Glycerophosphate Acyltransferase Research Articles

Lipoprotein lipase and glycerophosphate acyltransferase in ovine tissues are influenced by growth and energy intake regimen

The purpose of this study was to determine the effect of energy intake regimen on lipoprotein lipase (LPL) activity in muscle and adipose tissue, and on glycerophosphate acyltransferase (GPAT) activity in adipose tissue and liver of sheep. Six ewe lambs (31.3 ± 1.0 kg) per treatment consumed either a low- (LOW, 12.1 Mjoules ME/day) or a high-energy diet (HI, 19.2 Mjoules ME/day). Treatments LOW1 and HI were fed diets for 120 days. Treatment LOW2 was fed the low-energy diet until ewes attained the 120-day weight of HI ewes. Treatment CG (compensatory gain) was fed low-energy for 120 days and then fed high-energy for 30 more days. Growth rates and back fat thicknesses were consistent with energy intake regimen. Adipose tissue LPL activity was greater ( P < 0.05) in animals fed the diet containing the greatest energy density (HI) compared with all other treatment groups. Skeletal muscle LPL activity was greatest for LOW1 and lowest for CG ( P < 0.05); HI and LOW2 were intermediate. Cardiac muscle LPL activity was similar for LOW1 and CG, which were both greater ( P < 0.05) than HI and LOW2. Adipose tissue GPAT activity was lowest for LOW1 and highest for CG ( P < 0.05); HI and LOW2 were intermediate. Liver GPAT activity was similar for LOW1, HI, and CG, which were greater ( P < 0.05) than LOW2. Results indicate that ovine adipose tissue LPL may play a role in maintenance of adiposity, and that skeletal and cardiac muscle LPL appeared to be regulated differently. Adipose tissue GPAT activity was consistent with back fat thickness, indicating that this enzyme may play a significant role in regulating ovine adipose tissue lipid accretion. Coordinate regulation of adipose tissue LPL and GPAT was not apparent.

Stimulation of phospholipid synthesis in HeLa cells by epidermal growth factor and insulin: activation of choline kinase and glycerophosphate acyltransferase

The cultivation of HeLa cells in the absence of growth factors reduced proliferation with a considerable decrease in the phospholipid content. A single addition of EGF or insulin was not enough to stimulate proliferation, but still sufficient to efficiently restore the phospholipid content to the level of serum-grown cells. Both EGF and insulin stimulated the synthesis of major phospholipids in HeLa cells. Irrespective of whether cells were stimulated or not, the phospholipid composition and the phospholipid/protein ratio remained constant, suggesting the existence of a mechanism that keeps them constant under varying conditions. The degradation of phosphatidylcholine was not affected by EGF or insulin. Both EGF and insulin increased choline kinase activity equally and promoted the conversion of choline to cholinephosphate, accompanied by an expansion of the cholinephosphate pool in treated cells. The level of glycerophosphate acyltransferase was enhanced by EGF and insulin but EGF was more effective than insulin. The present results provide evidence that one of the roles of EGF and insulin during cell growth is to stimulate the synthesis of phospholipids.

Sources of triacylglycerol accumulation in livers of rats fed a cholesterol‐supplemented diet

The source of free fatty acids (FFA) and the pathways contributing to the accumulation of neutral fats in livers of rats fed a cholesterol-enriched diet were investigated in this report. Supplementation with 1% cholesterol in the diet for four weeks resulted in hepatomegaly in the rats. The contents of cholesterol and triacylglycerols (TG) per gram liver measured in rats fasted overnight increased by 48 mg (approximately tenfold) and 66 mg (approximately fourfold), respectively. The activities of glycerophosphate acyltransferase and diacylglycerol acyltransferase, the two key enzymes for TG synthesis in liver microsomes, were found to increase by 23 and 19%, respectively, in the cholesterol-fed rats. The secretion of plasma TG present predominantly in very low density lipoprotein was found to decrease by approximately 30%. The incorporation of tritium from tritiated water in liver FFA increased by twofold in rats fed the cholesterol-supplemented diet, whereas the activity of CPT I in liver mitochondria decreased by 23%. The uptake of plasma FFA in vivo in livers of fasted rats maintained on the cholesterol-supplemented diet decreased by 60%. Our data thus indicate that the excess TG accumulated in livers of rats fed the cholesterol-enriched diet resulted from increased synthesis and decreased secretion of TG. To meet the demand of fatty acids for this purpose, de novo lipogenesis increased, whereas beta-oxidation decreased. Although difference in the uptake of extrahepatic FFA may be discounted, a difference in the uptake of chylomicron remnants between the control and cholesterol-fed rats may not be ruled out.

Regulation of phosphatidic acid biosynthetic enzymes in Saccharomyces cerevisiae.

Phosphatidic acid is the biosynthetic precursor of all glycerolipids. To understand how phosphatidic acid biosynthesis is controlled in Saccharomyces cerevisiae, we studied the regulation of three enzyme activities involved in the synthesis of this glycerolipid precursor, i.e., glycerophosphate acyltransferase (GPAT), dihydroxyacetone phosphate acyltransferase (DHAPAT), and acyl DHAP reductase. GPAT activity was increased 3-fold, while DHAPAT activity was increased up to 9-fold in wild type cells grown in a nonfermentable carbon source compared to that of glucose-grown cells. The ratio of GPAT/DHAPAT activity was 12 in glucose-grown cells but only 4 in cells grown in glycerol/ethanol. In the previously characterized tpa1 mutant, (T. S. Tillman and R. M. Bell. 1986. J. Biol. Chem. 261: 9144-9149), GPAT was decreased 2-fold and DHAPAT 27-fold compared to activities in the wild type. Acyl DHAP reductase activity in both wild type and tpa1 cells grown on a nonfermentable carbon source was increased approximately 2-fold over that of glucose-grown cells. All three enzymatic activities increased as wild type cells grown on glucose entered the stationary phase of growth. Therefore, GPAT, DHAPAT, and acyl DHAP reductase activities appear to be regulated by the respiratory state of the cell. None of the activities was affected to a great extent by inositol, which is a key regulator of many enzymes involved in the synthesis of PtdOH-derived phospholipids in S. cerevisiae, nor by deletion of the mitochondrial genome.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of glycerophosphate acyltransferase from rat liver mitochondria.

Glycerophosphate acyltransferase (GAT) catalyzes the conversion of sn-glycerol 3-phosphate to lysophosphatidic acid (LPA), the first and committed step of triacylglycerol and phospholipid synthesis. In spite of the important regulatory roles GAT may play in this biosynthetic pathway, little information is available on the structure, biochemical properties, and regulation of GAT from eukaryotic cells. We solubilized GAT from rat liver mitochondrial membranes and purified it to an apparent homogeneity by hydroxylapatite chromatography, preparative isoelectric focusing, and gel filtration. The enzyme is composed of a single polypeptide of 85 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography of the native protein. The GAT activity was completely lost during the purification procedure and required addition of exogenous phospholipids for its reconstitution. Since a high phospholipid to detergent ratio was needed for full reactivation, it is concluded that GAT requires "lipid boundary" for reconstitution. The ability of different phospholipids to reactivate GAT decreased in the following order: phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), asolectin, phosphatidylinositol (PI), phosphatidylserine (PS), and cardiolipin. 1,2-Dioleoyl derivatives of PG and PE were more effective in reconstituting the GAT activity than corresponding dipalmitoyl derivatives. The GAT activation was further increased by using a combination of PG and PE or PG and PC. Regardless of the phospholipid used for reconstitution, palmitoyl-CoA was the best acyl donor and LPA was the only reaction product.

Starvation-realimentation overshoot in glycerophosphate acyltransferase in adipose tissue and liver of rats is influenced by type of dietary fat

The purpose of this study was to determine effects of dietary fat source on changes in glycerophosphate acyltransferase that occur with starvation-realimentation. Forty male Sprague-Dawley rats were randomly assigned to dietary treatments at 209 ± 3 g of body weight. Ten rats each were assigned to a 65 g/100 g sucrose diet that contained 5 g/100 g fat from the following sources: stearate (42% stearate, 41% oleate): oleate (high-oleate safflower oil); palm oil; or beef tallow. Five rats received free access to their diet for 4 days and five rats were starved for 2 days and then fed for 2 days. Rats were then killed and liver and epididymal adipose tissue dissected and homogenized for glycerophosphate acyltransferase assay. The starvation-realimentation regimen on the stearate diet stimulated adipose tissue glycerophosphate acyltransferase above non-starved controls. Realimentation on high-oleate and high-palmitate diets stimulated liver glycerophosphate acyltransferase most, as well as produced a starvation-realimentation overshoot in steady-state glycerolipid production. Results indicate that ad libitum consumption of diets containing high proportions of stearate after a period of starvation could overshoot liver glycerophosphate acyltransferase to a lesser extent than if the diet contained low proportions of this fatty acid. Diet fatty acid composition had less impact on adipose tissue than liver glycerophosphate acyltransferase activity in rats subjected to starvation-realimentation.

Development of pancellular toxicity in guinea pig lung by ingestion of oleylanilide

Toxic oil syndrome (TOS), characterized by widespread thromboembolism, vasculotoxicity, and ARDS, develops in humans ingesting denatured edible oils. The mechanism(s) involved in targeted vasculocentric damage in this multi-system disorder is not known. Oleylanilide (OA) was synthesized and fed to male, young adult guinea pigs by gavage for 30 days at doses of 35, 50, and 100 mg/kg/day in groups of six animals each respective to weight. Controls were fed olive oil. Oleylanilide fed animals gained less weight than controls. At the end of experiment, right lungs were inflation fixed in appropriate fixative for histology and transmission electron microscopy (TEM) and left lungs were frozen at -70 degrees C for biochemical analyses. The activity of glycerophosphate acyltransferase (GAT) and cholinephosphotransferase (CPT), two key enzymes involved in phospholipid biosynthesis, were decreased in lung due to OA ingestion. All doses of OA induced marked perivascular and peribronchoiolar monocytic infiltrates that often formed prominent nodules; segmental vascular smooth muscle cell proliferation and derangement of myocytic polarity, subendothelial foamy infiltrates, and edema; nuclear pyknosis and dropout in vascular and bronchial targetoid myocytes; and denudation of bronchiolar epithelial cells. Alveoli contained large numbers of monocytes, macrophages, red cells, edema, and debris. Transmission electron microscopy showed type I cell cytoplasmic ballooning and disintegration of type I cell; contracted and blebbed endothelial cells, fibrin thrombi in capillaries, intracellular megalamellar bodies in type II cells, and surfactant lamellae; and liposomes and fine granular precipitates within alveoli, and contraction and lift off of bronchiolar epithelial cells. Monocytes, mast cells, and eosinophils infiltrated bronchial walls. Furthermore, there was deposition of electron dense particles on the surface of the alveolar wall.(ABSTRACT TRUNCATED AT 400 WORDS)

Palmitate esterification and glycerophosphate acyltransferase activity in adipose tissue of growing lambs

The purposes of this study were to determine the effects of BW in growing lambs on rates of palmitate esterification (PE) and activity of glycerophosphate acyltransferase (GPAT) in subcutaneous and intermuscular adipose tissue in vitro and to determine the relationship between these in vitro measurements and carcass lipid accretion. At 37, 49, 60, and 71 kg of live weight, nine, nine, eight, and seven Columbia-Rambouillet-cross ewe lambs, respectively, were slaughtered and adipose tissue was dissected for glycerolipid biosynthesis assays. One-half of each carcass was boned, and carcass soft tissue lipid was measured. In both adipose tissues, PE and GPAT activity were highest at 37 kg (P = .0001); the greatest accumulation of carcass fat occurred between 37 and 49 kg (P = .0001). Differences in PE and GPAT were less well marked between 49-, 60-, and 71-kg lambs, but both were generally decreased with increased BW. Significant R2 from regressions of mass and percentage of carcass fat on either PE, GPAT, or steady-state glycerolipid synthesis (GLS) were from .39 to .44 (PE), .40 to .58 (GPAT), .50 to .75 (GLS), and .60 to .77 (PE, GPAT, and GLS combined). Changes in PE, GPAT, and GLS with BW were consistent with changes in rates of carcass fat accretion, which indicated a close relationship between glycerolipid biosynthesis in vitro and lipid deposition in vivo in growing-finishing lambs.

Effects of dietary fatty acid composition on glycerophosphate acyltransferase activity in rat adipose tissue and liver

1. 1. How source of dietary fat influences glycerophosphate acyltransferase (GPAT) activity in homogenates of adipose tissue and liver was determined using 5 rats per diet that contained 12% fat from either corn- (CO), high-oleate-safflower (HOSO), palm- or high-stearate-oils. 2. 2. Activity was greatest in adipose tissue homogenates of rats fed HOSO and CO whereas no treatment effects were observed in liver homogenates. 3. 3. No differences in fat pad weights were observed; thus HOSO- and CO-diets likely depressed other lipid synthetic rates. 4. 4. Data indicate that mono- and diunsaturated fatty acid diets supported greater adipose tissue GPAT activity than did saturated fat diets when fed to rats.

The hypotriglyceridemic effect of eicosapentaenoic acid in rats is reflected in increased mitochondrial fatty acid oxidation followed by diminished lipogenesis.

The effect of eicosapentaenoic acid (EPA) on fatty acid oxidation and on key enzymes of triglyceride metabolism and lipogenesis was investigated in the liver of rats. Repeated administration of EPA to normolipidemic rats resulted in a time-dependent decrease in plasma triglycerides, phospholipids and cholesterol. The triglyceride-lowering effect was observed after one day of feeding whereas lowering of plasma cholesterol and phospholipids was observed after five days of treatment. The triglyceride content of liver was reduced after two-day treatment. At that time, increased mitochondrial fatty acid oxidation occurred whereas mitochondrial and microsomal glycerophosphate acyltransferase was inhibited. The phosphatidate phosphohydrolase activity was unchanged. Adenosine triphosphate:citrate lyase, acetyl-CoA carboxylase, fatty acid synthetase and glucose-6-phosphate dehydrogenase were inhibited during the 15 d of EPA treatment whereas peroxisomal beta-oxidation was increased. At one day of feeding, however, when the hypotriglyceridemic effect was established, the lipogenic enzyme activities were reduced to the same extent in palmitic acid-treated animals as in EPA-treated rats. In cultured rat hepatocytes, the oxidation of [14C]palmitic acid to carbon dioxide and acid-soluble products was stimulated in the presence of EPA. These results suggest that the instant hypolipidemia in rats given EPA could be explained at least in part by a sudden increase in mitochondrial fatty acid oxidation, thereby reducing the availability of fatty acids for lipid synthesis in the liver for export, e.g., in the form of very low density lipoproteins, even before EPA induced peroxisomal fatty acid oxidation, reduced triglyceride biosynthesis and diminished lipogenesis.

Accelerated hepatic lipid synthesis in fasted septic rats.

We examined changes in the enzyme activities and metabolites related to hepatic fatty acid synthesis in fasted rats with sepsis produced by cecal ligation and puncture. Sepsis stimulated the in vivo incorporation of tritiated water into hepatic fatty acids and nonsaponifiable lipids. The activities of acetyl-CoA carboxylase, ATP-citrate lyase, and NADPH-generating enzymes (glucose-6-phosphate dehydrogenase and malic enzyme), the tissue levels of citrate and malonyl-CoA, and the dephosphorylation of carboxylase were increased in the livers of fasted septic rats compared with fasted sham-operated control rats. These results indicate that sepsis stimulated hepatic lipogenesis and sterologenesis in fasting rats. Furthermore, sepsis reduced the specific activity of hepatic mitochondrial carnitine palmitoyltransferase and raised that of glycerophosphate acyltransferase, suggesting an increased diversion of cytosolic acyl-CoA towards esterification. These intrahepatic metabolic changes strongly suggest that sepsis causes anabolic action on hepatic lipid metabolism.

Glycerolipid biosynthesis in adipose tissue of the bovine during growth

1. 1. Rates of palmitate esterification in tissue slices and glycerophosphate acyltransferase activity in homogenates were determined in bovine subcutaneous and intermuscular adipose tissue at 340, 418 or 498 kg of live weight. 2. 2. Lower rib section fat accretion rates were observed from 340 to 481 kg than from 418 to 498 kg. 3. 3. Changes in palmitate esterification rates at different body weights were consistent with reduced rib section fat accretion as well as with reported differences in fat accretion in subcutaneous and intermuscular fat depots. 4. 4. Glycerophosphate acyltransferase activity was increased at 418 kg and remained elevated whereas palmitate esterification was decreased at 418 and then increased at 498 kg. 5. 5. Differences between palmitate esterification and glycerophosphate acyltransferase in vitro may have been related to differences in substrate supply.

Regulation of mitochondrial and microsomal phospholipid synthesis by liver fatty acid-binding protein.

Recently we have detected and partially purified a 15-kDa cytosolic L-alpha-lysophosphatidic acid (LPA)-binding protein (LPABP), which stimulates export of LPA from mitochondria (Vancura, A., Carroll, M. A., and Haldar, D. (1991) Biochem. Biophys. Res. Commun. 175, 339-343). Now we have purified this protein to homogeneity. By Western immunoblot analysis, amino acid sequence analysis, and binding characteristics we have shown that LPABP is identical with liver fatty acid-binding protein (L-FABP). This protein binds LPA, and stimulates mitochondrial and microsomal glycerophosphate acyltransferase (GAT) and the export of LPA from both the organelles. The mitochondrially synthesized LPA exported by L-FABP can be converted to phosphatidic acid by microsomes. L-FABP also stimulates microsomal conversion of LPA to phosphatidic acid but strongly inhibits this reaction in mitochondria. However, in the absence of L-FABP mitochondria predominantly synthesize PA. Taken together, these findings are suggestive that L-FABP plays a major role in mitochondrial and microsomal phospholipid metabolism by regulating both the synthesis and utilization of LPA.

Factors influencing triacylglycerol synthesis in permeabilized rat hepatocytes.

Rat hepatocytes were treated with Staphylococcus aureus alpha-toxin to permeabilize their plasma membrane for low-molecular-mass compounds. During incubation with 1 mM labelled fatty acid, phosphatidate and, less clearly, lysophosphatidate rapidly reached a steady state, whereas labelled diacylglycerol accumulated to some extent, at least in the absence of exogenous CDP-choline. Esterification and oxidation were linearly related to the fatty acid concentration, and there was no indication for saturation with acyl-CoA. However, when permeabilized cells were incubated with labelled sn-glycerol 3-phosphate and 1 mM unlabelled fatty acid, glycerolipid synthesis and the level of esterification intermediates reached a plateau between 0.25 and 0.50 mumol of the triose phosphate/ml. The synthesis of phosphatidylcholine was dependent on addition of CDP-choline. In presence of the latter, diacylglycerol no longer accumulated and triacylglycerol synthesis was suppressed, although the sum of synthesized diacylglycerol, triacylglycerol and phosphatidylcholine remained constant. This indicates that the same pool of diacylglycerol is shared by choline-phosphotransferase and diacylglycerol acyltransferase and that the relative activity of these enzymes depends on the CDP-choline supply. Comparison of the levels of the esterification intermediates with the activity of the respective steps of the pathway reveals that, at a fixed fatty acid concentration, glycerophosphate acyltransferase determines the esterification rate, whereas lysophosphatidate acyltransferase and, at low CDP-choline levels, diacylglycerol acyltransferase approach saturation at elevated sn-glycerol 3-phosphate concentration. There is, however, no indication for a regulatory role of phosphatidate phosphohydrolase in this system. The significance of these findings for the regulation of triacylglycerol synthesis under conditions in vivo is discussed.

5] Glycerophosphate acyltransferase from Escherichia coli

This chapter discusses strategies of purifying and characterizing the glycerophosphate acyltransferase. These are (1) cloning of the plsB structural gene encoding the glycerophosphate acyltransferase and construction of strains, which overproduce the enzyme; (2) effective use of nonionic detergents to solubilize the glycerophosphate acyltransferase from membranes and the development of chromatographic steps, which resolve the solubilized, hydrophobic proteins; and (3) design of a quantitatively efficient reconstitution methodology to restore activity to enzyme preparations, which are stable, but inactive, in the presence of detergent. The plsB structural gene encoding the 83,000 M r glycerophosphate acyltransferase polypeptide has been cloned by monitoring the ability of DNA from a total E. coli library to overcome the glycerol 3-phosphate growth requirement of a plsB- mutant strain. Purification is divided into three steps of fractionation based on hydrophobic dye interaction, combined sizing and hydrophobic interaction, and ion exchange. The efficiency of 83,000 M r polypeptide recovery and reconstitution of activity under conditions is tabulated in the chapter.

6] Glycerophosphate acyltransferase from liver

Publisher Summary In mammalian organs, glycerophosphate acyltranferase is present in both the endoplasmic reticulum (microsomes) and mitochondrial outer membrane. In liver, the glycerophosphate acyltransferase activity is nearly equal in these two organelles, whereas in other organs the microsomal acyltransferase is approximately 10 times more active than the mitochondrial enzyme. The enzyme activity can be measured by following the conversion of sn -[2- 3 H]glycerol 3-phosphate to l-butanol-extractable lipid. The liver microsomal glycerophosphate acyltransferase is inhibited by all proteases. The mitochondrial enzyme, on the other hand, is not inhibited by trypsin and chymotrypsin. Non-site-specific proteases, such as proteinase K and subtilisin, inhibit the mitochondrial enzyme. The degree of inhibition increases if the ionic strength of the incubation medium is lowered. Both microsomal and mitochondrial glycerophosphate acyltransferase have been only partially purified. In contrast, the Escherichia coli glycerophosphate acyltranferase has been completely purified, its properties studied, and the gene cloned and sequenced. The successful purification of the prokaryotic enzyme has been facilitated by massive overproduction of the enzyme owing to molecular cloning.

Effect of acylation stimulating protein on the triacylglycerol synthetic pathway of human adipose tissue

Acylation stimulating protein (ASP) is a 14 kDa plasma protein which causes in vitro triacylglycerol synthesis in human adipocytes and fibroblasts to increase substantially. ASP was found to stimulate human adipose tissue microsomal glycerophosphate acyltransferase and diacylglycerol acyltransferase activities by 23% and 90%, respectively. However, phosphatidate phosphohydrolase activity showed no increase in activity, nor did microsomal acyl-CoA synthetase activity. Moreover, ASP did not decrease the apparent Km of diacylglycerol acyltransferase (DGAT), but rather increased its apparent Vmax suggesting direct interaction of ASP with DGAT.

A lysophosphatidic acid-binding cytosolic protein stimulates mitochondrial glycerophosphate acyltransferase

Rat liver cytosolic fraction caused up to five fold stimulation of mitochondrial glycerophosphate acyltransferase apparently by removing the lysophosphatidic acid formed by the acyltransferase. When mitochondria were incubated with palmityl-CoA, [2-3H]-sn-glycerol 3-phosphate and the cytosolic fraction and the supernatant fluid of the incubated mixture was passed through a Sephadex G-100 column, labeled lysophosphatidic acid eluted in three peaks with Mrs (i) 60-70 kDa, (ii) 10-20 kDa, and (iii) less than 5 kDa. Proteins, responsible for binding of lysophosphatidic acid in peaks (i) and (ii), were purified to near homogeneity as judged by electrophoretic analysis. The lysophosphatidic acid binding protein in peak (i) appears to be serum albumin and peak (iii) represents largely unbound lysophosphatidic acid. The 15 kDa protein, purified from peak (ii), bound lysophosphatidic acid, stimulated the acyltransferase and export of lysophosphatidic acid from mitochondria.

Biosynthesis of phosphatidic acid by glycerophosphate acyltransferases in rat liver mitochondria and microsomes

The acyltransferases that catalyze the synthesis of phosphatidic acid from labelled sn-[14C]glycero-3-phosphate and fatty acyl carnitine or coenzyme A derivatives have been shown to be present in both isolated mitochondria and microsomes from rat liver. The major reaction product was phosphatidic acid in both subcellular fractions. A small quantity of lysophosphatidic acid and neutral lipids were produced as by-products. Divalent cations had significant effects on both mitochondrial and microsomal fractions in stimulating acylation using palmitoyl CoA, but not when palmitoyl carnitine was used as the acyl donor. Palmitoyl CoA and palmitoyl carnitine could be used for acylation by both mitochondria and microsomes. Mitochondria were more permeable to palmitoyl carnitine and readily used it as the substrate for acylation. On the other hand, microsomes yielded a better rate with palmitoyl CoA and the rate of acylation from palmitoyl carnitine in microsomes was correlated with the degree of mitochondrial contamination. The enzymes were partially purified from Triton X-100 extracts of subcellular fractions. Based on the differences of substrate utilization, products formed, divalent cation effects, molecular weights, and polarity, the mitochondrial and microsomal acyltransferases appeared to be different enzymes.

Effect of 3- and 4-thia-substituted fatty acids on glycerolipid metabolism and mitochondrial β-oxidation in rat liver

Treatment of normolipidemic rats by alkylthiopropionic acid (CETTD), resulted in a dose- and time-dependent increase in total dihydroxyacetone phosphate acyltransferase (DHAPAT) activity, in extent comparable to that of 3-thiadicarboxylic acid (BCMTD) and alkylthioacetic acid (CMTTD). Thus, in CETTD- and CMTTD-treated rats, the specific DHAPAT activity increased in the microsomal, peroxisomal and mitochondrial fractions. In contrast, repeated administration of the peroxisome proliferator, BCMTD, decreased the specific DHAPAT activity both in the peroxisomal fraction and in purified peroxisomes. A three-fold increase in specific activity was, however, revealed in the mitochondrial fraction. Whether the variation of the DHAPAT activity in the mitochondrial and microsomal fractions among the feeding groups can be explained by increased number of enlarged and small peroxisomes sedimenting in the fractions, are to be considered. Subcellular fractionation studies confirmed previous findings that rat liver glycerophosphate acyltransferase (GPAT) was located both in mitochondria and the microsomal fraction. BCMTD was considerably more potent than CMTTD in stimulating the microsomal and mitochondrial GPAT activities. Administration of CETTD marginally affected the isoenzymes of GPAT. Diacylglycerol acyltransferase (DGAT) activity was increased by 35% in BCMTD and CMTTD treated rats, but by administration of CETTD the enzyme activity was decreased by more than 80%. The acyl-CoA cholesterol acyltransferase (ACAT) activity was marginally affected in animals treated with BCMTD, CMTTD and CETTD. Thus, the results indicate that the initial steps in the synthesis of triacylglycerols and ether glycerolipids as well as the last step in triacylglycerol synthesis could not be identified as mediating the fat accumulation or the lowering of triacylglycerol content in liver of CETTD, or BCMTD and CMTTD treated rats. On the other hand, CMTTD increased the palmitoyl-CoA oxidation in mitochondria, and CETTD considerably inhibited the activity. Therefore, it is conceivable that the development of fatty liver with CETTD is mostly due to inhibition of mitochondrial β-oxidation.