Glycerol Phosphate Acyltransferase Activity Research Articles

Membrane Disruption by Antimicrobial Fatty Acids Releases Low-Molecular-Weight Proteins from Staphylococcus aureus

The skin represents an important barrier for pathogens and is known to produce fatty acids that are toxic toward gram-positive bacteria. A screen of fatty acids as growth inhibitors of Staphylococcus aureus revealed structure-specific antibacterial activity. Fatty acids like oleate (18:1Δ9) were nontoxic, whereas palmitoleate (16:1Δ9) was a potent growth inhibitor. Cells treated with 16:1Δ9 exhibited rapid membrane depolarization, the disruption of all major branches of macromolecular synthesis, and the release of solutes and low-molecular-weight proteins into the medium. Other cytotoxic lipids, such as glycerol ethers, sphingosine, and acyl-amines blocked growth by the same mechanisms. Nontoxic 18:1Δ9 was used for phospholipid synthesis, whereas toxic 16:1Δ9 was not and required elongation to 18:1Δ11 prior to incorporation. However, blocking fatty acid metabolism using inhibitors to prevent acyl-acyl carrier protein formation or glycerol-phosphate acyltransferase activity did not increase the toxicity of 18:1Δ9, indicating that inefficient metabolism did not play a determinant role in fatty acid toxicity. Nontoxic 18:1Δ9 was as toxic as 16:1Δ9 in a strain lacking wall teichoic acids and led to growth arrest and enhanced release of intracellular contents. Thus, wall teichoic acids contribute to the structure-specific antimicrobial effects of unsaturated fatty acids. The ability of poorly metabolized 16:1 isomers to penetrate the cell wall defenses is a weakness that has been exploited by the innate immune system to combat S. aureus.

Cardiac diacylglycerol accumulation in high fat-fed mice is associated with impaired insulin-stimulated glucose oxidation

the molecular processes leading to cardiac insulin resistance induced via a high-fat diet (HFD) remain unclear. We examined the changes in cardiac insulin sensitivity and the potential mechanism(s) involved following HFD in mice. C57BL/6 mice were fed either a low-fat diet (LFD, 4% kcal fat) or a HFD (60% kcal fat) for 3 or 10 weeks. Insulin-stimulated glucose oxidation in isolated working hearts was decreased at 10 weeks of HFD compared with mice on LFD (249 ± 19 to 399 ± 46 vs. 551 ± 97 to 1464 ± 243 nmol/g dry wt/min; P < 0.05). The accumulation of myocardial diacylglycerol (DAG; 479 ± 174 vs. 266 ± 29 micromol/g wet wt; P < 0.05), but not long-chain acyl CoA, ceramide, or triacylglycerol, correlated with the development of insulin resistance. The accumulation of DAG occurred concomitantly with an increase in glycerol phosphate acyltransferase activity, a decrease in DAG acyltransferase activity, as well as an increase in the translocation of protein kinase C-α (PKCα) and phosphorylation of p70s6k. Neither HFD-induced accumulation of cardiac DAG nor up-regulation of phosphorylated p70s6k occurred in mice lacking malonyl CoA decarboxylase which are resistant to the development of HFD-induced insulin resistance. the activation of myocardial p70s6k and PKCα is closely associated with cardiac insulin resistance in which the accumulation of intra-myocardial DAG could be responsible.

Depot-specific effects of the PPARγ agonist rosiglitazone on adipose tissue glucose uptake and metabolism

We investigated mechanisms whereby peroxisome proliferator-activated receptor gamma (PPARgamma) agonism redistributes lipid from visceral (VF) toward subcutaneous fat (SF) by studying the impact of PPARgamma activation on VF and SF glucose uptake and metabolism, lipogenesis, and enzymes involved in triacylglycerol (TAG) synthesis. VF (retroperitoneal) and SF (inguinal) of rats treated or not for 7 days with rosiglitazone (15 mg/kg/day) were evaluated in vivo for glucose uptake and lipogenesis and in vitro for glucose metabolism, gene expression, and activities of glycerolphosphate acyltransferase (GPAT), phosphatidate phosphatase-1 (or lipin-1), and diacylglycerol acyltransferase. Rosiglitazone increased SF glucose uptake, GLUT4 mRNA, and insulin-stimulated glucose oxidation, conversion to lactate, glycogen, and the glycerol and fatty acid components of TAG. In VF, only glucose incorporation into TAG-glycerol was stimulated by rosiglitazone and less so than in SF (1.5- vs. 3-fold). mRNA levels of proteins involved in glycolysis, Krebs cycle, glycogen synthesis, and lipogenesis were markedly upregulated by rosiglitazone in SF and again less so in VF. Rosiglitazone activated TAG-glycerol synthesis in vivo (2.8- vs. 1.9-fold) and lipin activity (4.6- vs. 1.5-fold) more strongly in SF than VF, whereas GPAT activity was increased similarly in both depots. The preferential increase in glucose uptake and intracellular metabolism in SF contributes to the PPARgamma-mediated redistribution of TAG from VF to SF, which in turn favors global insulin sensitization.

The PPARγ agonist rosiglitazone enhances rat brown adipose tissue lipogenesis from glucose without altering glucose uptake

We investigated the mechanisms whereby peroxisome proliferator-activated receptor-gamma (PPARgamma) agonism affects glucose and lipid metabolism in brown adipose tissue (BAT) by studying the impact of PPARgamma activation on BAT glucose uptake and metabolism, lipogenesis, and mRNA levels plus activities of enzymes involved in triacylglycerol (TAG) synthesis. Interscapular BAT of rats treated or not with rosiglitazone (15 mg*kg(-1).day(-1), 7 days) was evaluated in vivo for glucose uptake and lipogenesis and in vitro for glucose metabolism, gene expression, and activities of glycerolphosphate acyltransferase (GPAT), phosphatidate phosphatase-1 (PAP or lipin-1), and diacylglycerol acyltransferase (DGAT). Rosiglitazone increased BAT mass without affecting whole tissue glucose uptake. BAT glycogen content (-80%), its synthesis from glucose (-50%), and mRNA levels of UDP-glucose pyrophosphorylase (-40%), which generates UDP-linked glucose for glycogen synthesis, were all reduced by rosiglitazone. In contrast, BAT TAG-glycerol synthesis in vivo and glucose incorporation into TAG-glycerol in vitro were stimulated by the agonist along with the activities and mRNA levels of glycerol 3-phosphate-generating phosphoenolpyruvate carboxykinase and glycerokinase. Furthermore, rosiglitazone markedly increased the activities of GPAT and DGAT but not those of lipin-1-mediated PAP-1, enzymes involved in the sequential acylation of glycerol 3-phosphate and TAG synthesis. Because an adequate supply of fatty acids is essential for BAT nonshivering thermogenesis, the enhanced ability of BAT to synthesize TAG under PPARgamma activation may constitute an important mechanism by which lipid substrates are stored in preparation for an eventual thermogenic activation.

The level and compartmentalization of phosphatidate phosphatase-1 (lipin-1) control the assembly and secretion of hepatic VLDL

Phosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1alpha and 1beta), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1alpha or -1beta increased the synthesis and secretion of [(3)H]glycerol-labeled lipids under either basal- or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL(1) (S(f) > 100) and VLDL(2) (S(f) 20-100). Expression of lipin-1alpha or -1beta increased secretion efficiency and decreased intracellular degradation of [(35)S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [(3)H]glycerolipids and [(35)S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1alpha not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1alpha mutant failed to promote [(35)S]apoB100 synthesis or secretion, and showed compromised stimulation in [(3)H]TAG synthesis and secretion. Thus, alteration in hepatic expression of lipin-1 and its compartmentalization control VLDL assembly/secretion.

Glucocorticoids and cyclic AMP selectively increase hepatic lipin-1 expression, and insulin acts antagonistically

Glucocorticoids (GCs) increase hepatic phosphatidate phosphatase (PAP1) activity. This is important in enhancing the liver's capacity for storing fatty acids as triacylglycerols (TAGs) that can be used subsequently for beta-oxidation or VLDL secretion. PAP1 catalyzes the conversion of phosphatidate to diacylglycerol, a key substrate for TAG and phospholipid biosynthesis. PAP1 enzymes in liver include lipin-1A and -1B (alternatively spliced isoforms) and two distinct gene products, lipin-2 and lipin-3. We determined the mechanisms by which the composite PAP1 activity is regulated using rat and mouse hepatocytes. Levels of lipin-1A and -1B mRNA were increased by dexamethasone (dex; a synthetic GC), and this resulted in increased lipin-1 synthesis, protein levels, and PAP1 activity. The stimulatory effect of dex on lipin-1 expression was enhanced by glucagon or cAMP and antagonized by insulin. Lipin-2 and lipin-3 mRNA were not increased by dex/cAMP, indicating that increased PAP1 activity is attributable specifically to enhanced lipin-1 expression. This work provides the first evidence for the differential regulation of lipin activities. Selective lipin-1 expression explains the GC and cAMP effects on increased hepatic PAP1 activity, which occurs in hepatic steatosis during starvation, diabetes, stress, and ethanol consumption.

Causes and prevention of tamoxifen-induced accumulation of triacylglycerol in rat liver

Tamoxifen can induce hepatic steatosis in women. In this study, we wanted to elucidate the mechanism behind the tamoxifen-induced accumulation of triacylglycerol in liver in female rats, and we hoped to prevent this development by combination treatment with the modified fatty acid tetradecylthioacetic acid (TTA). The increased hepatic triacylglycerol level after tamoxifen treatment was accompanied by decreased acetyl-coenzyme A carboxylase (ACC) and FAS activities, increased glycerol-3-phosphate acyltransferase (GPAT) activity, and a tendency to increased diacylglycerol acyltransferase (DGAT) activity. The activities and mRNA levels of enzymes involved in beta-oxidation, ketogenesis, and uptake of lipids from liver were unaffected by tamoxifen, whereas the uptake of lipoproteins was unchanged and the uptake of fatty acids was decreased. Combination treatment with tamoxifen and TTA (Tam+TTA) normalized the hepatic triacylglycerol level and increased the activities of ACC, FAS, GPAT, and DGAT compared with tamoxifen-treated rats. The activities and mRNA levels of enzymes involved in beta-oxidation, ketogenesis, and uptake of lipids were increased after Tam+TTA treatment. In conclusion, tamoxifen increased the hepatic triacylglycerol level, probably as a result of increased triacylglycerol biosynthesis combined with unchanged beta-oxidation. The tamoxifen-induced accumulation of triacylglycerol was prevented by cotreatment with TTA, through mechanisms of increased mitochondrial and peroxisomal beta-oxidation.

The C-terminal region of mitochondrial glycerol-3-phosphate acyltransferase-1 interacts with the active site region and is required for activity

Glycerol phosphate acyltransferase (GPAT) catalyzes the formation of 1-acyl- sn-glycerol-3-phosphate from glycerol-3-phosphate and long chain fatty acyl-CoA substrates. We previously determined the topography of the mitochondrial GPAT1 isoform (mtGPAT1, 828 amino acids). mtGPAT1 has two transmembrane domains (TMDs) (aa 472–493 and aa 576–592) with both the N- and C-termini facing the cytosol and a loop (aa 494–575) facing the intermembrane space. Alignment of amino acid sequences from mtGPAT1 and other acyltransferases and site directed mutagenesis studies have demonstrated that the active site of the enzyme resides in the N-terminal domain of the protein. In this study, we sequentially truncated the C-terminal domain and characterized the properties of the resulting mutants expressed in CHO cells. Although the mutants were overexpressed, none of them conferred GPAT activity. The loss of activity was not due to the miss-targeting of the proteins since immunofluorescence experiments demonstrated their mitochondrial localization. Instead, chemical crosslinking and protein cleavage studies demonstrated that the N- and C-termini of the protein interact. These results suggest that the C-terminal domain is necessary for mtGPAT1 activity, and probably contributes to catalysis or substrate binding.

A protein tyrosine kinase associated with the ATP-dependent inactivation of adipose diacylglycerol acyltransferase.

The activity that has been previously reported to reversibly inactivate adipose glycerolphosphate acyltransferase (GPAT) and diacylglycerol acyltransferase (DGAT) in vitro in the presence of ATP is shown here to be partially purified from adipose tissue with an apparent molecular weight of 68 kDa. The activity responsible for inactivating DGAT is associated with a kinase activity as determined by phosphate incorporation both into microsomal proteins and into a synthetic tyrosine-containing peptide as substrate for protein tyrosine kinase. Two microsomal polypeptides of 53 and 69 kDa are major substrates of this kinase. Both DGAT inactivating and kinase activities assayed from the purified sample have been found to be insensitive to the Ser/Thr kinase inhibitor H-7 while being sensitive to genistein and tyrphostin-25. A crude protein phosphatase preparation from liver was capable of reversing the effects of both activities. The purified sample was also shown to inactivate GPAT in the presence of ATP. These results suggest that a protein tyrosine kinase, in concert with a protein tyrosine phosphatase, may regulate the activities of DGAT and GPAT by a phosphorylation-dephosphorylation mechanism.

Higher postpartum hepatic triacylglycerol concentrations in dairy cows with free rather than restricted access to feed during the dry period are associated with lower activities of hepatic glycerolphosphate acyltransferase.

We investigated the activities of hepatic glycerolipid synthesizing enzymes during postpartum fatty liver development in 10 high-producing dairy cows that had free access to feed during the dry period; a parallel group of 8 control cows was fed according to recommended energy requirements. After calving, both test and control cows had free access to feed. In the period of 10-14 wk before calving, voluntary dry matter intake of the test cows was 20.6 kg/d (SEM 0.42); the restricted control cows received 7 kg/d. Postpartum triacylglycerol concentrations in liver biopsies were one- to twofold higher in the test than in the control cows. The higher plasma nonesterified fatty acid (NEFA) concentrations after parturition in the test vs. the control group were probably caused by a more negative energy balance in the test cows, which was associated with a slightly lower postpartum dry matter intake. After calving, hepatic mitochondrial glycerolphosphate acyltransferase (GPAT) activities were significantly lower in the test than in the restricted control cows. A low GPAT activity may divert fatty acids from esterification to beta-oxidation to protect the hepatocytes against further accumulation of triacylglycerols. The activities of hepatic phosphatidate phosphohydrolase, diacylglycerol acyltransferase, and cholinephosphate cytidylyltransferase were not different in the two groups. This study indicates that in cows given free instead of restricted access to feed during the dry period have a postpartum hepatic triacylglycerol accumulation that is mainly determined by a raised hepatic uptake of plasma NEFA.

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.

The response of brown adipose tissue mitochondrial glycerolphosphate acyltransferase to cold-exposure in hypothyroidism, after adrenalectomy and after treatment with cycloheximide

1. 1. Exposure to cold has previously been shown to considerably increase the activity of the mitochondrial form of glycerolphosphate acyltransferase (GPAT) in brown adipose tissue (A.C. Darnley C.A. Carpenter and E. D Saggerson, Biochem.J. 253, 351–355, 1988; J.R.D. Mitchell and E.D. Saggerson. P Biochem.J. 277, 665–669, 1991). 2. 2. Both adrenalectomy and chemically-induced hypothyroidism increased mitochondrial GPAT activity in rats maintained at 21°C. This increase was similar to that caused by exposing rats to the cold (4°C) for three days. Whereas exposure of hypothyroid rats to cold (4°C) resulted in a further increase in GPAT activity, no further increase in activity was observed after exposure of adrenalectomized rats to the cold. 3. 3. Administration of triiodothyronine (T 3) to rats maintained at 21°C had no effect on mitochondrial GPAT activity. 4. 4. Prior treatment with cycloheximide abolished 60–70% of the increase in GPAT activity caused by cold-exposure.

Effect of disulfiram (DS) on mitochondria from rat hippocampus: Metabolic compartmentation of DS neurotoxicity

This experiment was designed to study the acute effects of disulfiram on mitochondrial enzymes in nonsynaptic and synaptic mitochondria from rat hippocampus. Cytochrome c oxidase, monoamine oxidase-B, glycerolphosphate acyltransferase and beta-hydroxybutyrate dehydrogenase were studied. Differences in enzyme activity were seen in controls. Cytochrome c oxidase activity was higher in synaptic mitochondria whereas glycerolphosphate acyltransferase activity was higher in nonsynaptic mitochondria. Mitochondria from disulfiram treated rats, particularly synaptic mitochondria, exhibited lower specific activities of cytochrome c oxidase and monoamine oxidase-B. These alterations were not limited to either the inner or outer mitochondrial membrane. Transmission electron microscopy revealed that mitochondria from disulfiram treated rats were severely altered in isolated preparations as well as in those from whole tissue. This study shows that disulfiram exerts a differential effect on mitochondrial subpopulations.

Activities of enzymes of glycerolipid synthesis in brown adipose tissue after treatment of rats with the adrenergic agonists BRL 26830A and phenylephrine, after exposure to cold and in streptozotocin-diabetes

1. Measurements were made, relative to tissue DNA, of the activities of enzymes of glycerolipid synthesis in homogenates of interscapular brown adipose tissue. These were: mitochondrial and microsomal forms of glycerolphosphate acyltransferase (GPAT), Mg(2+)-dependent phosphatidate phosphohydrolase (PPH) and fatty acyl-CoA synthetase (FAS). 2. In normal animals, 3 days of cold-exposure (4 degrees C) increased all activities. The increase in mitochondrial GPAT activity was particularly pronounced (5-fold). Administration of the beta-adrenergic agonist BRL 26830A mimicked the effect of cold on microsomal GPAT activity. Mitochondrial GPAT, PPH and FAS activities were unresponsive to BRL 26830A. The alpha-adrenergic agonist phenylephrine significantly decreased activities of GPAT and PPH. 3. Streptozotocin-diabetes decreased mitochondrial GPAT activity, but did not abolish the effect of cold to increase this activity or the activity of microsomal GPAT. Diabetes abolished the effect of cold on PPH and FAS activities. 4. The findings are relevant to signals that drive early events in mitochondriogenesis and cell proliferation in brown adipose tissue on exposure to cold.

Microsomal glycerolphosphate acyltransferase inactivation by fatty acids

Glycerolphosphate acyltransferase activity in microsomes from rat adipose tissue is shown to decrease with time upon incubation with adipose tissue cytosolic fraction. The inactivation can be prevented with serum albumin and seems to be caused by an increase in endogenous free fatty acid as a consequence of the action of cytosolic lipase(s) on the membrane lipids. Similar inactivation can be observed after short incubation of microsomes with oleic acid at micromolar concentrations. Diacylglycerol acyltransferase is also inhibited by oleic acid, although to a lesser degree. In contrast, glucose-6-phosphatase and NADPH-cytochrome reductase activities are not changed. The oleic acid effect appears to occur upon binding to the microsomal membranes and can be prevented by bovine serum albumin at protein/fatty acid molar ratios above one. These results suggest that free fatty acids may be involved in the modulation of triacylglycerol synthetic enzymes.

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.

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.

Hepatic triacylglycerol synthesis during a period of fatty liver development in sheep.

Fatty liver was induced in six sheep by fasting and treatment with phloridzin and epinephrine. Treatment was associated with a rise in the concentration in serum and hepatic uptake of nonesterified fatty acid (NEFA) compared with pretreatment (P less than .01). At 24 h after the start of the treatment, concentrations of serum lipoprotein in each density class were not different from baseline, but all were elevated (P less than .01) by 120 h of treatment. Hepatic triacylglycerol (TG) concentration increased (P less than .01) 17-fold within the first 48 h of treatment, with no additional increase during the remaining 72 h. The activity of phosphatidate phosphohydrolase (PAP) in hepatic microsomes increased (P less than .01) fourfold over baseline by 48 h of treatment, then declined slightly by 120 h. The activities of diacylglycerol acyltransferase (DGAT) and glycerolphosphate acyltransferase (GPAT) in hepatic microsomes increased during treatment but appeared to follow a slightly different pattern from that of PAP. Activity of GPAT was not above baseline at 48 h, but was at 120 h (P less than .05); DGAT activity was increased (P less than .05) twofold at 48 h, with an apparent continued increase (P less than .01) to threefold over baseline by 120 h of treatment. Fatty liver appeared to develop during a period of rapid hepatic uptake of NEFA without a corresponding increase in serum lipoprotein concentrations. The activities of PAP, GPAT and DGAT, putative regulators of TG synthesis rate, all increased in liver microsomes during a period of high hepatic NEFA uptake, but that of PAP appeared to coincide most closely with the development of fatty liver.

Phosphatidylcholine synthesis in isolated type II pneumocytes from ozone-exposed rats

Phosphatidylcholine (PC) synthesis by alveolar type II cells, as an indicator for the production of pulmonary surfactant, was studied after a 4-h exposure of rats to 4 mg ozone/m3 (2 ppm). Lung ravage fluid analysis after exposure revealed significant increases in proteins, which is indicative for pulmonary injury. When type II cells were isolated immediately and thereafter cultured for 20 h, the rate of PC synthesis in cells derived from ozone-exposed rats was not significantly different from that in cells from unexposed controls. Yet, a decreased rate of PC synthesis was observed when these cells were subsequently exposed to ozone in vitro. The activity of the enzyme glycerolphosphate acyltransferase (GPAT) was slightly enhanced in cultured type II cells isolated from ozone-exposed rats, while the lysophosphatidylcholine acyltransferase (LPCAT) activity was unchanged. However, ozone exposure of rats did result in a significant decrease of PC synthesis when measured in freshly prepared type II cell suspensions, although both GPAT and LPCAT activities were not affected. It is concluded that a decrease in pulmonary surfactant related PC synthesis after ozone exposure of rats can be demonstrated in freshly isolated type II pneumocytes. Cultured type II cells from exposed rats lack this effect and are therefore less useful to study changes in phospholipid biosynthesis after in vivo ozone exposure. The data on in vitro ozone exposure of cultured type II cells, however, support the view that ozone may impair pulmonary surfactant production.

Interactions of insulin, glucagon and dexamethasone in controlling the activity of glycerol phosphate acyltransferase and the activity and subcellular distribution of phosphatidate phosphohydrolase in cultured rat hepatocytes.

Rat hepatocytes were incubated in monolayer culture for 8 h. Glucagon (10nM) increased the total phosphatidate phosphohydrolase activity by 1.7-fold. This effect was abolished by adding cycloheximide, actinomycin D or 500 pM-insulin to the incubations. The glucagon-induced increase was synergistic with that produced by an optimum concentration of 100 nM-dexamethasone. Theophylline (1mM) potentiated the effect of glucagon, but it did not affect the dexamethasone-induced increase in the phosphohydrolase activity. The relative proportion of the phosphohydrolase activity associated with membranes was decreased by glucagon when 0.15 mM-oleate was added 15 min before the end of the incubations to translocate the phosphohydrolase from the cytosol. This glucagon effect was not seen at 0.5 mM-oleate. Since glucagon also increased the total phosphohydrolase activity, the membrane-associated activity was maintained at 0.15 mM-oleate and was increased at 0.5 mM-oleate. This activity at both oleate concentrations was also increased in incubations that contained dexamethasone, particularly in the presence of glucagon. Insulin increased the relative proportion of phosphatidate phosphohydrolase that was associated with membranes at 0.15 mM-oleate, but not at 0.5 mM-oleate. It also decreased the absolute phosphohydrolase activity on the membranes at both oleate concentrations in incubations that also contained glucagon and dexamethasone. None of the hormonal combinations significantly altered the total glycerol phosphate acyltransferase activity. However, glucagon significantly increased the microsomal activities, and insulin had the opposite effect. Glucagon also decreased the mitochondrial acyltransferase activity. There was a highly significant correlation between the total phosphatidate phosphohydrolase activity and the synthesis of neutral lipids from glycerol phosphate and 0.5 mM-oleate in homogenates of cells from all of the hormonal combinations. Phosphatidate phosphohydrolase activity is increased in the long term by glucocorticoids and also by glucagon through cyclic AMP. In the short term, glucagon increases the concentration of fatty acid required to translocate the cytosolic reservoir of activity to the membranes on which phosphatidate is synthesized. Insulin opposes the combined actions of glucagon and glucocorticoids. The long-term events explain the large increases in the phosphohydrolase activity that occur in vivo in a variety of stress conditions. The expression of this activity depends on increases in the net availability of fatty acids and their CoA esters in the liver.