Subcellular distribution and some properties of N-ethylmaleimide-sensitive and-insensitive forms of glycerol phosphate acyltransferase in rat adipocytes.

1. Glycerol phosphate acyltransferase (GPAT) activities were measured in subcellular fractions obtained from rat epididymal adipocytes. These contained both N-ethylmaleimide-sensitive and N-ethylmaleimide-insensitive forms of the enzyme. 2. As shown by parallel measurements of marker enzymes, N-ethylmaleimide-insensitive GPAT is most probably a mitochondrial activity, whereas N-ethylmaleimide-sensitive GPAT is the microsomal enzyme. 3. Subcellular distributions are also reported for dihydroxyacetone phosphate acyltransferase (DHAPAT) (assayed with and without N-ethylmaleimide), monoacylglycerol phosphate acyltransferase (MGPAT) and Mg2+-dependent and Mg2+-independent forms of phosphatidate phosphohydrolase (PPH).     

A trypsin-sensitive, heat-labile, N-ethylmaleimide-sensitive factor in adipocyte post-microsomal supernatant which affects the assay of adipocyte glycerol phosphate acyltransferase activities.

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.     

Identification of a new glycerol-3-phosphate acyltransferase isoenzyme, mtGPAT2, in mitochondria

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step of glycerolipid synthesis. Two distinct GPAT isoenzymes had been identified in mammalian tissues, an N-ethylmaleimide (NEM)-sensitive isoform in the endoplasmic reticulum membrane (microsomal GPAT) and an NEM-resistant form in the outer mitochondrial membrane (mtGPAT). Although only mtGPAT has been cloned, the microsomal and mitochondrial GPAT isoforms can be distinguished, because they differ in acyl-CoA substrate preference, sensitivity to inhibition by dihydroxyacetone phosphate and polymixin B, temperature sensitivity, and ability to be activated by acetone. The preponderance of evidence supports a role for mtGPAT in synthesizing the precursors for triacylglycerol synthesis. In mtGPAT(-/-) mice, PCR genotyping and Northern analysis showed successful knockout of mtGPAT; however, we detected a novel NEM-sensitive GPAT activity in mitochondrial fractions and an anti-mtGPAT immunoreactive protein in liver mitochondria, but not in microsomes. Rigorous analysis using two-dimensional gel electrophoresis revealed that the anti-mtGPAT immunoreactive proteins in wild type and mtGPAT(-/-) liver mitochondria have different isoelectric points. These results suggested the presence of a second GPAT in liver mitochondria from mtGPAT(-/-) mice. Characterization of this GPAT activity in liver from mtGPAT null mice showed that, unlike the mtGPAT activity in wild type samples, activity in mtGPAT knockout mitochondria did not prefer palmitoyl-CoA, was sensitive to inactivation by NEM, was inhibited by dihydroxyacetone phosphate and polymixin B, was temperature-sensitive, and was not activated by acetone. We conclude that a novel GPAT (mtGPAT2) with antigenic epitopes similar to those of mtGPAT is detectable in mitochondria from the livers of mtGPAT(-/-) mice.     

Aging and acyl-CoA binding protein alter mitochondrial glycerol-3-phosphate acyltransferase activity

It is well known that cellular function declines with age. Since phosphatidic acid (PtdOH) biosynthesis is central to the generation of membrane phospholipids, the hypothesis that aging decreases PtdOH biosynthesis was tested. Glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LAT) activities were examined in isolated mitochondria and microsomes from young and old rat liver. The results show that mitochondrial GPAT preference for palmitoyl-CoA over oleoyl-CoA was only observed if albumin or acyl-CoA binding protein (ACBP) were present in the assay in the young rats. Furthermore, mitochondrial GPAT activity was significantly reduced in the presence of albumin and ACBP in aged mitochondria using palmitoyl-CoA as the substrate. These data show, for the first time, that mitochondrial GPAT acyl-CoA preference is due to the presence of a protein that binds acyl-CoAs, not the enzyme itself, and that aging significantly reduces mitochondrial GPAT activity.     

A study of glycerolphosphate acyltransferase in rat liver mitochondria-submitochondrial localization and some properties of the solubilized enzyme

• 1.1. Glycerolphosphate acyltransferase (GPAT) was solubilized from the rat liver mitochondrial membranes using sodium cholate. Dithiothreitol was necessary to stabilize the solubilized enzyme on storage.
• 2.2. Unlike the enzyme in situ in mitochondrial membranes, the solubilized mitochondrial GPAT was susceptible to inhibition by N-ethylmaleimide; a property more characteristic of the distinct microsomal form of GPAT.
• 3.3. Solubilized mitochondrial GPAT retained its very high preference for saturated acyl-CoA substrate (palmitoyl-CoA) and had no activity whatever with any tested concentration of the unsaturated substrate oleoyl-CoA.
• 4.4. Solubilization increased the affinity of mitochondrial GPAT for palmitoyl-CoA whilst decreasing the K_m for glycerol phosphate.
• 5.5. After separation of liver mitochondrial outer and inner membranes and estimation of cross-contamination by appropriate markers it was concluded that the mitochondrial inner membrane contains significant GPAT activity. This was established with preparations from fed, 48 hr-starved and streptozotocin-diabetic rats.

     

Participation of peroxisomes in lipid biosynthesis in the harderian gland of guinea pig.

Peroxisomal enzyme activities in the guinea-pig harderian gland, which has a unique lipid composition, were studied. Activities of catalase, acyl-CoA oxidase and the cyanide-insensitive acyl-CoA beta-oxidation system in this tissue were comparable with those in rat liver. The activities of dihydroxyacetone phosphate acyltransferase (DHAPAT, EC 2.3.1.42) and alkyl-DHAP synthase (EC 2.5.1.26) were appreciable, and the distributions of both activities were consistent with that of sedimentable catalase activity. Glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15), which is localized in both microsomes (microsomal fractions) and mitochondria in the rat liver, was a peroxisomal enzyme in the harderian gland, though the activity was only about one-tenth of the DHAPAT activity. These enzymes had different pH profiles and substrate specificity. The existence of high activities of enzymes of the acyl-DHAP pathway in peroxisomes suggests the physiological significance of peroxisomes in the biosynthesis of glycerol ether phospholipid and 1-alkyl-2,3-diacylglycerol in the guinea-pig harderian gland.     

Regulation by noradrenaline of the mitochondrial and microsomal forms of glycerol phosphate acyltransferase in rat adipocytes.

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.     

Evidence for the existence of isoenzymes of glycerol phosphate acyltransferase.

Subcellular-fractionation studies confirmed previous findings that rat liver glycerol phosphate acyltransferase was located in both mitochondria and the microsomal fraction. Studies of the two activities revealed several differences between them. The mitochondrial enzyme had a lower Km for sn-glycerol 3-phosphate and was more resistant to heat inactivation than was the microsomal enzyme. Some preparations of the mitochondrial enzyme were inhibited by high concentrations of glycerol phosphate. The mitochondrial enzyme was not inactivated by thiol-group reagents, whereas the microsomal enzyme was very rapidly inactivated by these compounds. However, the microsomal enzyme could be specifically protected against this inactivation by low concentrations of palmitoyl-CoA. The results indicate the existence of distinct isoenzymes of glycerol phosphate acyltransferase with different intracellular locations.     

The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae.

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 1.1.1.101) 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.     

Comparison of triacylglycerol synthesis in rat brown and white adipocytes. Effects of hypothyroidism and streptozotocin-diabetes on enzyme activities and metabolic fluxes.

1. Adipocytes were isolated from the interscapular brown fat and the epididymal white fat of normal, streptozotocin-diabetic and hypothyroid rats. 2. Measurements were made of the maximum rate of triacylglycerol synthesis by monitoring the incorporation of [U-14C]glucose into acylglycerol glycerol in the presence of palmitate (1 mM) and insulin (4 nM) and of the activities of the following triacylglycerol-synthesizing enzymes: fatty acyl-CoA synthetase (FAS), mitochondrial and microsomal forms of glycerolphosphate acyltransferase (GPAT), dihydroxyacetonephosphate acyltransferase (DHAPAT), monoacylglycerol phosphate acyltransferase (MGPAT), Mg2+-dependent phosphatidate phosphohydrolase (PPH) and diacylglycerol acyltransferase (DGAT). 3. FAS activity in brown adipocytes was predominantly localized in the mitochondrial fraction, whereas a microsomal localization of this enzyme predominated in white adipocytes. Subcellular distributions of the other enzyme activities in brown adipocytes were similar to those shown previously with white adipocytes [Saggerson, Carpenter, Cheng & Sooranna (1980) Biochem. J. 190, 183-189]. 4. Relative to cell DNA, brown adipocytes had lower activities of triacylglycerol-synthesizing enzymes and showed lower rates of metabolic flux into acylglycerols than did white adipocytes isolated from the same animals. 5. Diabetes decreased both metabolic flux into acylglycerols and the activities of triacylglycerol-synthesizing enzymes in white adipocytes. By contrast, although diabetes decreased metabolic flux into brown-adipocyte acylglycerols by 80%, there were no decreases in the activities of triacylglycerol-synthesizing enzymes, and the activity of PPH was significantly increased. 6. Hypothyroidism increased metabolic flux into acylglycerols in both cell types, and increased activities of all triacylglycerol-synthesizing enzymes in brown adipocytes. By contrast, in white adipocytes, although hypothyroidism increased the activities of FAS, microsomal GPAT and DGAT, this condition decreased the activities of mitochondrial GPAT and PPH. 7. It was calculated that the maximum capabilities for fatty acid oxidation and esterification are approximately equal in brown adipocytes. In white adipocytes esterification is predominant by approx. 100-fold. 8. Diabetes almost abolished incorporation of [U-14C]glucose into fatty acids in both adipocyte types. Hypothyroidism increased fatty acid synthesis in white and brown adipocytes by 50% and 1000% respectively.     

Studies of rat adipose-tissue microsomal glycerol phosphate acyltransferase.

Incubation of rat adipose-tissue microsomal fractions with iodoacetate caused an inactivation of glycerol phosphate acyltransferase that could be prevented by the presence of palmitoyl-CoA. A microsomal protein of subunit Mr 54 000 was found to react with radioactively labelled iodoacetate in the absence, but not in the presence, of palmitoyl-CoA. It is suggested that this protein is a component of glycerol phosphate acyltransferase. Incubation of rat adipose-tissue microsomal fractions with the catalytic subunit of cyclic AMP-dependent protein kinase, ATP and Mg2+ caused an inactivation of glycerol phosphate acyltransferase whose magnitude depended on the conditions used for assay of the acyltransferase. Rat adipose tissue microsomal proteins were phosphorylated by using protein kinase and [gamma-32P]ATP. One of the phosphorylated proteins was very similar, but not identical, in mobility to the Mr-54 000 protein labelled by iodoacetate. In contrast with a previous report [Sooranna & Saggerson (1976) FEBS Lett. 64, 36-39], no changes could be detected in the activity of glycerol phosphate acyltransferase in adipocytes treated with adrenaline. Adipocytes were labelled with [32P]Pi and treated with adrenaline, but no 32P was incorporated into the Mr-54000 protein labelled by iodoacetate. The results suggest that the activity of adipose-tissue microsomal glycerol phosphate acyltransferase is not directly controlled by phosphorylation.     

Rapid stimulation of liver palmitoyl-CoA synthetase, carnitine palmitoyltransferase and glycerophosphate acyltransferase compared to peroxisomal beta-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 beta-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 beta-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 beta-oxidation is proceeding before the enhancement of peroxisomal beta-oxidation.     

Carnitine acyltransferase and acyl-coenzyme A hydrolase activities in human liver. Quantitative analysis of their subcellular localization.

The subcellular localizations of carnitine acyltransferase and acyl-CoA hydrolase activities with different chain-length substrates were quantitatively evaluated in human liver by fractionation of total homogenates in metrizamide density gradients and by differential centrifugation. Peroxisomes were found to contain 8-37% of the liver acyltransferase activity, the relative amount depending on the chain length of the substrate. The remaining activity was ascribed to mitochondria, except for carnitine octanoyltransferase, for which 25% of the activity was present in microsomal fractions. In contrast with rat liver, where the activity in peroxisomes is very low or absent, human liver peroxisomes contain about 20% of the carnitine palmitoyltransferase. Short-chain acyl-CoA hydrolase activity was found to be localized mainly in the mitochondrial and soluble compartments, whereas the long-chain activity was present in both microsomal fractions and the soluble compartment. Particle-bound acyl-CoA hydrolase activity for medium-chain substrates exhibited an intermediate distribution, in mitochondria and microsomal fractions, with 30-40% of the activity in the soluble fraction. No acyl-CoA hydrolase activity appears to be present in human liver peroxisomes.     

Induction of the peroxisomal glycerolipid-synthesizing enzymes during differentiation of 3T3-L1 adipocytes. Role in triacylglycerol synthesis

The glycerophosphate backbone for triglyceride synthesis is commonly believed to be created through the conversion of dihydroxyacetone phosphate (DHAP) by glycerophosphate dehydrogenase (GPD) to sn-glycerol 3-phosphate (GP), which is then converted by glycerophosphate acyltransferase (GPAT) to 1-acyl-GP. Consistent with this, GPD and GPAT are highly induced during differentiation of mouse 3T3-L1 preadipocytes. While the acyl dihydroxyacetone phosphate (acyl-DHAP) pathway for glycerolipid synthesis is commonly believed to be involved only in glycerol ether lipid synthesis, we report here that during conversion of 3T3-L1 preadipocytes to adipocytes, the specific activity of peroxisomal DHAP acyltransferase (DHAPAT) is increased by 9-fold in 6 days, while acyl-DHAP:NADPH reductase is induced by 5-fold. A parallel increase in the catalase (the peroxisomal marker enzyme) activity is also seen. In contrast, the specific activity of alkyl-DHAP synthase, the enzyme catalyzing the synthesis of the ether bond, is decreased by 60% during the same period. Unlike microsomal GPAT, the induced DHAPAT is found to have high activity at pH 5.5 and is resistant to inhibition by sulfhydryl agents, heat, and proteolysis. On subcellular fractionation, DHAPAT is found to be associated with microperoxisomes whereas GPAT activity is mainly present in microsomes. Northern blot analyses reveal that induction of DHAPAT can be largely explained through increases in DHAPAT mRNA. A comparison of microsomal and peroxisomal glycerolipid synthetic pathways, using D-[3-(3)H, U-(14)C]glucose as the precursor of the lipid glycerol backbone shows that about 40-50% of triglyceride is synthesized via the acyl-DHAP pathway. These results indicate that the acyl-DHAP pathway is important not only for the synthesis of ether lipids, but also for the synthesis of triacylglycerol and other non-ether glycerolipids.     

Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2)

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.     

Dual localization of long-chain acyl-CoA hydrolase in rat liver: one in the microsomes and one in the mitochondrial matrix.

Subcellular fractionation studies of rat liver localized the activity of palmitoyl-L-carnitine hydrolase to the microsomal fraction whereas palmitoyl-CoA hydrolase activity was found both in the microsomal fraction and in mitochrondria. An unusual biphasic sataration curve for palmitoyl-CoA was observed when intact mitochondrial hydrolase activity. Disruption of the mitochondrial structure doubled the palmitoyl-CoA hydrolysis. Discontinuous sucrose gradient centrifugation and digitonin fractionation of rat liver mitochondria demonstrated that a palmitoyl-CoA hydrolase was associated with the matrix fraction. Pure matrix and microsomal fractions showed that the two hydrolase activities were differently affected by the presence of divalent cations. Both the specific activity and the saturation concentration of palmitoyl-CoA were higher for the microsomal enzyme than for the matrix-associated enzyme.     

Co-induction by peroxisome proliferators of microsomal 1-acylglycerophosphocholine acyltransferase with peroxisomal beta-oxidation in rat liver

Administration of clofibric acid, 2,2'-(decamethylenedithio)diethanol, di(2-ethylhexyl)phthalate or perfluorooctanoic acid to male rates increased markedly microsomal 1-acylglycerophosphocholine (a-acyl-GPC) acyltransferase in a dose-dependent manner in liver. Simultaneous administration of actinomycin D or cycloheximide completely abolished the increase in the enzyme activity. The treatment of rats with clofibric acid did not affect the rate of decay of 1-acyl-GPC acyltransferase. Regardless of a great difference in the chemical structures of the peroxisome proliferators, high correlation was observed between the induced activities of microsomal 1-acyl-GPC acyltransferase and peroxisomal beta-oxidation. Stearoyl-CoA desaturase was induced by peroxisome proliferators in a dose-dependent manner; nevertheless, high correlation was not seen between the induced activities of desaturase and peroxisomal beta-oxidation. Hormonal (adrenalectomy, diabetes, hyperthyroidism and hypothyroidism) and nutritional (starvation, starvation-refeeding, fat-free diet feeding and high-fat diet feeding) alterations hardly affected the activity of 1-acyl-GPC acyltransferase. The present results indicate that microsomal 1-acyl-GPC acyltransferase is a useful parameter responsive to the challenges by peroxisome proliferators and suggest that a similar regulatory mechanism operates for the inductions of microsomal 1-acyl-GPC acyltransferase and peroxisomal beta-oxidation.     

Stimulation of rat liver glycerol-3-phosphate acyltransferase activity by acid- and heat-stable low-molecular-weight substances from skeletal muscle of rats treated with insulin

Experiments were conducted to examine a possible mechanism of activation of rat liver microsomal glycerol-3-phosphate acyltransferase (GPAT) by insulin. Fractions of Mr 1100 were prepared from hind-limb muscles of rats, which had been given intravenous injections of insulin or saline, by a procedure which involved acidification (pH 3.8) and heating (100 degrees C), followed by chromatography on Sephadex G-25 in 50 mM formic acid. These fractions were shown to modify the activity of microsomal GPAT from the livers of fed rats which had not been treated with insulin. The difference in GPAT activity between microsomes supplemented with the Mr 1100 material and those treated with an equal volume of 50 mM formic acid from before the void volume of the column was determined. Relative to the formic acid control, the Mr 1100 material from saline-treated rats decreased GPAT activity, whereas Mr 1100 material from insulin-treated rats increased GPAT activity and the difference of 0.64 nmol/min/mg microsomal protein was significant (P less than 0.01). Fractions of approximately 3000 Da were found to behave in a similar manner and caused a significant (P less than 0.01) increase in GPAT activity of 0.46 nmol/min/mg microsomal protein. These substances, which stimulate GPAT activity, may be related to the putative insulin mediator substance.     

Changes in activities of some enzymes of glycerolipid synthesis in brown adipose tissue of cold-acclimated rats.

1. Measurements were made of the activities of the following enzymes of glycerolipid synthesis in homogenates of interscapsular brown adipose tissue obtained from rats subjected to a 4 degrees C environment for time periods of 6 h up to 12 days: fatty acyl-CoA synthetase (FAS), mitochondrial and microsomal forms of glycerolphosphate acyltransferase (GPAT), monoacylglycerolphosphate acyltransferase (MGPAT) and Mg2+-dependent phosphatidate phosphohydrolase (PPH). 2. Relative to tissue DNA content, the activities of mitochondrial GPAT, MGPAT and Mg2+-dependent PPH were significantly increased after 1 day of exposure to cold, and continued to increase thereafter. By contrast, FAS and microsomal GPAT activities were unchanged relative to tissue DNA. 3. The time profile of the increase in MGPAT activity correlated well with a concomitant increase in the microsomal marker NADP+-cytochrome c reductase. Changes in mitochondrial GPAT and in Mg2+-dependent PPH activities were larger in amplitude than that of MGPAT. 4. It is proposed that these selective changes in enzyme activity may be associated with the onset of brown-adipose-tissue hyperplasia or possibly with an increase in triacylglycerol synthesis during cold-acclimation.     

Phospholipid composition and acyltransferase activity of lamellar bodies isolated from rat lung.

The role of the lamellar body of the type II pneumocyte in the synthesis and storage of the phospholipids of the surfactant lipoprotein lining the alveolar surface has been investigated. Electron microscopy has been used to establish the purity of the isolated lamellar body, microsomal, and mitochondrial fractions. Additional proof of lamellar body purity was obtained by enzyme marker studies. The phospholipid:protein ratio of each of the above fractions was determined as well as that of surfactant lipoprotein isolated from rat lung. Lamellar body phospholipid:protein ratio was highest, 3.7 μmol of lipid phosphorus/mg of lung protein. The phospholipid composition of the lamellar body fraction was found to be similar to that of the isolated surfactant lipoprotein. Lamellar body phosphatidylcholine and phosphatidylglycerol each contained over 90% saturated fatty acids. The lamellar body fraction was found to possess significant acyltransferase activity between [1-¹⁴C]palmitoyl-CoA and phosphatidylcholine. This activity was somewhat higher than in the microsomal fraction and much greater than in the mitochondrial fraction. The activity in all fractions was stimulated by Ca²⁺ and Mg²⁺. [1-¹⁴C]oleoyl-CoA did not serve as an effective acyl donor. When 1-palmitoyl-2-lysophosphatidylcholine was used as the acceptor molecule and [1-¹⁴C]palmitoyl-CoA the donor, acyltransferase activity was increased over that found with phosphatidylcholine as donor in all fractions. The microsomal fraction had the greatest activity and the lamellar body fraction the least. The data obtained support the hypothesis that the lamellar body is involved in the synthesis and storage of the phospholipids of the surfactant lipoprotein complex.