Inhibition of gluconeogenesis in isolated rat hepatocytes after chronic treatment with phenobarbital.

International audienceGluconeogenesis was studied in hepatocytes isolated from phenobarbital-pretreated rats fasted for 24 h. In closed vial incubations, glucose production from lactate (20 mmol/l) and pyruvate (2 mmol/l), alanine (20 mmol/l) or glutamine (20 mmol/l) was suppressed by about 30-45%, although glycerol metabolism was not affected. In hepatocytes perifused with lactate and pyruvate (ratio 10:1), glucose production was inhibited by 50%, even at low gluconeogenic flux. From the determination of gluconeogenic intermediates at several steady states of gluconeogenic flux, we have found a single relationship between phosphoenolpyruvate and the rate of glucose production (Jglucose), and two different curves between cytosolic oxaloacetate and Jglucose in controls and in phenobarbital-pretreated hepatocytes. By using 3-mercaptopicolinate to determine the flux control coefficient of phosphoenolpyruvate carboxykinase we found that phenobarbital pretreatment led to an increase in this coefficient from 0.3 (controls) to 0.8 (phenobarbital group). These observations were confirmed by the finding that the activity of phosphoenolpyruvate carboxykinase was decreased by 50% after phenobarbital treatment. Hence we conclude that the inhibitory effect of phenobarbital on gluconeogenesis is due, at least partly, to a decrease in the flux through phosphoenolpyruvate carboxykinase

Octanoate affects 2,4-dinitrophenol uncoupling in intact isolated rat hepatocytes.

International audienceWhen intact isolated rat hepatocytes, either incubated or perifused, were uncoupled by 2,4-dinitrophenol, we found that the effect on glucose and lactate+pyruvate fluxes, cytosolic and mitochondrial redox states and ATP/ADP ratios were dependent on the nature of the exogenous substrate added. 2,4-Dinitrophenol addition (0.25 mmol/l) to cells perifused with dihydroxyacetone (10 mmol/l) resulted in a modest and transient activation of oxygen uptake accompanied by a surprising rise in lactate/pyruvate ratio indicating an increase in the cytosolic NADH/NAD+ ratio. In addition, such uncoupling, fully abolished glucose production, enhanced lactate+pyruvate flux, and strongly decreased cytosolic and mitochondrial ATP/ADP ratios. In these steady-state conditions, further addition of octanoate (0.4 mmol/l) induced a large and sustained enhancement of respiration with a concomitant decrease in the lactate/pyruvate ratio, whereas glucose flux was restored to some extent and cytosolic and mitochondrial ATP/ADP ratios increased. Inhibition of the malate-aspartate shuttle by the transaminase inhibitor aminooxyacetate (0.3 mmol/l) did not modify the effect of 2,4-dinitrophenol with dihydroxyacetone alone whereas it decreased the maximal stimulation of oxygen uptake after octanoate addition. In view of these results we propose the following conclusions. The uncoupling of intact cells by 2,4-dinitrophenol inhibits the translocation of reducing equivalents into the mitochondrial matrix probably by impairing the malate-aspartate shuttle. This explains the increase in the cytosolic NADH/NAD+ ratio and the transient activation of respiration with dihydroxyacetone. Fatty acid addition to cells uncoupled with 2,4-dinitrophenol appears to restore a mitochondrial membrane potential, probably by providing the respiratory chain with reduced cofactors directly in the matrix, thus allowing the transfer of reducing equivalents across the mitochondrial membrane. The restoration, to some extent, of a protonmotive force to uncoupled cells by fatty acid addition is also supported by an increase in ATP synthesis as evidenced by a glucose synthesis with dihydroxyacetone as gluconeogenic substrate

The inhibition of phosphoenolpyruvate carboxykinase following in vivo chronic phenobarbital treatment in the rat is due to a post-translational event.

International audienceChronic treatment of rats with phenobarbital has been reported to decrease gluconeogenesis in rat hepatocytes by a 50% inhibition of phosphoenolpyruvate (P-pyruvate) carboxykinase activity [Argaud, D., Halimi, S., Catelloni, F. & Leverve, X. (1991) Biochem. J. 280, 663-669]. Contrary to the current knowledge of P-pyruvate carboxykinase regulation, we failed to find a diminution of either P-pyruvate carboxykinase protein (by using a polyclonal antibody) or P-pyruvate carboxykinase mRNA, in the liver of rats treated with phenobarbital for 2 weeks. Kinetic studies of P-pyruvate carboxykinase activity, measured by either carboxylation of P-pyruvate or decarboxylation of oxaloacetate, revealed a decrease in both V(max) and Km after phenobarbital treatment, whereas the nutritional state affected only the V(max), as expected. Assessment of P-pyruvate carboxykinase specificity was confirmed by the full inhibition of the enzyme with its specific inhibitor 3-mercaptopicolinate in the micromolar range. P-Pyruvate carboxykinase, purified either by ammonium sulfate fractionation or by immunoprecipitation, exhibited a similar decrease in affinity after phenobarbital treatment. Although the molecular mass does not appear to be altered, the pH sensitivity to 3-mercaptopicolinate inhibition and the enzyme recovery after immunoprecipitation both seemed to be affected. This leads us to propose that the effect of chronic phenobarbital treatment on P-pyruvate carboxykinase activity is not the result of transcriptional regulation but is exerted at the post-translational level