Regulation of ketogenesis, gluconeogenesis and the mitochondrial redox state by dexamethasone in hepatocyte monolayer cultures.
ABSTRACT: The effects of the glucocorticoid dexamethasone on fatty acid and pyruvate metabolism were studied in rat hepatocyte cultures. Parenchymal hepatocytes were cultured for 24 h with nanomolar concentrations of dexamethasone in either the absence or the presence of insulin (10 nM) or dibutyryl cyclic AMP (1 microM BcAMP). Dexamethasone (1-100 nM) increased the rate of formation of ketone bodies from 0.5 mM-palmitate in both the absence and the presence of BcAMP, but inhibited ketogenesis in the presence of insulin. Dexamethasone increased the proportion of the palmitate metabolized that was partitioned towards oxidation to ketone bodies, and decreased the cellular [glycerol 3-phosphate]. The latter suggests that the increased partitioning of palmitate to ketone bodies may be associated with decreased esterification to glycerolipid. The Vmax. of carnitine palmitoyltransferase (CPT) and the affinity of CPT for palmitoyl-CoA were not affected by dexamethasone, indicating that the increased ketogenesis was not due to an increase in enzymic capacity for long-chain acylcarnitine formation. Dexamethasone and BcAMP, separately and in combination, increased gluconeogenesis. In the presence of insulin, however, dexamethasone inhibited gluconeogenesis. Changes in gluconeogenesis thus paralleled changes in ketogenesis. Dexamethasone decreased the [3-hydroxybutyrate]/[acetoacetate] ratio, despite increasing the rate of ketogenesis and presumably the mitochondrial production of reducing equivalents. The more oxidized mitochondrial NADH/NAD+ redox couple with dexamethasone is probably due either to an increased rate of electron transport or to increased transfer of mitochondrial reducing equivalents to the cytoplasm.
Project description:The contribution of pyruvate to ketogenesis was determined in rat hepatocyte suspensions by using [14C]pyruvate. The rates of conversion of pyruvate into ketone bodies in hepatocytes from fed and 24 h-starved rats were 10 and 17 mumol/h per g wet wt. respectively, and accounted for 50 and 29% of the total ketone bodies formed. In hepatocytes from fed rats, the addition of palmitate (0.25-1 mM) increased the rate of conversion of pyruvate into ketone bodies (80-140%), but decreased the relative contribution of pyruvate to total ketogenesis. In hepatocytes from starved rats, palmitate did not increase pyruvate conversion into ketone bodies.
Project description:During starvation for 72 h, tumour-bearing rats showed accelerated ketonaemia and marked ketonuria. Total blood [ketone bodies] were 8.53 mM and 3.34 mM in tumour-bearing and control (non-tumour-bearing) rats respectively (P less than 0.001). The [3-hydroxybutyrate]/[acetoacetate] ratio was 1.3 in the tumour-bearing rats, compared with 3.2 in the controls at 72 h (P less than 0.001). Blood [glucose] and hepatic [glycogen] were lower at the start of starvation in tumour-bearing rats, whereas plasma [non-esterified fatty acids] were not increased above those in the control rats during starvation. After functional hepatectomy, blood [acetoacetate], but not [3-hydroxybutyrate], decreased rapidly in tumour-bearing rats, whereas both ketone bodies decreased, and at a slower rate, in the control rats. Blood [glucose] decreased more rapidly in the hepatectomized control rats. Hepatocytes prepared from 72 h-starved tumour-bearing and control rats showed similar rates of ketogenesis from palmitate, and the distribution of [1-14C] palmitate between oxidation (ketone bodies and CO2) and esterification was also unaffected by tumour-bearing, as was the rate of gluconeogenesis from lactate. The carcinoma itself showed rapid rates of glycolysis and a poor ability to metabolize ketone bodies in vitro. The results are consistent with the peripheral, normal, tissues in tumour-bearing rats having increased ketone-body and decreased glucose metabolic turnover rates.
Project description:Isolated liver cells prepared from starved sheep converted palmitate into ketone bodies at twice the rate seen with cells from fed animals. Carnitine stimulated palmitate oxidation only in liver cells from fed sheep, and completely abolished the difference between fed and starved animals in palmitate oxidation. The rates of palmitate oxidation to CO2 and of octanoate oxidation to ketone bodies and CO2 were not affected by starvation or carnitine. Neither starvation nor carnitine altered the ratio of 3-hydroxybutyrate to acetoacetate or the rate of esterification of [1-14C]palmitate. Propionate, lactate, pyruvate and fructose inhibited ketogenesis from palmitate in cells from fed sheep. Starvation or the addition of carnitine decreased the antiketogenic effectiveness of gluconeogenic precursors. Propionate was the most potent inhibitor of ketogenesis, 0.8 mM producing 50% inhibition. Propionate, lactate, fructose and glycerol increased palmitate esterification under all conditions examined. Lactate, pyruvate and fructose stimulated oxidation of palmitate and octanoate to CO2. Starvation and the addition of gluconeogenic precursors stimulated apparent palmitate utilization by cells. Propionate, lactate and pyruvate decreased cellular long-chain acylcarnitine concentrations. Propionate decreased cell contents of CoA and acyl-CoA. It is suggested that propionate may control hepatic ketogenesis by acting at some point in the beta-oxidation sequence. The results are discussed in relation to the differences in the regulation of hepatic fatty acid metabolism between sheep and rats.
Project description:Labelled ketone bodies were produced readily from [U-(14)C]palmitate, [2-(14)C]palmitate and [1-(14)C]glycerol by sheep rumen-epithelial and liver tissues in vitro. On a tissue-nitrogen basis, both tissues had similar capacities for ketogenesis. Palmitate was a ketogenic substrate in both rumen-epithelial tissue and liver, and more of its (14)C appeared in ketone bodies than in the (14)CO(2) liberated. Glycerol was actively metabolized to ketone bodies, but more readily underwent complete oxidation to carbon dioxide; this complete oxidation was most pronounced in rumen-epithelial tissue from ketotic ewes. These experiments with labelled compounds confirm earlier observations that rumen-epithelial tissue, like liver, actively forms ketone bodies from long-chain fatty acids and show further that normal rumen-epithelial tissue can convert palmitate into ketone bodies as readily as into carbon dioxide. Free glycerol, which is metabolized only by liver tissue in non-ruminants, is also metabolized by rumen epithelium. The rumen epithelium thus has unique metabolic capacity among extrahepatic tissues.
Project description:High levels of ketone bodies are associated with improved survival as observed with regular exercise, caloric restriction, and-most recently-treatment with sodium-glucose linked transporter 2 inhibitor antidiabetic drugs. In heart failure, indices of ketone body metabolism are upregulated, which may improve energy efficiency and increase blood flow in skeletal muscle and the kidneys. Nevertheless, it is uncertain how ketone bodies affect myocardial glucose uptake and blood flow in humans. Our study was therefore designed to test whether ketone body administration in humans reduces myocardial glucose uptake (MGU) and increases myocardial blood flow.Eight healthy subjects, median aged 60 were randomly studied twice: (1) During 390 minutes infusion of Na-3-hydroxybutyrate (KETONE) or (2) during 390 minutes infusion of saline (SALINE), together with a concomitant low-dose hyperinsulinemic-euglycemic clamp to inhibit endogenous ketogenesis. Myocardial blood flow was measured by 15O-H2O positron emission tomography/computed tomography, myocardial fatty acid metabolism by 11C-palmitate positron emission tomography/computed tomography and MGU by 18F-fluorodeoxyglucose positron emission tomography/computed tomography. Similar euglycemia, hyperinsulinemia, and suppressed free fatty acids levels were recorded on both study days; Na-3-hydroxybutyrate infusion increased circulating Na-3-hydroxybutyrate levels from zero to 3.8±0.5 mmol/L. MGU was halved by hyperketonemia (MGU [nmol/g per minute]: 304±97 [SALINE] versus 156±62 [KETONE], P<0.01), whereas no effects were observed on palmitate uptake oxidation or esterification. Hyperketonemia increased heart rate by ≈25% and myocardial blood flow by 75%.Ketone bodies displace MGU and increase myocardial blood flow in healthy humans; these novel observations suggest that ketone bodies are important cardiac fuels and vasodilators, which may have therapeutic potentials.
Project description:Hepatic ketogenesis provides a vital systemic fuel during fasting because ketone bodies are oxidized by most peripheral tissues and, unlike glucose, can be synthesized from fatty acids via mitochondrial beta-oxidation. Since dysfunctional mitochondrial fat oxidation may be a cofactor in insulin-resistant tissue, the objective of this study was to determine whether diet-induced insulin resistance in mice results in impaired in vivo hepatic fat oxidation secondary to defects in ketogenesis. Ketone turnover (micromol/min) in the conscious and unrestrained mouse was responsive to induction and diminution of hepatic fat oxidation, as indicated by an eightfold rise during the fed (0.50+/-0.1)-to-fasted (3.8+/-0.2) transition and a dramatic blunting of fasting ketone turnover in PPARalpha(-/-) mice (1.0+/-0.1). C57BL/6 mice made obese and insulin resistant by high-fat feeding for 8 wk had normal expression of genes that regulate hepatic fat oxidation, whereas 16 wk on the diet induced expression of these genes and stimulated the function of hepatic mitochondrial fat oxidation, as indicated by a 40% induction of fasting ketogenesis and a twofold rise in short-chain acylcarnitines. Together, these findings indicate a progressive adaptation of hepatic ketogenesis during high-fat feeding, resulting in increased hepatic fat oxidation after 16 wk of a high-fat diet. We conclude that mitochondrial fat oxidation is stimulated rather than impaired during the initiation of hepatic insulin resistance in mice.
Project description:We examined the potential of overt carnitine palmitoyltransferase (CPT I) to control the hepatic catabolism of palmitoyl-CoA in suckling and adult rats, using a conceptually simplified model of fatty acid oxidation and ketogenesis. By applying top-down control analysis, we quantified the control exerted by CPT I over total carbon flux from palmitoyl-CoA to ketone bodies and carbon dioxide. Our results show that in both suckling and adult rat, CPT I exerts very significant control over the pathways under investigation. However, under the sets of conditions we studied, less control is exerted by CPT I over total carbon flux in mitochondria isolated from suckling rats than in those isolated from adult rats. Furthermore the flux control coefficient of CPT I changes with malonyl-CoA concentration and ATP turnover rate.
Project description:1. In livers from fed rats perfused with homologous whole blood of a haematocrit value of 37%, insulin decreased the perfusate concentrations of glucose and amino acids, production of ketone bodies (3-hydroxybutyrate + acetoacetate) and increased bile flow. 2. Perfusion with blood diluted with buffer to a haematocrit value of 17% decreased hepatic O2 consumption by 40-50%. Perfusate concentrations of glucose and lactate, the rate of ketogenesis and the ratios [lactate]/[pyruvate] and [3-hydroxybutyrate]/[acetoacetate] were all increased. 3. In livers perfused with blood of diminished haematocrit, effects of insulin on perfusate glucose an amino acids, ketogenesis and bile flow were abolished.
Project description:Rat parenchymal hepatocytes in monolayer culture were used to study the metabolic effects of epidermal growth factor (EGF) and insulin on ketogenesis, gluconeogenesis and glycogen metabolism. EGF, unlike insulin, did not inhibit ketogenesis from palmitate or gluconeogenesis from pyruvate in hepatocyte cultures. It also had no effect on these pathways in the presence of insulin. In contrast, EGF potently counteracted the stimulation of [14C]pyruvate incorporation into glycogen by insulin, and also glycogen deposition from both gluconeogenic precursors and glucose. The EGF concentration causing half-maximal effect was about 0.1 nM. The anti-glycogenic effect of EGF was observed after both long-term (24 h) and short-term (1 h) exposure to EGF, and was more marked in the presence of insulin than in its absence. EGF did not displace bound insulin, suggesting that it neither competes for the insulin receptor nor affects the affinity of the receptor for insulin. EGF did not alter cellular cyclic AMP; and inhibition of cyclic AMP phosphodiesterase activity did not prevent the anti-glycogenic effect of EGF. In liver-derived dividing epithelial cells, Hep-G2 cells and fibroblasts, which have no capacity for gluconeogenesis, EGF did not counteract the stimulatory effect of insulin on [14C]glucose incorporation into glycogen, and in the epithelial cells EGF increased [14C]glucose incorporation into glycogen. The counter-effect of EGF on the glycogenic action of insulin in parenchymal hepatocytes may be due to a direct effect on glycogen metabolism or to an interaction with the post-receptor events in insulin action.
Project description:1. The formation of acetoacetate, beta-hydroxybutyrate and glucose was measured in the isolated perfused rat liver after addition of fatty acids. 2. The rates of ketone-body formation from ten fatty acids were approximately equal and independent of chain length (90-132mumol/h per g), with the exception of pentanoate, which reacted at one-third of this rate. The [beta-hydroxybutyrate]/[acetoacetate] ratio in the perfusion medium was increased by long-chain fatty acids. 3. Glucose was formed from all odd-numbered fatty acids tested. 4. The rate of ketone-body formation in the livers of rats kept on a high-fat diet was up to 50% higher than in the livers of rats starved for 48h. In the livers of fat-fed rats almost all the O(2) consumed was accounted for by the formation of ketone bodies. 5. The ketone-body concentration in the blood of fat-fed rats rose to 4-5mm and the [beta-hydroxybutyrate]/[acetoacetate] ratio rose to 11.5. 6. When the activity of the microsomal mixed-function oxidase system, which can bring about omega-oxidation of fatty acids, was induced by treatment of the rat with phenobarbitone, there was no change in the ketone-body production from fatty acids, nor was there a production of glucose from even-numbered fatty acids. The latter would be expected if omega-oxidation occurred. Thus omega-oxidation did not play a significant role in the metabolism of fatty acids. 7. Arachidonate was almost quantitatively converted into ketone bodies and yielded no glucose, demonstrating that gluconeogenesis from poly-unsaturated fatty acids with an even number of carbon atoms does not occur. 8. The rates of ketogenesis from unsaturated fatty acids (sorbate, undecylenate, crotonate, vinylacetate) were similar to those from the corresponding saturated fatty acids. 9. Addition of oleate together with shorter-chain fatty acids gave only a slightly higher rate of ketone-body formation than oleate alone. 10. Glucose, lactate, fructose, glycerol and other known antiketogenic substances strongly inhibited endogenous ketogenesis but had no effects on the rate of ketone-body formation in the presence of 2mm-oleate. Thus the concentrations of free fatty acids and of other oxidizable substances in the liver are key factors determining the rate of ketogenesis.