Inhibition of lipogenesis by halothane in isolated rat liver cells.
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ABSTRACT:
SUBMITTER: Mapes JP
PROVIDER: S-EPMC1164568 | biostudies-other | 1977 Jan
REPOSITORIES: biostudies-other
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Project description:The livers of insulin-resistant, diabetic mice manifest selective insulin resistance, suggesting a bifurcation in the insulin signaling pathway: Insulin loses its ability to block glucose production (i.e., it fails to suppress PEPCK and other genes of gluconeogenesis), yet it retains its ability to stimulate fatty acid synthesis (i.e., continued enhancement of genes of lipogenesis). Enhanced lipogenesis is accompanied by an insulin-stimulated increase in the mRNA encoding SREBP-1c, a transcription factor that activates the entire lipogenic program. Here, we report a branch point in the insulin signaling pathway that may account for selective insulin resistance. Exposure of rat hepatocytes to insulin produced a 25-fold increase in SREBP-1c mRNA and a 95% decrease in PEPCK mRNA. Insulin-mediated changes in both mRNAs were blocked by inhibitors of PI3K and Akt, indicating that these kinases are required for both pathways. In contrast, subnanomolar concentrations of rapamycin, an inhibitor of the mTORC1 kinase, blocked insulin induction of SREBP-1c, but had no effect on insulin suppression of PEPCK. We observed a similar selective effect of rapamycin in livers of rats and mice that experienced an insulin surge in response to a fasting-refeeding protocol. A specific inhibitor of S6 kinase, a downstream target of mTORC1, did not block insulin induction of SREBP-1c, suggesting a downstream pathway distinct from S6 kinase. These results establish mTORC1 as an essential component in the insulin-regulated pathway for hepatic lipogenesis but not gluconeogenesis, and may help to resolve the paradox of selective insulin resistance in livers of diabetic rodents.
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Project description:Fat-cells were prepared from rat and guinea-pig epididymal adipose tissue and compared on the basis of the intracellular distributions and activities of enzymes and with respect to their utilization of various U-(14)C-labelled substrates for lipogenesis. 1. Compared with the rat, guinea-pig extramitochondrial enzyme activities differed in that aconitate hydratase, alanine aminotransferase, ATP-citrate lyase, lactate dehydrogenase, NAD-malate dehydrogenase, NADP-malate dehydrogenase and phosphoenolpyruvate carboxykinase activities were appreciably lower, whereas aspartate aminotransferase, glucose 6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase activities were appreciably higher. Mitochondrial activities of citrate synthase, NADP-isocitrate dehydrogenase and pyruvate carboxylase were appreciably lower, whereas mitochondrial activities of aspartate aminotransferase, glutamate dehydrogenase, NAD-malate dehydrogenase and phosphoenolpyruvate carboxykinase were higher in the guinea pig compared with the rat. 2. In general guinea-pig fat-cells incorporated acetate and lactate into fatty acids more readily than rat fat-cells, whereas rat fat-cells incorporated glucose and pyruvate more readily than guinea-pig fat-cells. 3. Acetate stimulated the incorporation of glucose into fatty acids in rat fat-cells, but had no appreciable effect upon this process in guinea-pig fat-cells. Acetate greatly decreased the incorporation of lactate into fatty acids in cells from both species. 4. Lactate/pyruvate ratios produced by incubation of guinea-pig cells with glucose+insulin were very low compared with those found with rat cells under the same conditions. 5. With glucose (+insulin) or with glucose+acetate (+insulin) as substrates guinea-pig cells produced enough NADPH by the hexose monophosphate pathway to satisfy the NADPH requirements of lipogenesis. In rat fat-cells under the same conditions, hexose monophosphate-pathway NADPH provision was not sufficient to meet the requirements of lipogenesis. 6. These results are discussed, particularly in relationship to the disposition of cytosolic reducing equivalents in the cells.
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Project description:Severe halothane (HAL)-induced hepatotoxicity occurs in one in 6000-30,000 patients by an unknown mechanism. Female sex is a risk factor in humans and rodents. We tested the hypothesis that a sex difference in natural killer (NK) cell activity contributes to HAL-induced liver injury. HAL (15 mmol/kg, ip) treatment resulted in severe liver injury by 12 h in female, wild-type BALB/cJ mice, and the magnitude of liver injury varied with stage of the estrous cycle. Ovariectomized (OVX) mice developed only mild liver injury. Plasma interferon-gamma (IFN-?) was elevated 10-fold in HAL-treated females compared with similarly treated male mice or with OVX female mice. IFN-? knockout mice were resistant to severe HAL-induced liver injury. The deactivation of NK cells with anti-asialo GM1 treatment attenuated liver injury and the increase in plasma IFN-? compared with immunoglobulin G-treated control mice. Mice with a mutated form of perforin, a protein involved in granule-mediated cytotoxicity, were protected from severe liver injury. Furthermore, HAL increased the activity of NK cells in vivo, as indicated by increased surface expression of CD69, an early activation marker. In response to HAL, NK cell receptor ligands on the surface of hepatocytes were expressed in a manner that can activate NK cells. These results confirm the sexual dimorphic hepatotoxic response to HAL in mice and suggest that IFN-? and NK cells have essential roles in the development of severe HAL-induced hepatotoxicity.
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Project description:The metabolism of proline was studied in liver cells isolated from starved rats. The following observations were made. 1. Consumption of proline could be largely accounted for by production of glucose, urea, glutamate and glutamine. 2. At least 50% of the total consumption of oxygen was used for proline catabolism. 3. Ureogenesis and gluconeogenesis from proline could be stimulated by partial uncoupling of oxidative phosphorylation. 4. Addition of ethanol had little effect on either proline uptake or oxygen consumption, but strongly inhibited the production of both urea and glucose and caused further accumulation of glutamate and lactate. Accumulation of glutamine was not affected by ethanol. 5. The effects of ethanol could be overcome by partial uncoupling of oxidative phosphorylation. 6. The apparent K(m) values of argininosuccinate synthetase (EC 6.3.4.5) for aspartate and citrulline in the intact hepatocyte are higher than those reported for the isolated enzyme. 7. 3-Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase (EC 4.1.1.32), greatly enhanced cytosolic aspartate accumulation during proline metabolism, but inhibited urea synthesis. 8. It is concluded that when proline is provided as a source of nitrogen to liver cells, production of ammonia by oxidative deamination of glutamate is inhibited by the highly reduced state of the nicotinamide nucleotides within the mitochondria. 9. Conversion of proline into glucose and urea is a net-energy-yielding process, and the high state of reduction of the nicotinamide nucleotides is presumably maintained by a high phosphorylation potential. Thus when proline is present as sole substrate, the further oxidation of glutamate by glutamate dehydrogenase (EC 1.4.1.3) is limited by the rate of energy expenditure of the cell.
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Project description:Background & aimsHepatic inflammation occurs immediately after cells are transplanted to the liver, but the mechanisms that underlie this process are not fully defined. We examined cyclooxygenase pathways that mediate hepatic inflammation through synthesis of prostaglandins, prostacyclins, thromboxanes, and other prostanoids following transplantation of hepatocytes.MethodsWe transplanted F344 rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient F344 rats. Changes in cyclooxygenase pathways were analyzed, and specific pathways were blocked pharmacologically; the effects on cell engraftment and native liver cells were determined.ResultsTransplantation of hepatocytes induced hepatic expression of prostaglandin-endoperoxide synthases 1 and 2, which catalyze production of prostaglandin H2, as well as the downstream factor thromboxane synthase, which produces thromboxane A2 (a regulator of vascular and platelet responses in inflammation). Transplanted hepatocytes were in proximity with liver cells that expressed prostaglandin-endoperoxide synthases. The number of engrafted hepatocytes increased in rats given naproxen or celecoxib before transplantation but not in rats given furegrelate (an inhibitor of thromboxane synthase) or clopodigrel (an antiplatelet drug). Naproxen and celecoxib did not prevent hepatic ischemia or activation of neutrophils, Kupffer cells, or inflammatory cytokines, but they did induce hepatic stellate cells to express cytoprotective genes, vascular endothelial growth factor and hepatocyte growth factor, and matrix-type metalloproteinases and tissue inhibitor of metalloproteinase-1, which regulate hepatic remodeling.ConclusionsActivation of cyclooxygenase pathways interferes with engraftment of transplanted hepatocytes in the liver. Pharmacologic blockade of prostaglandin-endoperoxide synthases stimulated hepatic stellate cells and improved cell engraftment.
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Project description:1. With reference to the post-operative dysfunction of the liver observed after halothane anaesthesia, the effects of the anaesthetic on some metabolic functions were studied in the isolated perfused rat liver. Oxygen uptake, glycolysis, gluconeogenesis and urea synthesis were affected by halothane at a concentration (2.5% of the gas phase) within the range used in clinical anaesthesia. 2. At this concentration of halothane uptake of oxygen was inhibited in livers from both fed and starved rats. 3. In livers from fed rats there was a 16-fold increase in lactate production. This was accompanied by a fivefold decrease in the tissue content of 2-oxoglutarate and a more than twofold decrease in citrate. The calculated [free NAD(+)]/[free NADH] ratio in both cytoplasm and mitochondria was lower in the halothane-exposed livers than in controls. 4. In livers of starved rats the rate of gluconeogenesis from lactate was decreased by halothane to 30% of the control rate. 5. Halothane inhibited gluconeogenesis from alanine and propionate to the same extent as from lactate, whereas glucose formation from dihydroxyacetone, glycerol, fructose and sorbitol was relatively unaffected. 6. During gluconeogenesis from 10mm-lactate the tissue content of ATP was decreased by 50%, glutamate by 50% and 2-oxoglutarate was decreased eightfold in the halothane-exposed livers. 7. Halothane decreased urea synthesis in the presence of 10mm-NH(4)Cl and 2mm-ornithine to 15% of the control rate. 8. The inhibitions of gluconeogenesis and urea synthesis were completely abolished within 15min of withdrawal of the anaesthetic. 9. The stimulation of uptake of oxygen brought about by the addition of lactate or precursors of urea was abolished by halothane. 10. Effects on gluconeogenesis similar to those of halothane occurred in livers exposed to the anaesthetic methoxyflurane, although normal rates were not restored on withdrawal of the drug. Other anaesthetic agents tested (ketamine-HCl and trichloroethylene) decreased gluconeogenesis to 66% of the control rate. 11. The inhibitory effects of halothane are consistent with an interference at the stage of the NADH dehydrogenase of the electron-transport chain.
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