The Transcriptional Effects of PCB118 and PCB153 on the Liver, Adipose Tissue, Muscle and Colon of Mice: Highlighting of Glut4 and Lipin1 as Main Target Genes for PCB Induced Metabolic Disorders.

Epidemiological studies have associated environmental exposure to polychlorinated biphenyls (PCBs) with an increased risk of type 2 diabetes; however, little is known about the underlying mechanisms involved in the metabolic side-effects of PCB. Our study evaluated the transcriptional effects of a subchronic exposure (gavage at Day 0 and Day 15 with 10 or 100 µmol/Kg bw) to PCB118 (dioxin-like PCB), PCB153 (non-dioxin-like PCB), or an equimolar mixture of PCB118 and PCB153 on various tissues (liver, visceral adipose tissue, muscle, and colon) in mice. Our results showed that a short-term exposure to PCB118 and/or PCB153 enhanced circulating triglyceride levels but did not affect glycemia. Among the studied tissues, we did not observe any modification of the expression of inflammation-related genes, such as cytokines or chemokines. The main transcriptional effects were observed in visceral adipose and liver tissues. We found a downregulation of lipin1 and glut4 expression in these two target organs. In adipose tissue, we also showed a downregulation of Agpat2, Slc25a1, and Fasn. All of these genes are involved in lipid metabolism and insulin resistance. In muscles, we observed an induction of CnR1 and Foxo3 expression, which may be partly involved in PCB metabolic effects. In summary, our results suggest that lipin1 and glut4, notably in adipose tissue, are the main targeted genes in PCB-induced metabolic disorders, however, further studies are required to fully elucidate the mechanisms involved.

Phosphatidic acid and diacylglycerol generation is regulated by insulin in cerebral cortex synaptosomes from adult and aged rats.

Insulin receptor associated with the cerebral cortex (CC) has been shown to be involved in brain cognitive functions. Furthermore, deterioration of insulin signaling has been associated with age-related brain degeneration. We have reported previously that aging stimulates phospholipase D/phosphatidate phosphohydrolase 2 (PLD/PAP2) pathway in CC synaptosomes from aged rats, generating a differential availability of their reaction products: diacylglycerol (DAG) and phosphatidic acid (PA). The aim of this work was to determine the effect of aging on DAG kinase (DAGK), as an alternative pathway for PA generation, and to evaluate the effect of insulin on PLD/PAP2 pathway and DAGK. PLD, PAP2, and DAGK activities were measured using specific radiolabeled substrates in CC synaptosomes from adult (4 months old) and aged rats (28 months old). In adult animals, in the presence of the tyrosine phosphatase inhibitor (sodium o-vanadate), insulin stimulated PLD activity at 5 min incubation. DAGK activity was also increased at the same time of incubation and PAP2 was inhibited. In aged animals, PLD activity was not modified by the presence of insulin plus vanadate, PAP2 was inhibited, and DAGK was stimulated by the hormone. Insulin, vanadate, and the combination of both induced protein tyrosine phosphorylation in adult CC synaptosomes. Aged rats showed a lower level of protein phosphorylation with respect to adult rats. Our results show that insulin modulates PA and DAG availability through the regulation of PLD/PAP2 and DAGK pathways in adult rat CC synaptosomes. Additionally, we demonstrated that PA and DAG generation is regulated differentially by insulin during aging.

Phosphoinositides, ezrin/moesin, and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors.

The post-Golgi trafficking of rhodopsin in photoreceptor cells is mediated by rhodopsin-bearing transport carriers (RTCs) and regulated by the small GTPase rab8. In this work, we took a combined pharmacological-proteomic approach to uncover new regulators of RTC trafficking toward the specialized light-sensitive organelle, the rod outer segment (ROS). We perturbed phospholipid synthesis by activating phospholipase D with sphingosine 1-phosphate (S1P) or inhibiting phosphatidic acid phosphohydrolase by propranolol (Ppl). S1P stimulated the overall rate of membrane trafficking toward the ROS. Ppl stimulated budding of RTCs, but blocked membrane delivery to the ROS. Ppl caused accumulation of RTCs in the vicinity of the fusion sites, suggesting a defect in tethering, similar to the previously described phenotype of the rab8T22N mutant. Proteomic analysis of RTCs accumulated upon Ppl treatment showed a significant decrease in phosphatidylinositol-4,5-bisphosphate-binding proteins ezrin and/or moesin. Ppl induced redistribution of moesin, actin and the small GTPase rac1 from RTCs into the cytosol. By confocal microscopy, ezrin/moesin and rac1 colocalized with rab8 on RTCs at the sites of their fusion with the plasma membrane; however, this distribution was lost upon Ppl treatment. Our data suggest that in photoreceptors phosphatidylinositol-4,5-bisphosphate, moesin, actin, and rac1 act in concert with rab8 to regulate tethering and fusion of RTCs. Consequentially, they are necessary for rhodopsin-laden membrane delivery to the ROS, thus controlling the critical steps in the biogenesis of the light-detecting organelle.

Cycloalliin, a cyclic sulfur imino acid, reduces serum triacylglycerol in rats.

Allium species such as onions and garlic are used as foodstuff, condiment, flavoring, and folk medicine. Onions may decrease hyperlipidemia and improve atherosclerosis. However, the ingredients in onion that are responsible for this phenomenon are not known. In the present study, we investigated the effects of cycloalliin, a sulfur-containing imino acid in onions, on lipid metabolism in Sprague-Dawley rats. When supplemented at the 0.1% and 0.3% levels to the atherogenic diet, cycloalliin reduced serum triacylglycerol (TAG) concentration by approximately 40% compared to the control. Serum cholesterol ester level also showed a tendency to decrease in cycloalliin groups. Hepatic lipid levels were comparable among the groups, although TAG and phospholipid contents were slightly higher in both cycloalliin groups. Dietary cycloalliin had no significant effect on hepatic enzyme activities responsible for TAG synthesis (phosphatidate phosphohydrolase, malic enzyme, and glucose-6-phosphate dehydrogenase (G6PDH)). In conclusion, dietary cycloalliin has serum TG-lowering effect without affecting hepatic TAG synthesis and content in rats, suggesting an alteration of lipoprotein assembly and secretion processes in the liver.

Hepatic drug metabolizing enzyme induction and serum triacylglycerol elevation in rats treated with chlordiazepoxide, griseofulvin, rifampicin and phenytoin.

Five days intraperitoneal administration of rats with chlordiazepoxide (0.4 mg/kg), griseofulvin (7 mg/kg), rifampicin (8. 6 mg/kg), phenytoin (4.3 mg/kg) and phenobarbitone (1.4 mg/kg; an established inducer of microsomal enzymes) caused a significant increase in serum triacylglycerol (P<0.001) and the activities of aniline hydroxylase, aminopyrine N-demethylase and p-nitroanisole O-demethylase (P<0.001). Aniline hydroxylase, aminopyrine N-demethylase and p-nitroanisole O-demethylase activities were increased 1.48-, 1.15- and 1.47-fold, respectively, in chlordiazepoxide-treated rats, 1.65-, 1.20- and 1.38-fold in griseofulvin-treated rats, 1.74-, 1.36- and 1.44-fold in rifampicin-treated rats, 1.56-, 1.29- and 1.62-fold in phenytoin-treated rats and 2.26-, 1.72- and 1.93-fold in phenobarbitone-treated rats. Chlordiazepoxide, griseofulvin, rifampicin, phenytoin and phenobarbitone increased the activity of cytosolic phosphatidate phosphohydrolase by 52, 58, 67, 73 and 82%, respectively, while the drugs elicited 50, 60, 60, 73 and 87% increases in the activity of the microsomal phosphatidate phosphohydrolase. Similarly, chlordiazepoxide, griseofulvin, rifampicin, phenytoin and phenobarbitone elicited 2.4-, 2.39-, 2.34-, 1.69- and 3.75-fold increases in serum triacylglycerol concentrations. The correlations between serum triacylglycerol concentrations and the activities of aniline hydroxylase, aminopyrine N-demethylase and p-nitroanisole O-demethylase were significant in all treatment groups (r=0.83, r=0.92 and r=0.87, respectively, n=30, P<0.001). Our results suggest that induction of hepatic enzymes by the administered drugs may lead to hypertriglyceridaemia as an adverse effect, possibly by inducing the activity of regulatory enzymes in the biosynthesis of triglyceride.

Erythromycin A-derived macrolides modify the functional activities of human neutrophils by altering the phospholipase D-phosphatidate phosphohydrolase transduction pathway: L-cladinose is involved both in alterations of neutrophil functions and modulation of this transductional pathway.

All erythromycin A derivatives, irrespective of the size of the lactone ring and the nature of the substituent, inhibit oxidant production by neutrophils and promote their degranulation. We demonstrate in this study that the L-cladinose at position 3 of the lactone ring is a key structure in the modulation of these two neutrophil functions, suggesting that this sugar (alone or combined with a lactone structure) interferes with cell target(s) involved in both oxidant production and exocytosis. Taking roxithromycin as an example of erythromycin A derivatives, we also found that these molecules interfered with the phospholipase D (PLD)-phosphatidate phosphohydrolase pathway in two ways. In nonstimulated neutrophils, roxithromycin and all L-cladinose-bearing molecules activated PLD, as reflected by 1-O-[3H]alkyl-2-acyl-phosphatidyl-ethanol production. In addition, these drugs induced an accumulation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid (PA), but not 1-O-[3H]alkyl-2-acylglycerol. PA accumulation seems to be involved in the induction of exocytosis by macrolides, as the roxithromycin-induced release of granular enzymes was impaired strongly in the presence of ethanol. By contrast, in stimulated neutrophils, roxithromycin inhibited PLD activity and totally impaired 1-O-[3H]alkyl-2-acylglycerol production. The inhibition of diglyceride production by roxithromycin (not its descladinosyl derivative) could explain its inhibitory effect on oxidant production. The relevance of our data to the clinical situation, particularly the anti-inflammatory activity of these drugs, requires further investigation.

Phosphatidate phosphohydrolase catalyzes the hydrolysis of ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate.

A Mg2+-independent phosphatidate phosphohydrolase was purified from rat liver plasma membranes in two distinct forms, an anionic protein and a cationic protein. Both forms of the enzyme dephosphorylated phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate. When assayed at a constant molar ratio of lipid to Triton X-100 of 1:500, the apparent Km values of the anionic phosphohydrolase for the lipid substrates was 3.5, 1.9, 0.4, and 4.0 microM, respectively. The relative catalytic efficiency of the enzyme for phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate was 0.16, 0.14, 0.48, and 0.04 liter (min x mg)-1, respectively. The hydrolysis of phosphatidate was inhibited competitively by ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate. The Ki(app) values were 5.5, 5.9, and 4.0 microM, respectively. The hydrolysis of phosphatidate by the phosphohydrolase conformed to a surface dilution kinetic model. It is concluded that the enzyme is a lipid phosphomonoesterase that could modify the balance of phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate relative to diacylglycerol, ceramide, monoacylglycerol, and sphingosine, respectively. The enzyme could thus play an important role in regulating cell activation and signal transduction.

Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes.

Plasma membrane phosphatidic acid phosphohydrolase (PAPH) plays an important role in signal transduction by converting phosphatidic acid to diacylglycerol. PAPH-2, a Mg(2+)-independent, detergent-dependent enzyme involved in cellular signal transduction, is reportedly absent from the plasma membranes of neutrophilic leukocytes, a cell that responds to metabolic stimulation with abundant phospholipase D-dependent diacylglycerol generation. The present study was designed to resolve this discrepancy, focusing on the influence of cellular disruption techniques, detergent availability and cation sensitivity on the apparent distribution of PAPH in neutrophil subcellular fractions. The results clearly indicate the presence of two distinct types of PAPH within the particulate and cytosolic fractions of disrupted cells. Unlike the cytosolic enzyme, the particulate enzymes was not potentiated by magnesium and was strongly detergent-dependent. The soluble and particulate enzymes displayed dissimilar pH profiles. Separation of neutrophil particulate material into fractions rich in plasma membranes, specific granules and azurophilic granules by high speed discontinuous density gradient centrifugation revealed that the majority of the particulate activity was confined to plasma membranes. This activity was not inhibited by pretreatment with n-ethyl-maleimide in concentrations as high as 25 mM. PAPH activity recovered in the cytosolic fraction of disrupted neutrophils was almost completely inhibited by 5.0 mM n-ethylmaleimide. We conclude that resting neutrophils possess n-ethylmaleimide-resistant PAPH (type 2) within their plasma membranes. This enzyme may markedly influence the kinetics of cell activation by metabolizing second messengers generated as a result of activation of plasma membrane phospholipase D.

Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by nucleotides.

Regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by nucleotides was examined using pure enzyme and Triton X-100/phosphatidate-mixed micelles. Adenosine, guanosine, cytidine, and uridine nucleotides inhibited phosphatidate phosphatase activity in a dose-dependent manner. ATP and CTP were the most potent inhibitors of the enzyme. A kinetic analysis was performed to determine the mechanism of enzyme inhibition by nucleotides. The mechanism of inhibition by ATP and CTP with respect to phosphatidate (the substrate) was complex. The dependence of phosphatidate phosphatase activity on phosphatidate was cooperative, and nucleotides affected both Vmax and Km. ATP did not inhibit phosphatidate phosphatase activity by binding to the enzyme or to phosphatidate. Phosphatidate phosphatase dependence on Mg2+ ions (the cofactor) followed saturation kinetics, and the mechanism of nucleotide inhibition with respect to Mg2+ ions was competitive. Thus, the mechanism of enzyme inhibition by nucleotides was the chelation of Mg2+ ions. The inhibitor constant for ATP was lower than its cellular concentration in glucose-grown cells. However, the inhibitor constant for ATP was higher than its cellular concentration in glucose-starved cells. Changes in the cellular concentration of ATP affected the proportional synthesis of triacylglycerols and phospholipids. These results were consistent with the regulation of phosphatidate phosphatase activity by ATP through a Mg2+ ion chelation mechanism.

Low concentration of Triton X-100 inhibits diacylglycerol acyltransferase without measurable effect on phosphatidate phosphohydrolase in the human primordial placenta.

Agents causing accumulation of endogenous diacylglycerols (DAG) may be helpful in studies on the intracellular regulatory effects and on the mechanisms of attenuation of this putative second messenger. In a previous study (Tóth et al., BBA 921 (1987) 417-425) we have shown a marked stimulatory effect of low micellar concentration (0.05%, v/v) of Triton X-100 on the labelling of phosphatidic acid with (32P)phosphate in minced human primordial placenta. The present results demonstrate that 0.05% Triton X-100 inhibits nearly completely the conversion of (3H)diacylglycerols into (3H)triacylglycerols in placenta fragments incubated with (3H)glucose. However, this concentration of the detergent does not have any effect on the appearance of label in the sum of acylglycerols (comprising mono-, di- and triacylglycerols) and phosphatidylcholine, indicating a lack of effect on phosphatidate phosphohydrolase. The about 5-fold elevation of (3H)diacylglycerols was attended by an approximately 3-fold rise in (3H)phosphatidic acid and a 1.3-fold increase in the labelling of phosphatidylcholine. These findings provide supportive evidence that 1,2-DAG was formed due to inhibition of DAG-acyltransferase and suggest that some of the DAG was transformed into phosphatidic acid by diacylglycerol-kinase.

Effects of okadaic acid on the activities of two distinct phosphatidate phosphohydrolases in rat hepatocytes.

Incubation of hepatocytes with okadaic acid displaced the N-ethylmaleimide-sensitive phosphatidate phosphohydrolase from the membrane fraction into the cytosol and partially prevented the oleate-induced movement of phosphohydrolase from cytosol to membranes. However, higher concentrations of oleate still caused translocation and activation of the phosphohydrolase. This enzyme is stimulated by Mg2+, and is probably involved in glycerolipid synthesis. Okadaic acid also decreased the concentration of diacylglycerol within the hepatocytes. Okadiac acid had no observable effect on the activity of an N-ethylmaleimide-insensitive phosphatidate phosphohydrolase which remained firmly attached to membranes. This activity is not stimulated by Mg2+ and is probably involved in signal transduction by the phospholipase D pathway.

Inhibitory effect of epinephrine on phosphatidate activity in isolated rat hepatocytes.

The variations in phosphatidate phosphohydrolase activity were investigated in the post-mitochondrial fraction of isolated rat hepatocytes incubated for short periods with epinephrine, dibutyryl cyclic AMP or oleate. Epinephrine decreased the enzyme activity by 42% at 1 microM concentration. The inhibitory effect was abolished in the presence of a beta-adrenoceptor antagonist, propranolol, but was not affected by the alpha-adrenoceptor agonist, phenylephrine, or the agonist, phentolamine. Dibutyryl-cAMP inhibited the enzyme activity by 49%. The presence of the cyclic-AMP phosphodiesterase inhibitor, aminophyline, together with epinephrine slightly increased the enzyme inhibition. Oleate stimulated the enzyme activity (100%) and its effect was antagonized by dibutyryl-cyclic AMP (24%) and by epinephrine (15%). The results indicate that epinephrine acts on rat hepatocytes via beta-adrenoceptor activation and cAMP may be involved in the mechanism by which phosphatidate phosphohydrolase is regulated.

Rat adipose tissue phosphatidic acid phosphatase: lack of effect of nucleotides on cytosolic enzyme activity.

The soluble phosphatidic acid phosphatase from rat adipose tissue was partially purified using ammonium sulfate fractionation and hydroxyapatite chromatography. Administration of ethanol has been found to increase phosphatidic acid phosphatase activity. The enzyme activity has been found to be dependent on magnesium ions with maximal activity at 2-5 mM magnesium. The enzyme displays an apparent pH optimum of 7.0. The activity of the enzyme is not affected by addition of ATP or ADP, in contrast with the results for hepatic phosphatidic acid phosphatase. The results suggest that these two enzymes may be regulated by different mechanisms and that they may thus represent two different types of isoenzyme.