Glycerophosphate Acyltransferase Activity Research Articles

Ethionine-induced alterations of enzymes involved in lipid metabolism and their possible relationship to induction of fatty liver

Changes of enzymes involved in the hepatic metabolism of long-chain fatty acids (palmitoyl-CoA synthetase (EC 6.2.1.3), carnitine palmitoyltransferase (EC 6.2.1.3), glycerophosphate acyltransferase (EC 2.3.1.15)) in the liver of male rats were examined after ethionine exposure. Ethionine administration resulted in a dose-and time-dependent enhancement of the palmitoyl-CoA synthetase activity both in the mitochondrial, peroxisomal and microsomal fractions. The total carnitine palmitoyltransferase activity in the mitochondrial fraction was enhanced. Ethionine administration was also associated with dose- and time-dependent changes of the microsomal glycerophosphate acyltransferase activity, whereas the mitochondrial enzyme activity was marginally affected. The hepatic triacylglycerol content of the enthionine-treated animals was increased. Hepatic lipids were accumulated in large droplets. Serum triacylglycerol and cholesterol were decreased. In particular, the serum HDL-cholesterol level was lowered. The concentration of ATP in the liver decreased. Accumulation of the metabolic product S-adenosylethionine (AdoEth) was observed for the first 2 days of exposure followed by a fall in S-adenosylmethionine (Ado-Met) during the next 10 days. Linear regression analysis of ATP content versus AdoEth and AdoMet showed highly significant correlations. A significant correlation between the hepatic triacyglycerol and AdoEth content was also observed upon ethionine treatment. The data show that ethionine perturbs the hepatic lipid metabolism. Enhanced esterification of long-chain fatty acids, but not a simple reduction of their oxidation, might contribute to enthionine-induced fatty liver in addition to a block in secretion of lipoproteins and decreased protein synthesis.

Rapid stimulation of liver palmitoyl-CoA synthetase, carnitine palmitoyltransferase and glycerophosphate acyltransferase compared to peroxisomal β-oxidation and palmitoyl-CoA hydrolase in rats fed high-fat diets

Key enzymes involved in oxidation and esterification of long-chain fatty acids were investigated in male rats fed different types and amounts of oil in their diet. A diet with 20% (w/w) fish oil, partially hydrogenated fish oil (PHFO) and partially hydrogenated soybean oil (PHSO) was shown to stimulate the mitochondrial and microsomal palmitoyl-CoA synthetase activity (EC 6.2.1.3) compared to soybean oil-fed animals after 1 week of feeding. Rapeseed oil had no effect. Partially hydrogenated oils in the diet resulted in significantly higher levels of mitochondrial glycerophosphate acyltransferase compared to unhydrogenated oils in the diet. Rats fed 20% (w/w) rapeseed oil had a decreased activity of this mitochondrial enzyme, whereas the microsomal glycerophosphate acyltransferase activity was stimulated to a comparable extent with 20% (w/w) rapeseed oil, fish oil or PHFO in the diet. Increasing the amount of PHFO (from 5 to 25% (w/w)) in the diet for 3 days led to increased mitochondrial and microsomal palmitoyl-CoA synthetase and microsomal glycerophosphate acyltransferase activities with 5% of this oil in the diet. The mitochondrial glycerophosphate acyltransferase was only marginally affected by increasing the oil dose. Administration of 20% (w/w) PHFO increased rapidly the mitochondrial and microsomal palmitoyl-CoA synthetase, carnitine palmitoyltransferase and microsomal glycerophosphate acyltransferase activities almost to their maximum value within 36 h. In contrast, the glycerophosphate acyltransferase and palmitoyl-CoA hydrolase (EC 3.1.2.2) activities of the mitochondrial fraction and the peroxisomal β-oxidation reached their maximum activities after administration of the dietary oil for 6.5 days. This sequence of enzyme changes (a) is in accordance with the proposal that an increased cellular level of long-chain acyl-CoA species act as metabolic messages for induction of peroxisomal β-oxidation and palmitoyl-CoA hydrolase i.e., these enzymes are regulated by a substrate-induced mechanism, and (b) indicates that, with PHFO, a greater part of the activated fatty acids are directed from triacylglycerol esterification and hydrolysis towards oxidation in the mitochondria. It is also conceivable that the mitochondrial β-oxidation is proceeding before the enhancement of peroxisomal β-oxidation.

Effect of methotrexate on long-chain fatty acid metabolism in liver of rats fed a standard or a defined, choline-deficient diet

The effect of methotrexate on lipids in serum and liver and key enzymes involved in esterification and oxidation of long-chain fatty acids were investigated in rats fed a standard diet and a defined choline-deficient diet. Hepatic metabolism of long-chain fatty acids were also studied in rats fed the defined diet with or without choline. When methotrexate was administered to the rats fed the standard diet there was a slight increase in hepatic lipids and a moderate reduction in the serum level. The palmitoyl-CoA synthetase activity and the microsomal glycerophosphate acyltransferase activity in the liver of rats were increased by methotrexate. The data are consistent with those where the liver may fail to transfer the newly formed triacylglycerols into the plasma with a resultant increase in liver triacylglycerol content and a decrease in serum lipid levels. Fatty liver of methotrexate-exposed rats can not be attributed simply to a reduction of fatty acid oxidation as the carnitine palmitoyltransferase activity was increased. The methotrexate response in the rats fed the defined choline-deficient diet was different. There was a reduction in both serum and hepatic triacylglycerol and the glycerophosphate acyltransferase and palmitoyl-CoA synthetase activities. The carnitine palmitoyltransferase activity was unchanged. Hepatomegaly and increased hepatic fat content, but decreased serum triacylglycerol, total cholesterol and HDL cholesterol were found to be related to the development of choline deficiency as the pleiotropic responses were almost fully prevented by addition of choline to the choline-deficient diet. Addition of choline to the choline-deficient diet normalized the total palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities. In contrast to methotrexate exposure, choline deficiency increased the mitochondrial glycerophosphate acyltransferase activity. The data are consistent with those of where fatty liver induction of choline deficiency may be related to an enhanced esterification of long-chain fatty acids concomitant with a reduction of their oxidation.

Effects of dietary selenium on lipid peroxidation, mitochondrial function and protein profiles in the heart of the myopathic syrian golden hamster (BIO 14.6)

Male, weanling myopathic Syrian Golden Hamsters (BIO 14.6 strain) were fed a selenium-adequate diet (controls) or this diet supplemented with 1.0 ppm selenium (treated) for 30 days. Se-treated animals exhibited a 50% reduction in lipid peroxidation in heart homogenates relative to controls and a 61% increase in mitochondrial glycerophosphate acyltransferase activity. Gel electrophoresis revealed no alterations in cardiac protein profiles from treated or control animals. These data suggest that the selenium prevents peroxidative injury and maintains mitochondrial function in the absence of alterations in cardiac proteins.

Effect of polymorphonuclear leukocytes (PMNs) generated oxidants on the glycerophosphate acyltransferase activity of rat sarcoplasmic reticulum

Effect of polymorphonuclear leukocytes (PMNs) generated oxidants on the glycerophosphate acyltransferase activity of rat sarcoplasmic reticulum

Role of Adipocyte Precursors in the Onset of Obesity Induced by Overfeeding in Suckling Rats

Rats were overfed during the suckling period by litter size manipulation in order to investigate the possible contribution of preadipocytes from the stromavascular compartment of adipose tissue to the development of obesity. Rats raised in litters of four pups were overfed; for normal feeding we assigned eight pups per litter. As early as 10 d of age, overfed rats became fatter than controls, and showed an increase in both plasma insulin and triacylglycerol levels. At this age, adipose tissue overdevelopment arose only from adipocyte hypertrophy, since hyperplasia occurred only at 15 d of age. Concurrently, compared to normal feeding, overfeeding led to significantly higher activities of lipoprotein lipase (LPL), glycerophosphate dehydrogenase (GPDH) and glycerophosphate acyltransferase (GPAT) in mature fat cells; 10-d-old overfed pups exhibited a higher stromal cell number. Further separation of this heterogeneous fraction by density gradient centrifugation showed a higher preadipocyte number as compared to that of controls. In stromal cells, LPL, GPDH, GPAT and acyl CoA ligase activities were detected during the suckling period. As compared to controls, overfeeding induced an increase in both LPL and GPDH activities in 10-d-old pups. Results indicate that overfeeding in early life induced an excess of fat storage capacity through a simultaneous increase in proliferation and differentiation rates of adipocyte precursors.

Evolution de la capacité d'estérification des acides gras dans les adipocytes de rat avant le sevrage : effet de la suralimentation lactée

During the suckling period, lipid storage in rat adipose tissue arises from exogenous triglycerides, as milk contains a large amount of lipid. In the present work, we studied the effect of overfeeding during the suckling period on the developmental pattern of the fatty acid esterification pathway in fat cells. Early overfeeding was induced by restricting litter size; at 2 days of age, the pups were randomly distributed to form litters of 4 (overfed group) or 8 (controls). During the suckling period the activity of lipoprotein lipase (LPL), glycerophosphate dehydrogenase (G3PDH), glycerophosphate acyl transferase (GPAT) and acyl CoA ligase were measured in isolated fat cells from the inguinal adipose tissue. As early as 10 days of age, plasma triglyceride levels were increased in overfed pups as compared to the controls and, in these animals, the adipose tissue was overdeveloped, mainly due to fat cell hypertrophy. At that age, overfeeding induced a significant increase in LPL and GPAT activities, but acyl CoA ligase and GPDH were not modified in overfed pups. Fat cell hypertrophy appeared to be responsible for the increased GPAT activity in the overfed pups. On the contrary, the effect of overfeeding on LPL activity was not only related to fat cell enlargement, as the enzyme activity continued to increase in the overfed rats when expressed per unit of cell surface area. These results show that overfeeding during the suckling period rapidly induced an increase in the fat storage capacity of the adipose tissue. This suggests that circulating triglyceride levels could regulate directly or through hormonal control the activity of the fatty acid esterification pathway in fat cells.

Acute effect of bromodichloromethane on lipid metabolism in rat liver microsomes

Acute effects of CHBrCl 2 on microsomal lipid metabolism were studied in the liver of male Wistar rats administered a single oral dose (215 mg/kg). 3 h after administration, the modulation of phospholipid metabolism and the accumulation of triacylglycerol (TG) were noted in the microsomal membranes. A marked decrease in the synthetic rate of phospatidylcholine (PC) could be explained by a fall in the microsomal activity of CTP.-phosphocholine cytidylyltransferase. A decrease in glycerophosphate acyltransferase activity and an enhancement in phosphatidate phosphatase activity seemed to offset each other, and consequently no significant change was observed in the synthetic rate of microsomal TG. These alterations in microsomal lipid metabolism can be regarded as one of the manifestations of hepatotoxicity of CHBrCl 2.

Effect of a high cholesterol diet on lipid metabolizing enzymes in spontaneously hypertensive rats

A high cholesterol diet induced a fatty liver and an increase in cholesterol oleate in spontaneously hypertensive rats. The activity of microsomal glycerophosphate acyltransferase in liver increased 2–3-fold to meet the increased supply of oleate, the synthesis of which was stimulated by a 10-fold increase in microsomal Δ 9-desaturase activity. Hepatic fatty acid synthetase and diacylglycerol acyltransferase activities were decreased somewhat. These results, together with the fact that the large increases in hepatic cholesterol ester and triacylglycerol were not correspondingly reflected in plasma, indicated that the fatty liver resulted from decreased secretion of lipoprotein rather than increased lipogenesis. Endogenous cholesterol in liver microsomes increased 2-fold and hepatic acyl-CoA:cholesterol acyltransferase activity increased 3-fold, whereas plasma lecithin:cholesterol acyltransferase activity was unchanged. Thus, the increase in cholesterol oleate seen in spontaneously hypertensive rats fed a high cholesterol diet is due mainly to increases in acyl-CoA:choIesterol acyltransferase and Δ 9-desaturase activities.

A kinetic study of glycerophosphate acyltransferase of rat adipocytes in relation to its control by noradrenaline.

Glycerophosphate acyltransferase present in an extract of rat adipocytes is strongly inhibited by excess palmitoyl-CoA. This inhibition is released by serum albumin but an excess of serum albumin is inhibitory, particularly at low palmitoyl-CoA concentrations. An optimal activity is reached when the ratio palmitoyl-CoA/albumin is in the range of 3-6. In the absence of albumin, oleic acid inhibits the activity at all palmitoyl-CoA concentrations. This inhibition is released by albumin and, inversely, oleic acid releases the inhibition by high concentrations of albumin. Another effect of fatty acids is to favour the inactivation of the glycerophosphate acyltransferase in extracts of adipocytes kept at 0 degree C. This inactivation is time-dependent and cannot be reversed by the addition of albumin to the assay mixture. Treatment of adipocytes with noradrenaline had no effect on the activity of the enzyme as long as the cells had been separated from fatty acids and albumin. With extracts of unwashed cells, the effect of noradrenaline on both the activity and stability of glycerophosphate acyltransferase could be explained by the presence of fatty acids in the extract.

Development of glycerophosphate acytransferase in guinea pig lung mitochondria and microsomes

Development of mitochondrial and microsomal glycerophosphate acyltransferase in the fetal guinea pig lung was investigated. Mitochondrial and microsomal enzyme activity gradually increased from 45 days to 55 days of gestation. The specific activity in the microsomal fraction (8.2 nmol/min per mg protein) then declined until term, but increased again in the 24-h newborn from 2.5 to 6.1 nmol/min per mg protein. Glycerophosphate acyltransferase activity in the mitochondrial fraction declined after 55 days (3.5 nmol/min per mg) to a minimum level at 60 days (1.8 nmol/min per mg), but increased again in the 24-h newborn (4.0 nmol/min per mg). The specific activity of both mitochondrial and microsomal enzyme declined after 24 h after birth until adult levels were attained. Glycerophosphate acyltransferase activity in mitochondria and microsomes from adult lung was 0.8 and 2.0 nmol/min per mg, respectively. Microsomal enzyme activity was consistently inhibited (over 95%) throughout gestation and adulthood by exposure to any one of several proteinases: trypsin, chymotrypsin, papain, bromelain, pronase and nagarse. Although mitochondrial enzyme activity was also inhibited by these proteinases, there was a continuous increase in proteinase-resistant glycerophosphate acyltransferase activity between 45 days of gestation and term. In contrast, adult mitochondrial enzyme activity was stimulated by all the proteinases studied. These results suggest that early in gestation, glycerophosphate acyltransferase lies more exposed on the cytoplasmic side of the mitochondrial outer membrane and as gestation progresses it becomes embedded into the phospholipid bilayer.

Location of glycerol phosphate acyltransferase in the transverse plane of mitochondrial outer membrane of guinea pig lung

Incubation of guinea pig lung mitochondrial suspension in an isotonic low ionic strength buffer containing various proteolytic enzymes caused significant stimulation of the glycerophosphate acyltransferase activity. The maximal stimulation range between 20 and 105%, and the order was as follows: bromelain >chymotrypsin >pronase >trypsin >papain > nagarse. Under hypotonic conditions, over 85% of GAT was destroyed by all the proteolytic enzymes. Microsonal enzyme activity was consistently inhibited (>95%) by exposure to any of these proteases even under isotonic conditions. These results suggest that GAT is located on the inner aspect of the mitochondrial outer membrane. Also, it is likely that a portion of this enzyme or that of a modulator is present in the outer side of the outer membrane and proteolysis of this component causes stimulation.

Studies on reconstituted partially purified glycerophosphate acyltransferase from Escherichia coli.

Solubilized glycerophosphate acyltransferase from Escherichia coli was reconstituted in small unilamellar vesicles consisting of phosphatidylcholine/phosphatidylglycerol in a molar ratio of 4:1. Glycerol 3-phosphate, trapped inside these vesicles, cannot be acylated by the enzyme upon addition of extra-vesicular palmitoyl-CoA. Thus, substrate-binding sites and active sites are asymmetrically oriented in the model membrane. When up to 10 mol/100 mol lysophosphatidic acid was incorporated in the vesicles a decrease in glycerophosphate acyltransferase activity is observed at amounts exceeding 1 mol% lysophosphatidate. Similar experiments, using lysophosphatidylcholine and phosphatidic acid, suggest the decrease to result from an increase in negative surface charge. Reconstituted glycerophosphate acyltransferase exhibits a preference for palmitoyl-CoA over oleoyl-CoA. This preference increases considerably at elevated temperatures. The glycerophosphate acyltransferase could, therefore, participate in the temperature-dependent changes in the fatty acid composition of the phospholipids in E. coli.

An enzymatic rationale for the randomization of the positional distribution of fatty acids in phospholipids of ascites hepatoma AH 130

Fatty acids present in glycerophospholipids isolated from Yoshida ascites hepatoma AH 130 are more randomly distributed among the 1- and 2-positions than are fatty acids of normal liver phospholipids. The relative abundance of unsaturated fatty acids at the 1-position was ascribed to the lower palmitate-specific glycerophosphate acyltransferase activity in mitochondria of the hepatoma cells, an observation supporting the conclusion put forward for the similar randomization observed in Ehrlich ascites cells (Haldar D., Tso, W.-W. and Pullman M.E. (1979) J. Biol. Chem. 254, 4502–4509). The relative abundance of saturated fatty acids at the 2-position could be ascribed to the relatively lower acyl-CoA:l-acyl-glycerophosphocholine acyltransferase activity and to the change in the selectivity of the hepatoma acyl-CoA:l-acyl-glycerophosphate acyltransferase system into the lung type. The relatively lower selectivity for arachidonoyl-CoA as compared with oleoyl-CoA of the 1-acyl-glycerophosphocholine acyltransferase system is consistent with the decrease in polyenoic fatty acid content at the 2-position of the hepatoma phospholipids.

The rapid induction of liver glycerophosphate acyltransferase in mice by clofibrate, a hypolipidemic agent.

Administration of clofibrate to adult mice (20-25 mg of clofibrate/day) increased liver glycerophosphate acyltransferase (EC 2.3.1.15) activity by 2.3-fold within 24 h. This increased glycerophosphate acyltransferase activity was mainly localized in the liver microsomal fraction. Three other hypolipidemic drugs, i.e. Bezafibrate (Boehringer Mannheim Co.), Gemfibrozil (Warner-Lambert Co.) and Wy-14,643 (Wyeth Laboratories), when fed to mice, also increased the liver glycerophosphate acyltransferase activity by 2-3-fold in 24 h. Simultaneous administration of inhibitors of protein biosynthesis, such as cycloheximide or actinomycin D completely abolished such stimulation of liver glycerophosphate acyltransferase by clofibrate.

DNA synthesis and cell division related to adipose differentiation of 3T3 cells.

Adipose differentiation of 3T3-F442A cells in surface cultures depends on adipogenic factor present in the culture medium. We found that after stimulation with adipogenic serum, 3T3-F442A cell underwent a burst of DNA synthesis before adipose conversion was manifested by an augmented lipogenic enzyme activity. In differentiating cells, DNA synthesis, judged by a 100-fold higher rate of [3H]thymidine incorporation into TCA-insoluble material, was followed by a 100-fold increase in the activity of glycerophosphate acyltransferase. Cytosine arabinoside, added to the cultures at a concentration of 3 micrograms/ml, exerted 95% inhibition of [3H]thymidine incorporation and also inhibited adipose conversion. The burst of DNA synthesis correlated with a 2.5-fold increase in the amount of DNA and in the number of cells in the culture. The DNA content was the same in differentiated and nondifferentiated cells. We conclude that after the interaction with the adipogenic factor, the cells go through DNA synthesis and cell division essential for adipose conversion.

Glycerolipid biosynthesis in rat adipose tissue 10. Changes during a starvation and re-feeding cycle

Effects of starvation and re-feeding on adipocyte glycerolipid formation were investigated in young (age 45–55 days) and old rats (age 83–94 days). Adipocyte homogenates were used to assay glycerophosphate acyltransferase and Mg 2+-dependent phosphatidase phosphohydrolase. Glycerophosphate acyltransferase was measured in the presence of [ 14C]glycerol 3-phosphate , palmitate, ATP, CoA and Mg 2+. The release of inorganic phosphate from aqueous dispersed phosphatidase was taken as a measure of phosphatidate phosphohydrolase activity. Young rats starved for 48–72 h showed a 2-fold decline in the glycerophosphate acyltransferse activity. Older rats did not show any change in the glycerophosphate acyltransferase activity during 96 h starvation. Re-feeding of starved rats with chow for 48 h caused significant increases in the glycerophosphate acyltransferase activity. These changes were mainly limited to N-ethylmaleimide-sensitive glycerophosphate acyltransferase. Phosphatidate phosphohydrolase activity decreased significantly (2-fold) during starvation in both young and old rats. Phosphatidate phosphohydrolase activity was regained completely after re-feeding of starved rats. Initial changes in the glycerophosphate acyltransferase and phosphatidate phosphohydrolase activities were very slow. Most notable increases in the glycerophosphate acyltransferase and phosphatidate phosphohydrolase activities were observed between 24 and 48 h after initiation of a re-feeding schedule. Mean adipocyte size decreased during starvation of rats for 72 h. Although considerable increases in the activities of both glycerophosphate acyltransferase and phosphatidate phosphohydrolase were apparent by re-feeding of starved rats for 48 h, mean adipocyte size did not change during this period. Thus, enzyme changes which occurred after re-feeding were independent of the adipocyte size. To separate the effects of age from the cell size on adipocyte glycerolipid formation during starvation and re-feeding periods, adipocytes from older rats were subjected to filtration through a nylon screen to obtain adipocytes of similar sizes. These studies suggest that the age of the animal significantly influences the effects of starvation and re-feeding on adipocyte glycerolipid formation.

Localization of glycerophosphate acyltransferase in Escherichia coli

sn-Glycero-3-phosphate acyltransferase (EC 2.3.1.15) the first enzyme involved in phospholipid biosynthesis, is known to be associated with the cytoplasmic membrane of Escherichia coli. The localization of this enzyme in the transverse plane of the membrane was investigated by proteolysis of intact and lysed spheroplasts and by inhibition of glycerol 3-phosphate transport into intact cells in the presence of azide. Glycerophosphate acyltransferase was found to be resistent to proteolysis by trypsin in intact spheroplasts, whereas its enzymatic activity could be destroyed completely by trypsin in lysed spheroplasts. These results are in line with a localization of the acyltransferase at the inner aspect of the cytoplasmic membrane. Sodium azide was shown to have no inhibitory effect on glycerophosphate acyltransferase activity. Lack of incorporation of glycero phosphate into the phospholipids of glycerol phosphate transport-negative cells and inhibition of this incorporation in wild-type and glycerol 3-phosphate transport-constitutive cells by azide support a cytoplasmic-oriented localization of the glycerophosphate acyltransferase.

Glycerophosphate acyltransferase activity in perfused liver of normal and hyperlipemic rats: Glucagon effect

Mitochondrial glycerophosphate-acetyltransferase activity (GPAT) was determined in the isolated and perfused liver of diet-induced hyperlipemic rats, and was found to be significantly increased compared to normal rats, A positive correlation existed between hepatic triglyceride output and GPAT. Perfusion of 10(-5) M glucagon induced a significant reduction in GPAT levels. It is suggested that the lipid-lowering action of glucagon may be mediated also through an inhibition of GPAT activity.

MICROSOMAL-ASSOCIATED GLYCEROPHOSPHATE ACYLTRANSFERASE ACTIVITY IN GERMINATING SOYBEANS

Acyl CoA:sn-glycero-3-phosphate O-acyltransferase (glycerophosphate acyltransferase, EC 2.3.1.15) catalyzes the acylation of glycero-3-phosphate to form lysophosphatidic acid. This enzyme catalyzes the first committed step in the biosynthesis of phospholipids and acylglycerols in soybeans. Glycerophosphate acyltransferase was predominatly associated with the microsomal fraction of germinating soybeans. The pH optimum for the reaction was 7.0. The enzyme exhibited saturation kinetics for glycero-3-phosphate (Km of 0.2 mM) and palmitoyl CoA (Km of 6.4 μM). A variety of acyl CoA derivatives served as substrates for the enzyme. Palmitoyl CoA was the most effective acyl CoA substrate. The addition of the nonionic detergent Triton X-100 inhibited glycerophosphate acyltransferase activity. Thioreactive agents inhibited the enzyme indicating that a sulphydryl group is essential for activity. The activation energy for the reaction was 8.8 Kcal per mole. The microsomal enzyme was reasonably stable to temperatures up to 70° C.