochrome c reductase. Glycerol phosphate acyltransferase activity in microsomes from noradrenaline-treated cells is unstable, being rapidly lost on incubation at 30 degrees C. Bivalent metal ions (Mg2+, Ca2+) or post-microsomal supernatant protected against this inactivation. Glycerol phosphate acyltransferase activity in microsomes from noradrenaline-treated cells could not be re-activated by incubation with either alkaline phosphatase or phosphoprotein phosphatase-1. Addition of cyclic AMP-dependent protein kinase catalytic subunits to adipocyte microsomes incubated with [gamma-32P]ATP considerably increased the incorporation of 32P into microsomal protein, but did not cause inactivation of glycerol phosphate acyltransferase. These findings provide no support for the proposal that inactivation of adipocyte microsomal glycerol phosphate acyltransferase by noradrenaline is through a phosphorylation type of covalent modification.
| S-EPMC1152232 | biostudies-other
Project description:Addition of adipocyte 100 000 g post-microsomal supernatant to assays of glycerol phosphate acyltransferase in isolated mitochondria or microsomal fractions decreased activity at lower concentrations of palmitoyl-CoA. At higher concentrations of palmitoyl-CoA, activation was observed on addition of post-microsomal supernatant. The effect of post-microsomal supernatant to decrease activity at lower [palmitoyl-CoA] was abolished by heating or by trypsin treatment, and was also abolished by addition of N-ethylmaleimide to assays or by pretreatment of post-microsomal supernatant with N-ethylmaleimide. The stimulatory effect seen at higher [palmitoyl-CoA] was not sensitive to heat or trypsin treatment. The effect of post-microsomal supernatant at lower [palmitoyl-CoA] cannot be attributed to palmitoyl-CoA hydrolase activity. It was found that brief treatment of adipocyte mitochondria with low concentrations of trypsin was an effective way to remove contaminating microsomal glycerol phosphate acyltransferase activity. Adipocyte post-microsomal supernatant was more effective than an equivalent quantity of liver post-microsomal supernatant protein in decreasing adipocyte microsomal glycerol phosphate acyltransferase activity. The effects of the supernatants from both tissues were decreased by flavaspidic acid. Semi-purified Z-protein fraction from rat liver did not mimic the effect of adipocyte post-microsomal supernatant to decrease glycerol phosphate acyltransferase at lower [palmitoyl-CoA]. Post-microsomal supernatants obtained from noradrenaline-treated adipocytes were less effective than those from control cells in decreasing glycerol phosphate acyltransferase activity in microsomal fractions at lower [palmitoyl-CoA]. It is suggested that adipocyte cytosol may contain an acyl-CoA-binding protein or proteins differing from Z-protein in some respects. The physiological significance of the findings is briefly discussed.
| S-EPMC1152233 | biostudies-other
Project description: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 wild-type 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.
| S-EPMC2442282 | biostudies-literature
Project description:Synthesis of phospholipids can occur de novo or via remodeling of the existing phospholipids. Synthesis of triglycerides, a form of energy storage in cells, is an end product of these pathways. Several 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs) acylate lysophosphatidic acid (LPA) at the sn-2 (carbon 2) position to produce phosphatidic acid (PA). These enzymes are involved in phospholipids and triglyceride synthesis through an evolutionary conserved process involving serial acylations of glycerol-3-phosphate. We cloned a cDNA predicted to be an AGPAT isoform (AGPAT10). This cDNA has been recently identified as glycerol-3-phosphate-O-acyltransferase isoform 3 (GPAT3). When this AGPAT10/GPAT3 cDNA was expressed in Chinese Hamster ovary cells, the protein product localizes to the endoplasmic reticulum. In vitro enzymatic activity using lysates of human embryonic kidney-293 cells infected with recombinant AGPAT10/GPAT3 adenovirus show that the protein has a robust AGPAT activity with an apparent V(max) of 2 nmol/min per mg protein, but lacks GPAT enzymatic activity. This AGPAT has similar substrate specificities for LPA and acyl-CoA as shown for another known isoform, AGPAT2. We further show that when overexpressed in human Huh-7 cells depleted of endogenous AGPAT activity by sh-RNA-AGPAT2-lentivirus, the protein again demonstrates AGPAT activity. These observations strongly suggest that the cDNA previously identified as GPAT3 has AGPAT activity and thus we prefer to identify this clone as AGPAT10 as well.
| S-EPMC5070603 | biostudies-literature
Project description: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.
| S-EPMC3184552 | biostudies-literature
Project description:Epidemiological evidence indicates that thyrotropin (TSH) is positively correlated with the severity of obesity. However, the mechanism remains unclear. Here, we show that TSH promoted triglyceride (TG) synthesis in differentiated adipocytes in a thyroid hormone-independent manner. Mice with subclinical hypothyroidism, which is characterized by elevated serum TSH but not thyroid hormone levels, demonstrated a 35% increase in the total white adipose mass compared with their wild-type littermates. Interestingly, Tshr KO mice, which had normal thyroid hormone levels after thyroid hormone supplementation, resisted high-fat diet-induced obesity. TSH could directly induce the activity of glycerol-3-phosphate-acyltransferase 3 (GPAT3), the rate-limiting enzyme in TG synthesis, in differentiated 3T3-L1 adipocytes. However, following either the knockdown of Tshr and PPAR? or the constitutive activation of AMPK, the changes to TSH-triggered GPAT3 activity and adipogenesis disappeared. The over-expression of PPAR? or the expression of an AMPK dominant negative mutant reversed the TSH-induced changes. Thus, TSH acted as a previously unrecognized master regulator of adipogenesis, indicating that modification of the AMPK/PPAR?/GPAT3 axis via the TSH receptor might serve as a potential therapeutic target for obesity.
| S-EPMC4284501 | biostudies-literature
Project description: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 analogs 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.
| S-EPMC3184547 | biostudies-literature
Project description:Sequence analysis using the Promoser program predicted two promoter-like regions for rat mtGPAT: a distal promoter approximately 30kb upstream and a proximal promoter near the first translational codon. Rat liver cells transfected with pGL3-basic vector containing the distal and proximal promoter resulted in 10.8- and 4.8-fold increase in the luciferase activity, respectively. Results of electromobility shift assay and chromatin immunoprecipitation suggested binding of transcription factors to the distal and proximal promoter regions. 5' RACE PCR showed two transcripts with different transcriptional start sites. When transfected rat liver cells were starved and refed, there was about 2.7-fold increase in the luciferase activity with cells transfected with the distal promoter while the proximal promoter showed no change. Thus, the two promoters could be functionally distinguished. Taken together, the results suggest that there are two promoters for rat mtGPAT gene and that the transcriptional regulation is mediated through the distal promoter.
| S-EPMC2262852 | biostudies-literature
Project description:Microalgae are considered a suitable production platform for high-value lipids and oleochemicals. Several species including Nannochloropsis oceanica produce large amounts of essential [Formula: see text]-3 polyunsaturated fatty acids (PUFAs) which are integral components of food and feed and have been associated with health-promoting effects. N. oceanica can further accumulate high contents of non-polar lipids with chemical properties that render them a potential replacement for plant oils such as palm oil. However, biomass and lipid productivities obtained with microalgae need to be improved to reach commercial feasibility. Genetic engineering can improve biomass and lipid productivities, for instance by increasing carbon flux to lipids. Here, we report the overexpression of glycerol-3-phosphate acyltransferase (GPAT) in N. oceanica during favorable growth conditions as a strategy to increase non-polar lipid content. Transformants overproducing either an endogenous (NoGPAT) or a heterologous (Acutodesmus obliquus GPAT) GPAT enzyme targeted to the endoplasmic reticulum had up to 42% and 51% increased non-polar lipid contents, respectively, compared to the wild type. Biomass productivities of transformant strains were not substantially impaired, resulting in lipid productivities that were increased by up to 37% and 42% for NoGPAT and AoGPAT transformants, respectively. When exposed to nutrient stress, transformants and wild type had similar lipid contents, suggesting that GPAT enzyme exerts strong flux control on lipid synthesis in N. oceanica under favorable growth conditions. NoGPAT transformants further accumulated PUFAs in non-polar lipids, reaching a total of 6.8% PUFAs per biomass, an increase of 24% relative to the wild type. Overall, our results indicate that GPAT is an interesting target for engineering of lipid metabolism in microalgae, in order to improve non-polar lipid and PUFAs accumulation in microalgae.
| S-EPMC9844033 | biostudies-literature