Microsomal Phosphatidate Phosphohydrolase Activity Research Articles

Effect of hesperetin, a citrus flavonoid, on the liver triacylglycerol content and phosphatidate phosphohydrolase activity in orotic acid-fed rats.

The effect of dietary hesperetin on the hepatic lipid content and the enzyme activities involved in triacylglycerol (TG) synthesis in rats fed diets with or without 1% orotic acid (OA) was studied. Hepatic TG content was raised by approximately 5-fold after administration of OA for 10 days. The OA-feeding significantly increased the activity of hepatic microsomal phosphatidate phosphohydrolase (PAP), which is the rate-limiting enzyme for TG synthesis. Hepatic glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme activities were also increased. An addition of 1% hesperetin to the OA-supplemented diet resulted in the decrease of the hepatic TG content by 44% and of microsomal PAP activity. Dietary hesperetin alone neither affected liver TG content nor PAP activity significantly. OA-feeding caused an increased liver cholesterol level, whereas simultaneous addition of hesperetin and OA reduced its content to the control level. A slight reduction of hepatic cholesterol by hesperetin was also observed in the OA-free dietary group. The present study demonstrated that dietary hesperetin can reduce the hepatic TG accumulation induced by OA, and this was associated with the reduced activity of TG synthetic enzyme, PAP.

Dietary sunflower oil reduces plasma and liver triacylglycerols in fasting rats and is associated with decreased liver microsomal phosphatidate phosphohydrolase activity

Plasma and liver lipids were studied in male weanling rats fed diets containing moderate levels of fat (6% by weight) as sunflower oil (SF diet, rich in linoleic acid), salmon oil (SM diet, rich in long-chain n-3 fatty acids), or a blend of peanut and rapeseed oil (PR diet, rich in oleic acid). After nine weeks of feeding, the fasting plasma cholesterol concentrations were 49 and 24% lower in groups SM and SF, respectively, as compared to group PR. Both dietary salmon oil and sunflower oil lowered the triacylglycerol concentration of plasma and liver but, unexpectedly, the response was higher with sunflower oil. Indeed, in group SM the values were 15 and 30% lower in plasma and liver, whereas in group SF, they were 24 and 53% lower, respectively. As compared to group PR, liver triacylglycerols and microsomes contained 2.5- and 2.3-fold less oleic acid, respectively, in group SF, and they were 9.2- and 3.2-fold enriched in n-3 fatty acids, respectively, in group SM. The liver triacylglycerol concentrations were correlated with changes in the microsomal Mg(2+)-dependent phosphatidate phosphohydrolase activity (r = 0.47, P < 0.01). As oleic acid, unlike long-chain n-3 fatty acids, is considered to promote the triacylglycerol synthesis and secretion, our findings suggest that changes in the membrane fatty acid composition could affect the triacylglycerol content of liver and plasma. Moreover, the availability within the liver, of oleic acid, predominantly incorporated into triacylglycerols, might limit the triacylglycerol production in SF-fed rats.

Time Trends of Plasma Lipids and Enzymes Synthesizing Hepatic Triacylglycerol During Postpartum Development of Fatty Liver in Dairy Cows

We studied development of fatty liver in high producing dairy cows with free access to feed during the dry period and thus showed the combined effects of parturition and prepartum overfeeding. Postpartum liver triacylglycerol concentrations at 1 wk postpartum, as measured in liver biopsies, had increased more than 6-fold, which was preceded or accompanied by an increase in plasma NEFA concentrations. Concentrations of hepatic phospholipid changed only slightly. The amounts of total lipids in serum, very low density lipoproteins, and high density lipoproteins significantly decreased by .5 wk after parturition, and concentrations of high density lipoproteins rose steadily. The pattern was similar for concentrations of total cholesterol and phospholipid in serum. Total lipid concentrations in low density lipoproteins were not altered after parturition. The activity of microsomal phosphatidate phosphohydrolase in the liver showed a transient increase at .5 wk after calving, but activity of microsomal glycerolphosphate acyltransferase remained relatively constant. The activities of diacylglycerol acyltransferase had increased about twice at 1 wk after calving and remained at this high level until at least 4 wk after parturition. The rise in activity of diacylglycerol acyltransferase was probably a response to the extra influx of fatty acids to channel them into triacylglycerol. Activities of microsomal cholinephosphate cytidylyltransferase initially increased after calving and then decreased slightly. Activities of hepatic choline kinase had increased after calving. This study indicates that hepatic triacylglycerol accumulates because of the increased hepatic uptake of NEFA and the simultaneous increase in activity of diacylglycerol acyltransferase.

Docosahexaenoic acid shows no triglyceride-lowering effects but increases the peroxisomal fatty acid oxidation in liver of rats.

The effect of docosahexaenoic acid (DHA) on mitochondrial and peroxisomal fatty acid oxidation and on key enzymes in triglyceride metabolism was investigated in the liver of rats fed a standard diet, a cholesterol diet, and a pelleted chow diet. Unexpectedly, in all three rat models repeated administration of highly purified DHA (92% pure) at different doses and times, at a dose of 1000 mg/day per kg body weight, resulted in no significant decrease of hepatic and plasma concentration of triglycerides. The serum concentrations of cholesterol and phospholipids showed an increase in a time-dependent manner in rats fed the pelleted chow diet. The hepatic concentration of cholesterol was increased in rats fed the cholesterol diet and pelleted chow diet after administration of DHA compared to palmitic acid. In all rat models, treatment with DHA tended to increase the peroxisomal beta-oxidation. This was accompanied with a significant increase (1.5-fold) of fatty acyl-CoA oxidase activity. The mitochondrial fatty acid oxidation system and carnitine palmitoyl-transferase activity, however, were almost unchanged. Moreover, palmitoyl-CoA synthetase activity was increased, whereas the palmitoyl-CoA hydrolase activity was decreased. Neither microsomal phosphatidate phosphohydrolase activity nor cytosolic phosphatidate phosphohydrolase activity was affected by DHA feeding in the three rat models. Acyl-CoA:1,2-diacylglycerol acyltransferase activity was also unaffected. In contrast to docosahexanoic acid feeding, eicosapentaenoic acid (EPA) administration possessed a hypotriglyceridemic effect and resulted in an increase of mitochondrial and peroxisomal oxidation of fatty acids. Carnitine palmitoyltransferase activity was also stimulated. Phosphatidate phosphohydrolase activity was unaffected whereas diacylglycerol acyltransferase activity was increased by EPA treatment compared with palmitic acid feeding. The results indicate that docosahexaenoic acid, in contrast to eicosapentaenoic acid, does not inhibit the synthesis and secretion of triglycerides in the liver. In addition, the results emphasize the importance that stimulation of peroxisomal beta-oxidation by these n-3 fatty acids is not sufficient to decrease the serum levels of triglycerides. In addition, increased mitochondrial beta-oxidation of fatty acids and thereby decreased availability of nonesterified fatty acids may be a mechanism by which EPA inhibits triglyceride, and subsequently very low density lipoprotein-triglyceride, production. Whether DHA and EPA possess different metabolic properties should be considered.

Effects of age and diet on the lipid content and composition of gallbladder bile, liver and serum in laying strains of hen.

1. Changes in lipid content and composition of liver and bile during pre- and post-laying periods were investigated in hens of a laying strain. 2. The large increase in liver lipid concentration, particularly triacylglycerols, at the onset of laying was accompanied by the appearance of triacylglycerols in the bile. 3. Accumulation of triacylglycerol in the liver was increased by a diet containing maize and soyabean meal. This was associated with increases in the triacylglycerol concentration in both serum and bile. 4. Liver microsomal phosphatidate phosphohydrolase activity was positively correlated with liver triacylglycerol content in birds given the maize/soyabean meal diet. 5. The time course of triacylglycerol accumulation in the liver and associated changes in the bile triacylglycerol concentration in laying strain hens were different to those previously observed in broiler hens. 6. Over the lifetime of the bird, secretion of triacylglycerols into bile may play an important role in the prevention of fatty liver syndromes.

The molecular species of phosphatidic acid, diacylglycerol and phosphatidylcholine synthesized from sn-glycerol 3-phosphate in rat lung microsomes

The species pattern of phosphatidic acid, diacylglycerol and phosphatidylcholine synthesized from [ 14C]glycerol 3-phosphate was measured using a newly developed HPLC technique yielding 13 molecular species. A direct comparison of these species patterns presupposes determination of the lipolytic activity of lung microsomes. The lipolytic activity was quantitatively determined by measuring the changes of the endogenous concentration of diacylglycerol, triacylglycerol and free fatty acids. The species pattern of endogenous diacylglycerol measured in the time-course of lipolysis did not show any changes up to an incubation period of 20 min, suggesting that the lipolytic activity showed only a very low selectivity for individual substrate species. Diisopropylfluorophosphate (5 μmol/mg microsomal protein) strongly decreased the lipolytic activities as well as the microsomal phosphatidate phosphohydrolase activity, as measured by means of exogenous phosphatidic acid, and also the generation of phosphatidic acid from [ 14C]glycerol 3-phosphate. In lung microsomes, labeled phosphatidic acid and diacylglycerols were synthesized from the endogenous free fatty acids and sn-[ 14C)glycerol 3-phosphate, which had previously been added. By addition of CDPcholine to the prelabeled microsomes the synthesis of phosphatidylcholine was measured. After hydrolysis of phosphatidic acid and phosphatidylcholine with cytoplasmatic phosphatidate phosphohydrolase or phospholipase C, respectively, the de novo synthesized species patterns of these two lipids and of the diacylglycerol were determined. Comparison of the species pattern of de novo synthesized phosphatidic acid with that of diacylglycerol largely showed the same distribution of radioactivity among the individual species, except that the relative proportion of label was higher in the 16:0 16:0 and 16:0 18:0 species of phosphatidic acid and lower in the 16:0 20:4 and 18:0 20:4 species than in the corresponding species of diacylglycerol. The species pattern of de novo-synthesized diacylglycerol showed no differences from that of the phosphatidylcholine synthesized from it. From this result we concluded that the cholinephosphotransferase of lung microsomes is nonselective for individual species of the diacylglycerol substrate. The 16:0 18:1 and 16:0 18:2 species of phosphatidic acid, diacylglycerol and phosphatidylcholine showed a higher synthesis rate than their 18:0 counterparts, whereas the 16:0 or 18:0 analogues of species containing 20:4 and 22:6 fatty acids showed nearly the same synthesis rates. The comparison of the species pattern of the endogenous phosphatidylcholine with its species pattern resulting from de novo synthesis showed that the relative proportion of the 16:0 16:0 species of endogenous phosphatidylcholine is twice as high as the relative proportion of this species of the de novo-synthesized phosphatidylcholine. From this difference we calculated the relative contribution of the de novo synthesis in the total production of disaturated phosphatidylcholine to be 50%.

The acute effects of streptozotocin-induced diabetes on rat liver glycerolipid biosynthesis

Streptozotocin-induced diabetes produced a significant rise in rat serum and liver triacylglycerol content and hepatic triacylglycerol biosynthesis measured in vivo. Microsomes, isolated from the livers of streptozotocin-exposed animals (2–72 h), exhibited an increased capacity to incorporate sn-[1,3- 14C]glycerol 3-phosphate into neutral lipid (diacylglycerol and triacylglycerol) in the presence of ATP, CoA and palmitate. The streptozotocin-induced elevation of microsomal neutral lipid production was accompanied by a corresponding rise in the activity of microsomal phosphatidate phosphohydrolase (4-fold after 72 h of streptozotocin exposure). Diabetic-dependent increases in acylglycerol formation, phosphatidate phosphohydrolase activity and serum triacylglycerol and fatty acid levels were reversed by administering insulin (10 units protamine zinc/kg) at 16-h intervals (three separate doses) beginning 24 h after streptozotocin exposure. However, the diabetic-related rise in hepatic triacylglycerol content was only partially corrected by insulin administration. Streptozotocin-related increases in liver triacylglycerol biosynthesis and phosphatidate phosphohydrolase activity were associated with alterations in plasma factors, since homogenates of hepatocyte monolayers exposed (18 h) to plasma isolated from diabetic (72 h exposure to streptozotocin) animals exhibit an increased capacity to incorporate sn-[1,3- 14C]glycerol 3-phosphate into triacylglycerol compared to homogenates of cells exposed to plasma from control (non-fasted) animals. The importance of these plasma factors in altering hepatic acylglycerol formation was also supported by the observation that hepatocyte monolayers exposed to a mixture of plasma isolated from normal (non-fasted) animals and plasma components elevated in diabetes (glucagon, glucose, oleate and ketones) showed increases in triacylglycerol formation which were similar to those produced by exposure to diabetic plasma. Additional studies demonstrated that fatty acids (oleate) appeared to be the agent primarily responsible for the diabetic plasma-induced rise in monolayer triacylglycerol biosynthesis and phosphatidate phosphohydrolase activity.

Effect of ethanol on hepatic phosphatidate phosphohydrolase: Dose-dependent enzyme induction and its abolition by adrenalectomy and pyrazole treatment

The role of glucocorticoids and metabolites of hepatic intermediary metabolism as possible mediators of the ethanol-induced stimulation of hepatic phosphatidate phosphohydrolase activity was studied in intact, adrenalectomized, and pyrazole-treated rats. The ethanol-induced increase in the soluble and microsomal phosphatidate phosphohydrolase activity was dependent on the amount of ethanol administered up to a dose of 3 to 4 g/kg body wt, suggesting that the ethanol effect is largely due to an unspecific stress effect of ethanol, and possibly mediated by stress homones such as glucocorticoids. Adrenalectomy abolished the ethanol-induced increase in microsomal phosphatidate phosphohydrolase activity, but did not modify the effect of ethanol on the soluble enzyme. Pyrazole abolished the ethanol-induced increase in soluble phosphatidate phosphohydrolase activity in both intact and adrenalectomized rats, but did not modify the induction of the membrane-bound enzyme in intact rats. The amount of triacylglycerol accumulating in the liver after ethanol administration correlated better with the soluble than with the membrane-bound phosphatidate phosphohydrolase activity. The results suggest that the hepatic phosphatidate phosphohydrolase activities are subject to separate regulatory mechanisms, involving hormones and also low molecular weight metabolites. The soluble and membrane-bound enzymes may have different tasks in hepatic glycerolipid metabolism.

Enhancement of microsomal phosphatidate phosphohydrolase and diacylglycerol acyltransferase activity by insulin during growth of rat adipocyte precursors in culture

The availability of a propagating adipocyte precursor culture system has provided the opportunity to study biochemical processes under conditions in which any known interacting influences are controlled. We have studied the activity of various triacylglycerol-biosynthetic enzymes during maturation of rat epididymal adipocyte precursors and any possible effect of insulin on enzyme activity. At certain times in culture, the specific activity of microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) and diacylglycerol acyltransferase (EC 2.3.1.20) is significantly enhanced in cells grown in the presence of added insulin. Under the culture conditions used in this study, the adipocyte precursors acquire several small lipid inclusions and become rounder, but do not assume the signet-ring appearance of mature fat cells during the first 2 weeks of monolayer confluence. Consequently, the effects of the hormone on enzyme activity become evident prior to complete morphological maturation. Phosphatidate phosphohydrolase is believed to be a rate-controlling enzyme in triacylglycerol synthesis in adipose tissue and liver. The fact that the adipocyte precursor microsomal, rather than cytosolic, phosphohydrolase is influenced by insulin suggests that the membrane-bound enzyme is the regulatory phosphohydrolase in intact cells. The enhancement of diacylglycerol acyltransferase activity may be of significance in the reesterification of fatty acids with diacylglycerols, a reaction that by passes the phosphohydrolase step. Thus, in addition to the well-known mechanisms by which insulin promotes triacylglycerol accretion in adipocytes and their precursors, the hormone significantly enhances the specific activity of critical enzymes of triacylglycerol synthesis.

Phosphatidate Phosphohydrolase and the Regulation of Glycerolipid Biosynthesis

Phosphatidate is an intermediate in the biosynthesis of diacylglycerol, triacylglycerol, phosphatidylethanolamine, phosphatidylcholine and CDP-diacylglycerol. In addition, phosphatidate may be subject to degradation by particulate phospholipases. The factors regulating the disposition of phosphatidate have interested numerous investigators. We have studied the reactions of phosphatidate metabolism in several tissues and under various environmental conditions. Phosphatidate phosphohydrolase activity is found primarily in the microsomal and supernatant fractions of liver, adipose tissue and intestine. Presumably the physiologically important substrate for this reaction is the particulate-bound phosphatidate and methods for the preparation of this substrate have been developed (Fallon et al., 1975). Radioactively labelled phosphatidate bound to microsomal particles is prepared from sn-[1 ,3-14C]glycerol 3-phosphate. A wide range of microsomal phosphatidate concentrations may be prepared by this technique, although the usual microsomal concentration is approx. 5 nmol/mg of microsomal protein. This substrate has been used in most of our studies of phosphatidate phosphohydrolase, CDP-diacylglycerol formation and phospholipase A. There are several lines of evidence which support an important role for phosphatidate phosphohydrolase in the regulation of neutral-lipid formation by liver or adipose-tissue particulate preparations. (1) The reaction rate of phosphatidate phosphohydrolase under optimum conditions in vitro is the lowest in the overall synthetic pathway in liver microsomal fractions (Lamb & Fallon, 1974a). The rate of phosphohydrolase activity was determined as 0.6-0.8 nmol/min per mg of microsomal protein when radiolabelled particulate-bound phosphatidate was used as substrate. The overall rate of glycerolipid formation by these microsomal preparations was approx. l.Onmol/min per mg of microsomal protein. The optimum rate of microsomal sn-glycerol 3-phosphate acyltransferase was 5-7-fold higher. In the presence of added rat liver supernatant fraction the rate of microsomal phosphatidate phosphohydrolase is increased 5-20-fold. However, at ratios of microsomal protein to supernatant protein estimated for the intact cell, the increase in phosphohydrolase activity is much more modest. (2) Induction of increased liver triacylglycerol formation by administration of a high-fructose diet is accompanied by an equivalent rise in the activity of the phosphohydrolase, as shown in Table 1. These changes in lipid metabolism occur simultaneously over a 10-day period (Lamb & Fallon, 19746). Further, the phosphatidate content of liver microsomal preparation is diminished by approx. 15 %, and diacylglycerol increased by approx. 90 % under these dietary conditions. (3) The increase in liver triacylglycerol content after partial hepatectomy is accompanied by a corresponding increase in phosphatidate phosphohydrolase activity (Mangiapane et al., 1973). (4) Several drugs which cause substantial inhibition of phosphatidate phosphohydrolase activity in vitro also result in inhibition of neutral-lipid formation when administered to the intact animal (Brindley & Bowley, 1975; Lamb et al., 1977). A series of 1,3-bis(substituted phenoxy)-2-propanones inhibited microsomal phosphatidate phosphohydrolase activity as measured in vitro. The inhibition ranged from 8 to 92% total activity. In animals fed on these agents in laboratory chow the incorporation of [14C]glycerol into hepatic triacylglycerol was decreased by 3-75 %. Agents which caused a 50 % or greater decrease in phosphatidate phosphohydrolase activity in vitro also lowered

Metabolism of phospholipids IX. Phosphatidate phosphohydrolase in rat liver

1. 1. Rat-liver homogenates were fractionated into mitochondrial, lysosomal, microsomal, and particle-free supernatant fractions which were characterised by specific marker enzymes, and the intracellular distribution of phosphatidate phosphohydrolase ( l-α-phosphatidate phosphohydrolase, EC 3.1.3.4) was studied. 2. 2. The lysosomal fraction had the highest specific activity with respect to phosphatidate phosphohydrolase activity but the mitochondrial and microsomal fractions contained the relatively highest proportions of this enzyme. It was concluded that phosphatidate phosphohydrolase is a true constituent of all three particulate sub-cellular fractions, with some activity occurring in the supernatant. 3. 3. Repeated extraction of the three particulate subcellular fractions by freezing and thawing in 0.3 M sucrose solubilised 60, 25 and 16% of the mitochondrial, lysosomal, and microsomal phosphatidate phosphohydrolase activity, respectively. 4. 4. Investigation of Michaelis constants, pH optima, and the effect of detergents and cations revealed significant differences between the mitochondrial enzyme which had been extracted by freezing and thawing and that which remained insoluble. 5. 5. Examination of an extract obtained from a mitochondrial preparation by repeated freezing and thawing showed that of a number of important enzymes in glyceride and phospholipid biosynthesis only phosphatidate phosphohydrolase was extracted.