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Compared with triacylglycerol (TAG), dietary 1,3-diacylglycerol (1,3-DAG) is associated with reduced serum lipid and glucose levels. We investigated the metabolism of 1,3-DAG by assaying its intermediate metabolites during digestion and absorption in the rat small intestine. After gavage with TAG emulsion, TAG was digested mainly to 2-monoacylglycerol (2-MAG) and unesterified fatty acid (FFA) in the rat small intestinal lumen. 2-MAG was directly absorbed into the small intestinal epithelial cells and esterified to 1,2(2,3)-DAG, and further esterified to TAG. After gavage with 1,3-DAG emulsion, 1,3-DAG was digested mainly to 1(3)-MAG and FFA in the rat small intestinal lumen with subsequent significant increase of 1-MAG and 1,3-DAG concentrations in small intestinal mucosal epithelial cells, and the 2-MAG, 1,2(2,3)-DAG, and TAG concentrations in mucosal epithelial cells were not significantly different after 1,3-DAG than after TAG gavage, suggesting that the metabolic pathway of 1,3-DAG is different from that of TAG. In intestinal mucosal epithelial cells, we further assayed enzyme levels and gene expression of proteins in the phosphatidic acid (PtdOH) pathway. The glycerol kinase, phosphatidate phosphatase, and diacylglycerol acyltransferase-2 expression and the relative expression of mRNA of enzymes were significantly increased in the 1,3-DAG group compared with the TAG group, suggesting that TAG synthesis from dietary 1,3-DAG was mainly via PtdOH pathways, which may partially account for the effect of dietary DAG on postprandial serum TAG.

Lipid kinases and phosphatases play essential roles in signal transduction processes involved in cytoskeletal rearrangement, membrane trafficking, and cellular differentiation. Phosphatidic acid (PtdOH) is an important mediator lipid in eukaryotic cells, but little is known regarding its regulation in the parasite Trypanosoma cruzi, an agent of Chagas disease. In order to clarify the relationship between PtdOH metabolism and developmental stages of T. cruzi, epimastigotes in culture were subjected to hyperosmotic stress (~1,000 mOsm/L), mimicking the environment in the rectum of vector triatomine bugs. These experimental conditions resulted in differentiation to an intermediate form between epimastigotes and trypomastigotes. Morphological changes of epimastigotes were correlated with an increase in PtdOH mass accomplished by increased enzyme activity of diacylglycerol kinase (DAGK, E.C. 2.7.1.107) and concomitant decreased activity of phosphatidate phosphatases type 1 and type 2 (PAP1, PAP2, E.C. 3.1.3.4). Our results indicate progressive increases of PtdOH levels during the differentiation process, and suggest that the regulation of PtdOH metabolism is an important mechanism in the transition from T. cruzi epimastigote to intermediate form.

DAG derived from phosphatidylcholine (PtdCho) acts as a lipid second messenger. It can be generated by the activation of phospholipase D (PLD) and the phosphatidic acid phosphohydrolase type 2 (PAP2) pathway or by a PtdCho‐specific phospholipase C (PtdCho‐PLC). Our purpose was to study PtdCho‐PLC activity in rat cerebral cortex synaptosomes (CC Syn). DAG production was highly stimulated by detergents such as Triton X‐100 and sodium deoxycholate. Ethanol and tricyclodecan‐9‐yl‐xanthate potassium salt decreased DAG generation by 42 and 61%, respectively, at 20 min of incubation. These data demonstrate that both the PLD/PAP2 pathway and PtdCho‐PLC contribute to DAG generation in CC Syn. PtdCho‐PLC activity remained located mainly in the synaptosomal plasma membrane fraction. Kinetic studies showed Km and Vmax values of 350 μM and 3.7 nmol DAG × (mg protein × h)−1, respectively. Western blot analysis with anti‐PtdCho‐PLC antibody showed a band of 66 KDa in CC Syn. Our results indicate the presence of a novel DAG‐generating pathway in CC Syn in addition to the known PLD/PAP2 pathway. Fil: Mateos, Melina Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Uranga, Romina Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Salvador, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina

The nephrotic syndrome is associated with disturbances in plasma lipid pattern and metabolism. However, the reason for these perturbations is poorly understood. In the present study, we have investigated hepatic triglyceride metabolism in puromycin aminonucleoside-induced nephrotic syndrome in rats. Nephrotic rats displayed a 70% increase in hepatic triglyceride levels compared to controls (16.9 +/- 1.6 vs. 9.8 +/- 0.6 mumol/g liver; means +/- SEM, P0.01). The capacity for hepatic mitochondrial beta-oxidation of fatty acids was substantially elevated (80%). This was associated with a rise in the liver content of the fatty acid carrier carnitine (1.24 +/- 0.06 vs. 0.85 +/- 0.07 mumol/g dry weight, P0.05). A positive correlation between the levels of acetylcarnitine and acetyl-CoA was found in normal as well as in nephrotic rats, implying that carnitine plays an important role as an acetyl group acceptor in the liver under normo- and hyperlipidemic conditions. Changes in carnitine levels seem to be tightly coupled to the rate of fatty acid oxidation. There was a significant elevation in the activity of phosphatidate phosphohydrolase (E.C. 3.1.3.4) in liver microsomes from nephrotic rats (1.07 +/- 0.09 vs. 0.81 +/- 0.04 nmol/min.mg protein, P0.02). Hepatic very low density lipoprotein (VLDL)-triglyceride secretion rate was 18% higher in nephrotic rats than in controls. The results demonstrate a deranged hepatic triglyceride metabolism in nephrosis, with an increased hepatic triglyceride biosynthesis, a sizable accumulation of hepatic triglycerides, and only a modest increase in VLDL triglyceride secretion. In addition, mitochondrial beta-oxidation of fatty acids was enhanced, associated with an increased availability of carnitine.

Phosphatidate phosphatase (EC 3.1.3.4) catalyzes the hydrolysis of phosphatidate to yield sn-1,2-diacylglycerol and inorganic phosphate. In mammalian systems, forms of phosphatidate phosphatase involved in glycerolipid synthesis and signal transduction have been identified. Forms of the enzyme involved in signal transduction have been purified and partially characterized. In yeast, phosphatidate phosphatases associated with the endoplasmic reticulum and mitochondria have also been purified and partially characterized. Information on phosphatidate phosphatases from mammals and yeast is useful in characterizing the enzyme from plant systems. This review examines progress on the characterization of phosphatidate phosphatases from mammals, yeast, and higher plants. The purification and characterization of the phosphatidate phosphatase involved in glycerolipid synthesis in developing oilseeds may lead to the identification of the encoding gene. Increasing our understanding of the enzymes of lipid synthesis in developing seeds will aid in the development of biotechnological strategies for seed oil modification.

Translocation of long-chain acyl-coenzyme A hydrolase from the microsomal fraction to the cytosolic fraction was promoted in cell-free extracts of rat liver by palmitic acid, oleic acid, tetradecylthioacetic acid, and tetradecylthiopropionic acid, and by their CoA esters. The CoA esters were more effective than the non-esterified acids in the translocation of the enzyme. Treatment of normolipidemic rats with sulfur-substituted non-β-oxidizable fatty acid analogues resulted in a transitory increase in hepatic concentration of long-chain acyl-CoA. Longer feeding times almost normalized the hepatic long-chain acyl-CoA content. Microsomal long-chain acyl-CoA hydrolase activity was inhibited, whereas the activity of the cytosolic form was stimulated. The rise in enzyme activity coincided with a reduction in liver content of triglyceride and an increase in hepatic phospholipid content. The results suggest that the activity of long-chain acyl-CoA hydrolase in the cytosol may control the amount of acyl-CoA thioesters in the liver. Esterified and non-esterified fatty acids causedin vitro translocation of phosphatidate phosphohydrolase and cytidine 5′-triphosphate (CTP):phosphocholine cytidylyltransferase from the cytosolic fraction to the microsomal fraction. However, the translocation of these two enzyme systems was not obtainedin vivo. The activity of phosphatidate phosphohydrolase decreased in microsomal and cytosolic fractions while the activity of cytidylyltransferase in these fractions increased. The activities of soluble phosphatidate phosphohydrolase and long-chain acyl-CoA hydrolase appeared to be inversely correlated. The results imply that in cytoplasm, long-chain acyl-CoA hydrolase may compete with the biosynthetic enzymes for the acyl-CoA substrate, thus influencing the rate of lipid synthesis. The reduced hepatic triglyceride content observed in tetradecylthioacetic acid-treated rats is probably due to reduced triglyceride synthesis, which is mediated by an inhibition of phosphatidate phosphohydrolase accompanied with translocation and stimulation of long-chain acyl-CoA hydrolase. Development of fatty liver as an effect of tetradecylthiopropionic acid is probably due to accelerated triglyceride biosynthesis, which is mediated by a stimulation of phosphatidate phosphohydrolase and a decrease in cytosolic palmitoyl-CoA hydrolase activity.

The effect of long-chain n-3 fatty acids on hepatic key enzymes of cholesterol metabolism and triglyceride biosynthesis was investigated in two rat models. In the first model, rats were intravenously infused for two weeks with a fat emulsion containing 20% of triglycerides in which either n-6 or n-3 fatty acids predominated. The treatment with n-3 fatty acids led to a reduction primarily of serum cholesterol (45%), but also of serum triglycerides (18%). HMG-CoA reductase activity and cholesterol 7 alpha-hydroxylase activity were reduced by 45% and 36%, respectively. There were no significant effects on diacylglycerol acyltransferase (DGAT) or phosphatidate phosphohydrolase (PAP) activities. In the second model, rats were fed a diet enriched with sucrose, coconut oil and either sunflower oil (n-6 fatty acids) or fish oil (long-chain n-3 fatty acid ethyl esters). The treatment with n-3 fatty acids decreased serum triglycerides (41%) and, to a lesser extent, serum cholesterol (17%). Neither glycerol 3-phosphate acyltransferase (GPAT) or DGAT were affected by n-3 fatty acids. In contrast, PAP activity was reduced by 26%. HMG-CoA reductase was not significantly affected, whereas cholesterol 7 alpha-hydroxylase activity was reduced by 36%. The results indicate that part of the TG-lowering effect of long-chain n-3 fatty acids may be mediated by inhibition of the soluble phosphatidate phosphohydrolase. The effect on serum cholesterol may be partly due to inhibition of HMG-CoA reductase.

Aging is a process that affects different organs, of which the brain is particularly susceptible. PA and DAC are central intermediates in the phosphoglyceride as well as in the neutral lipid biosynthetic pathway, and they have also been implicated in signal transduction. Phospholipase D (PLD) and phosphatidate phosphohydrolase (PAP) are the enzymes that generate PA and DAG. The latter can be transformed into MAC by diacylglycerol lipase (DGL). In the present study, we examine how aging modulates the PLD, PAP, and DGL isoforms in cerebellar subcellular fractions from 4- (adult), 28-, and 33-mon-old (aged) rats. PI-4,5-bisphosphonate (PIP2)-dependent PLD, PAP1, and DCL1 were distributed in different percentages in all cerebellum subcellular fractions. On the other hand, PAP2 and DGL2 activities were observed in all subcellular fractions except in the cytosolic fraction. Aging modified the enzyme distribution pattern. In addition, aging decreased nuclear (45%), mitochondrial-synaptosomal (55%), and cytosolic (71%) PAP1 activity and increased (28%) microsomal PAP1 activity. DGL1 activity was decreased in nuclear (85%) and mitochondrial-synaptosomal (63%) fractions by aging. On the other hand, PIP2-dependent PLD activities were increased in the mitochondrial-synaptosomal fraction. PAP2 and DGL2 were increased in the microsomal fraction by 87 and 114%, respectively, and they were decreased in the nuclear fraction. The changes observed in cerebellum PAP1 and DCL1 activities from aged rats with respect to adult rats could be related to modifications in lipid metabolism. Differential PA metabolization during aging through PIP2-dependent PLD/PAP2/DGL2 activities could be related to alterations in the neural signal transduction mechanisms. Fil: Pasquaré, Susana Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Salvador, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina

In the starved state and during metabolic stress, free fatty acids (FFA) are the principal hepatic energy supply, undergoing beta-oxidation. Accordingly, it appears paradoxical that FFA have been reported to increase the liver's esterification capacity by translocating the rate-limiting enzyme phosphatidic acid phosphohydrolase (PAP-1) from the cytosol to the endoplasmic reticulum. We have therefore investigated the regulation of rat liver PAP-1. Oleic acid inhibited PAP activity in all subcellular fractions, with PAP-1 activity in cytosol being the most sensitive. Inhibition was also observed with oleoyl-CoA, linoleate, and palmitate. Fatty acids and their derivatives show detergent effects at high concentrations, and such effects can lead to enzyme inhibition. Inhibition by oleate, however, was reversed by phosphatidic acid and albumin and exhibited sigmoidal kinetics. These results demonstrate that PAP-1 is reversibly inhibited by FFA and their CoA esters, which may play a role in directing hepatic FFA to beta-oxidation during times of increased energy demand.

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, P0.01). 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.

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.

In view of previous reports that the activity of the Mg(++)-dependent phosphatidic acid phosphatase in adipose tissues of rat and mouse is elevated in obesity, we attempted to assay this activity in biopsies of human omental adipose tissue obtained from normal-weight and morbidly obese subjects in connection with operations. The major portion of the phosphatidic acid phosphatase activity was found in the cytosol, and the small amount found in the microsomal fraction was too low for accurate measurement. It was not possible to assay the activity in the crude cytosol. After precipitation with ammonium sulfate, however, the enzyme activity was linear with both the incubation time and the concentration of enzyme. It was not possible to obtain substrate saturation of the enzyme under the conditions employed. When assayed in the presence of a high concentration of substrate (0.6 mmol/l) the activity obtained in normal-weight patients, 7.8 +/- 2.4 nmol/mg protein/min (n = 10), was not significantly different from that in morbidly obese patients, 5.6 +/- 0.8 nmol/mg protein/min (n = 10). There was no relation between the size of adipose cells and phosphatidic acid phosphatase activity. Furthermore, there was no apparent relation between phosphatidic acid phosphatase activity in omental adipose tissue and that in the liver. The findings suggest that the increased biosynthesis of triglycerides in human obesity is not associated with an increased capacity of the soluble phosphatidic acid phosphatase in adipose tissue.

The microsomal bound phosphatidic acid phosphohydrolase from lactating rat mammary tissue had a specific activity of six nmoles per mg protein per minute. The optimum pH was 7.0; magnesium at 1.3 mM was required for maximum activity, and at low substrate concentrations magnesium lowered the Km of the enzyme for phosphatidic acid. Diglycerides exerted little effect while diglyceride ether stimulated enzyme activity. Inorganic salts, i.e., potassium phosphate and potassium chloride, enhanced rates of phosphatidic acid hydrolysis under standard assay conditions.

Phosphatidate phosphatase (EC 3.1.3.4) was detected in filamentous pathogenic fungi. In both dermatophytes,Microsporum gypseum andEpidermophyton floccosum, the enzyme was located in the mitochondrial and microsomal subcellular fractions with a pH optimum of 6.0. TheE. floccosum enzyme was more active than that ofM. gypseum. Although the enzyme in both fractions of the dermatophytes was susceptible to inhibition by Fe(2+), Mn(2+), Cu(2+), Ba(2+) and Hg(2+), theE. floccosum microsomal enzyme was completely inhibited by Mn(2+) and Fe(2+). The enzyme ofM. gypseum as well as ofE. floccosum exhibited a Mg(2+) dependency in presence of EDTA. Sulphydryl reagents did not inhibit either the mitochondrial or the microsomal enzyme of these fungi. Phosphorylcholine and lysophosphatidylcholine markedly inhibited the enzyme in the two dermatophytes; however, the inhibition was less pronounced with phosphatidylcholine.

Rats of weaning age were fed for a period of 1,3 or 6 weeks either a control diet (laboratory stock diet) or a semisynthetic diet containing 20% by weight of either mustard seed oil (1/3 of the total fatty acids were comprised of erucic acid) or corn oil (2/3 of the total fatty acids consisted of linoleic acid). Mitochondrial and microsomal fractions were isolated from the hearts and livers of these rats, and the rate of acylation ofsn-[U-14C] glycerol 3-phosphate (P) was examined using palmitoyl-CoA or erucoyl-CoA as the acyl donor. In addition, activities of phosphatidate phosphatase of the mitochondrial, microsomal and soluble fractions were assayed. Studies on the acylation of glycerol 3-P with palmitoyl-CoA demonstrated that feeding of the high fat/high erucic acid diet for 1,3 or 6 weeks significantly increased the rate of formation of monoacylglycerol 3-P by the cardiac subcellular fractions as compared to the control. The rate of formation of diacylglycerol 3-P also increased but to a lesser degree. Feeding the high fat/high linoleic acid diet tended to increase acylation of glycerol 3-P by cardiac subcellular fractions. However, neither high fat diet influenced acyltransferase activities of the hepatic subcellular fractions or phosphatase activities of the cardiac and hepatic fractions. Studies on the acylation of glycerol 3-P with erucoyl-CoA demonstrated that the rate of acylation was ca. 1/10 that measured using palmitoyl-CoA in all experiments; in particular, the formation of diacylglycerol 3-P was extremely slow, suggesting that erucoyl-CoA is an unsuitable substrate for the position-2 of the monoacylglycerol 3-P. The rate of acylation by the cardiac and hepatic subcellular fractions was not influenced by the feeding of the high-fat diets. The rate of glycerol 3-P acylation by both cardiac and hepatic mitochondrial fraction was ca. 2/3 of the rate of acylation by the respective microsomal fraction. In addition, the ratio of monoacyl-to diacylglycerol 3-P synthesized by the mitochondrial fraction was smaller than that by the microsomal fraction. These results suggest that acylation of glycerol 3-P by the mitochondrial cannot be attributed to the action of the contaminating microsomal enzymes.

Phosphatidate phosphohydrolase (PAPase) activity was measured in lamellar bodies purified from porcine lung tissue. After repeated freeze-thawing, only a negligible amount of PAPase activity was released into the soluble fractions, whereas there was release of 2 lysosomal marker enzyme activities, glucosaminidase and beta-galactosidase into the soluble fraction. In addition, a lysosomal-enriched fraction was prepared from adult rat lung tissue by prior treatment of the rats with Triton WR 1339. Treatment with Triton WR 1339 resulted in the significant shift of the activities of the lysosomal marker enzymes, glucosaminidase and beta-galactosidase, to less dense subcellular fractions. The highest specific activity of PAPase was found in a subcellular fraction which had a density that was intermediate between that of the mitochondrial and microsomal fractions and the distribution of PAPase activity was not affected by the prior treatment of the rats with Triton WR 1339.