Development and regulation of ketogenesis in hepatocytes isolated from newborn rats.
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ABSTRACT: The development of fatty acid metabolism was studied in isolated hepatocytes from newborn rats. Ketone-body production from oleate is increased 6-fold between 0 and 16 h after birth. This increase is related to an enhanced beta-oxidation rather than to a channeling of acetyl-CoA from the tricarboxylic acid cycle to ketone-body synthesis. The increase in oleate oxidation is not related to a decreased esterification rate, as the latter is already low at birth and does not decrease further. At birth, lipogenic rate is 2-3-fold lower than in fed adult rats and it decreases to undetectable values in 16 h-old rats. A 90% inhibition of lipogenesis in hepatocytes of newborn rats (0 h) by glucagon and 5-(tetradecyloxy)-2-furoic acid does not lead to an increased oxidation of non-esterified fatty acids. This suggests that the inverse relationship between lipogenesis and ketogenesis in the starved newborn rat is not responsible for the switch-on of fatty acid oxidation at birth. Moreover, ketogenesis from octanoate, a medium-chain fatty acid the oxidation of which is independent of carnitine acyltransferase, follows the same developmental pattern at birth as that from oleate.
Project description:In hepatocytes from 1-day-old rats, active gluconeogenesis occurs in parallel with active ketogenesis, although the carbon atoms of non-esterified fatty acids do not participate in glucose synthesis. Once a significant ketogenesis is established, a further increase does not enhance gluconeogenesis. Indeed, octanoate is more ketogenic than oleate, but stimulates gluconeogenesis to a similar extent.
Project description:The rates of ketogenesis from endogenous substrates, butyrate or oleate, have been measured in isolated hepatocytes from suckling and weanling rats. Ketogenesis from endogenous substrate and from oleate decreased on weaning, whereas the rate from butyrate remained unchanged. It is concluded that the major site of regulation of ketogenesis during this period of development involves the disposal of long-chain fatty acyl-CoA between the esterification and beta-oxidation pathways. Modulators of lipogenesis [dihydroxyacetone and 5-(tetradecyloxy)-2-furoic acid] did not alter the rate of ketogenesis in hepatocytes from suckling rats, and it is suggested that this is due to the low rate of lipogenesis in these cells. Hepatocytes from fed weanling rats have a high rate of lipogenesis and evidence is presented for a reciprocal relationship between ketogenesis and lipogenesis, and ketogenesis, and esterification in these cells. Dibutyryl cyclic AMP stimulated ketogenesis from oleate in hepatocytes from fed weanling rats, even in the presence of an inhibitor of lipogenesis [5-(tetradecyloxy)-2-furoic acid], but not in cells from suckling rats. It is suggested that cyclic AMP may act via inhibition of esterification and that in hepatocytes from suckling rats ketogenesis is already maximally stimulated by the high basal concentrations of cyclic AMP [Beaudry, Chiasson & Exton (1977) Am. J. Physiol. 233, E175--E180].
Project description:Vasopressin (10nM) inhibited ketogenesis (56%) in hepatocytes from fed rats when oleate (1 mM) was the substrate, but had no effect with butyrate (10mM). The hormone increased the accumulation of lactate and stimulated the esterification of [1(-14)C]oleate (70%). These effects of vasopressin were reversed by glucagon (10 nM). The physiological implications of these findings are discussed.
Project description:In hepatocytes from 48 h-starved rats identical glucagon dose-response curves were obtained for the stimulation of gluconeogenesis from lactate, for ketogenesis and for the decreasing of the C5-dicarboxylate pool. Glucagon (20 nM) caused a 5-fold increase in 3-hydroxybutyrate formation, but decreased acetoacetate production 50% of that of the control. In hepatocytes from biotin-deficient rats glucagon no longer stimulated gluconeogenesis from lactate, but still produced its effects on the mitochondrial redox state and the C5-dicarboxylate pool. The results suggest that the primary site of the hormone action on gluconeogenesis is located within the mitochondria rather than in the cytosol.
Project description:THE DEVELOPMENT OF THE INNER MITOCHONDRIAL MEMBRANE IN FOETAL AND NEONATAL RAT LIVER WAS STUDIED BY FOLLOWING THREE PARAMETERS: (1) the activity of several respiratory enzymes in homogenates and purified mitochondria, (2) the spectrophotometric determination of cytochrome content in the mitochondria and (3) the cardiolipin content in both homogenates and purified mitochondria. Respiratory-enzyme activities of homogenates of foetal liver were one-quarter to one-twentieth of those of homogenates of adult liver, and the enzyme specific activities in purified mitochondria from foetal liver were one-half to one-eighth of those in mitochondria from adult liver. The cardiolipin content of liver homogenates increased approximately twofold during the development period, but there was no significant change in the cardiolipin content of purified mitochondria. It is concluded that cell mitochondrial content approximately doubles in the immediate postnatal period. There was no evidence for an increase in the relative amount of cristae protein in mitochondria during this period to account for increases in mitochondrial enzyme specific activity, since cardiolipin and cytochrome concentrations remained unchanged and electron micrographs revealed no differences. The cause of the lower respiratory-enzyme specific activity in foetal liver mitochondria is unclear. Qualitative differences in respiratory units in foetal and mature animals are suggested.
Project description:Organs from nonheart-beating donors are attractive for use in cell therapy. Understanding the nature of molecular perturbations following reperfusion/reoxygenation would be highly significant for nonheart-beating donor cells. Nonheart-beating donor rats hepatocytes were studied for global gene expression with Affymetrix microarraays.
Project description:RationaleInhaled nitric oxide (NO) has been used to prevent bronchopulmonary dysplasia, but with variable results. Ethyl nitrite (ENO) forms S-nitrosothiols more readily than does NO, and resists higher-order nitrogen oxide formation. Because S-nitrosylation is a key pathway mediating many NO biological effects, treatment with inhaled ENO may better protect postnatal lung development from oxidative stress than NO.ObjectivesTo compare inhaled NO and ENO on hyperoxia-impaired postnatal lung development.MethodsWe treated newborn rats beginning at birth to air or 95% O(2) +/- 0.2-20.0 ppm ENO for 8 days, or to 10 ppm NO for 8 days. Pups treated with the optimum ENO dose, 10 ppm, and pups treated with 10 ppm NO were recovered in room air for 6 more days.Measurements and main resultsENO and NO partly prevented 95% O(2)-induced airway neutrophil influx in lavage, but ENO had a greater effect than did NO in prevention of lung myeloperoxidase accumulation, and in expression of cytokine-induced neutrophil chemoattractant-1. Treatment with 10 ppm ENO, but not NO, for 8 days followed by recovery in air for 6 days prevented 95% O(2)-induced impairments of body weight, lung compliance, and alveolar development.ConclusionsInhaled ENO conferred protection superior to inhaled NO against hyperoxia-induced inflammation. ENO prevented hyperoxia impairments of lung compliance and postnatal alveolar development in newborn rats.
Project description:Organs from non-heart-beating donors are attractive for use in cell therapy. Understanding the nature of molecular perturbations following reperfusion/reoxygenation will be highly significant for non-heart-beating donor cells. We studied non-heart-beating donor rats for global gene expression with Affymetrix microarrays, hepatic tissue integrity, viability of isolated hepatocytes, and engraftment and proliferation of transplanted cells in dipeptidyl peptidase IV-deficient rats. In non-heart-beating donors, liver tissue was morphologically intact for >24 hours with differential expression of 1, 95, or 372 genes, 4, 16, or 34 hours after death, respectively, compared with heart-beating donors. These differentially expressed genes constituted prominent groupings in ontological pathways of oxidative phosphorylation, adherence junctions, glycolysis/gluconeogenesis, and other discrete pathways. We successfully isolated viable hepatocytes from non-heart-beating donors, especially up to 4 hours after death, although the hepatocyte yield and viability were inferior to those of hepatocytes from heart-beating donors (P < 0.05). Similarly, although hepatocytes from non-heart-beating donors engrafted and proliferated after transplantation in recipient animals, this was inferior to hepatocytes from heart-beating donors (P < 0.05). Gene expression profiling in hepatocytes isolated from non-heart-beating donors showed far greater perturbations compared with corresponding liver tissue, including representation of pathways in focal adhesion, actin cytoskeleton, extracellular matrix-receptor interactions, multiple ligand-receptor interactions, and signaling in insulin, calcium, wnt, Jak-Stat, or other cascades.Liver tissue remained intact over prolonged periods after death in non-heart-beating donors, but extensive molecular perturbations following reperfusion/reoxygenation impaired the viability of isolated hepatocytes from these donors. Insights into molecular changes in hepatocytes from non-heart-beating donors offer opportunities for improving donor cell viability, which will advance the utility of non-heart-beating donor organs for cell therapy or other applications.
Project description:Preclinical studies have demonstrated that intrauterine growth retardation (IUGR) is associated with reduced lung development during the neonatal period and infancy. Uteroplacental insufficiency (UPI), affecting approximately 10% of human pregnancies, is the most common cause of IUGR. This study investigated the effects of UPI on lung development and the intestinal microbiota and correlations in newborn rats with IUGR, using bilateral uterine artery ligation to induce UPI. Maternal fecal samples were collected on postnatal day 0. On postnatal days 0 and 7, lung and intestinal microbiota samples were collected from the left lung and the lower gastrointestinal tract. The right lung was harvested for histological assessment and Western blot analysis. Results showed that UPI through bilateral uterine artery ligation did not alter the maternal gut microbiota. IUGR impaired lung development and angiogenesis in newborn rats. Moreover, on postnatal day 0, the presence of Acinetobacter and Delftia in the lungs and Acinetobacter and Nevskia in the gastrointestinal tract was negatively correlated with lung development. Bacteroides in the lungs and Rodentibacter and Romboutsia in the gastrointestinal tract were negatively correlated with lung development on day 7. UPI may have regulated lung development and angiogenesis through the modulation of the newborn rats' intestinal and lung microbiota.
Project description:Lipogenesis is increased in hepatocytes from fed lactating rats compared with virgin rats. Inhibition of lipogenesis with 5-(tetradecyloxy)-2-furoic acid resulted in increased ketogenesis from endogenous substrate, but not from oleate. Dihydroxyacetone increased ketogenesis from endogenous substrate, but not from oleate. Dihydroxyacetone increased lipogenesis and esterification of [1--14C]oleate and decreased ketogenesis; these changes were reversed by the inhibitor. The reciprocal relationship between lipogenesis and ketogenesis in hepatocytes from fed rats may be due to alterations in [malonyl-CoA] [McGarry, Mannaerts & Foster (1977) J. Clin. Invest. 60, 265--270; Cook, King & Veech (1978) J. Biol. Chem. 253, 2529--2531], but this mechanism is not considered to be sufficient to explain the increased ketogenesis in starvation completely.