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

Argaud, Doriane; Halimi, Serge; Catelloni, F.; Leverve, Xavier

doi:10.1042/bj2800663

Cited by 43 publications

(40 citation statements)

References 36 publications

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“…Hepatocytes were isolated by the method of Berry and Friend (16) (19,20). To study the metabolic effect of glucagon, perifused liver cells were titrated with DHA in the presence or absence of glucagon (10 Ϫ7 M).…”

Section: Methodsmentioning

confidence: 99%

Glucose 6-Phosphate Hydrolysis Is Activated by Glucagon in a Low Temperature-sensitive Manner

Ichaï

Guignot

El-Mir

et al. 2001

Journal of Biological Chemistry

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Glucagon affects liver glucose metabolism mainly by activating glycogen breakdown and by inhibiting pyruvate kinase, whereas a possible effect on glucose-6-phosphatase has also been suggested. Although such a target is of physiological importance for liver glucose production it was never proven. By using a model of liver cells, perifused with dihydroxyacetone, we show here that the acute stimulation of gluconeogenesis by glucagon (10 ؊7 M) was not related to the significant inhibition of pyruvate kinase but to a dramatic activation of the hydrolysis of glucose 6-phosphate. We failed to find an acute change in glucose-6-phosphatase activity by glucagon, but the increase in glucose 6-phosphate hydrolysis was abolished at 21°C; conversely the effect on pyruvate kinase was not affected by temperature. The activation of glucose 6-phosphate hydrolysis by glucagon was confirmed in vivo, in postabsorptive rats receiving a constant infusion of glucagon, by the combination of a 2-fold increase in hepatic glucose production and a 60% decrease in liver glucose 6-phosphate concentration. Besides the description of a novel effect of glucagon on glucose 6-phosphate hydrolysis by a temperature-sensitive mechanism, this finding could represent an important breakthrough in the understanding of type II diabetes, because glucose 6-phosphate is proposed to be a key molecule in the transcriptional effect of glucose.The metabolic effects of glucagon on liver glucose metabolism have long been described, mediated by both cAMP-and calciumdependent signalings (1-3). Besides a powerful and well documented effect on glycogen breakdown, glucagon also increases liver gluconeogenesis mainly by the allosteric inhibition of pyruvate kinase by its phosphorylation (4, 5). In addition, some effects of glucagon on the fructose phosphate cycle have been described, i.e. inhibition of 6-phosphofructo-1-kinase and activation of fructose-1,6-bisphosphatase (6, 7). Apart from these indisputable effects, a stimulation of glucose-6-phosphatase, a key enzyme in glucose production, has been proposed but never proven (8 -10). In view of a potential key role of glucose 6-phosphate as a cellular intermediate in the transcriptional effects of glucose (11,12), an effect of glucagon on glucose-6-phosphatase could be of major importance. Indeed, despite a prominent effect on liver glucose metabolism, the role of glucagon in the pathogenesis of type II diabetes is still unclear and a matter of debate (13).By studying glucose production under steady state conditions in a model of rat hepatocytes perifused with dihydroxyacetone (DHA) 1 as an exogenous source of carbohydrate, we show in the present work that despite a clear inhibitory effect of glucagon on pyruvate kinase, this effect was not responsible for the enhanced glucose production, indicating that this step is actually not controlling this pathway. Actually, the dramatic increase in the glucose production after glucagon addition was related to a substantial activation of glucose 6-phosphate hydrolysis in glucos...

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Section: Methodsmentioning

confidence: 99%

Glucose 6-Phosphate Hydrolysis Is Activated by Glucagon in a Low Temperature-sensitive Manner

Ichaï

Guignot

El-Mir

et al. 2001

Journal of Biological Chemistry

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“…After 40 min, when a steady state has been reached, glycerone was infused at a constant rate in order to obtain a new steady state (15-20 min) [19]. In the same way, several successive steady states were obtained at various concentrations of glycerone.…”

Section: Perifusion Of Hepatocytesmentioning

confidence: 99%

Decrease in Cytosolic ATP/ADP Ratio and Activation of Pyruvate Kinase after in vitro Addition of Almitrine in Hepatocytes Isolated from Fasted Rats

Leverve

Fontaine

Putod-Paramelle

et al. 1994

European Journal of Biochemistry

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Previously, we have shown in experiments with isolated mitochondria that almitrine, a drug used for patients with chronic lung disease, affects the H+/ATP stoichiometry of the F,F,-ATPase [Rigoulet, M., Fraisse, L., Ouhabi, R., GuCrin, B., Fontaine, E. & Leverve, X. M. (1990) Biochim. Biophys. Actu 1018, 91-97]. In the present study, we have investigated the effect of almitrine on gluconeogenesis and oxygen consumption in isolated hepatocytes. Almitrine decreased both the cytosolic and mitochondrial ATP/ADP ratios but had no effect on oxygen consumption in cells incubated with and without octanoate. This must have been due to a double effect. On the one hand, a decrease in the ATP/ADP ratio decreases ATP utilization; on the other hand, in the presence of almitrine more oxygen is required to synthesize ATP. Almitrine did affect gluconeogenesis from various substrates (lactate + pyruvate, glycerone or fructose), but had no effect on glycerol or glutamine metabolism. The effect on gluconeogenesis from glycerone was due to an increase in glycolytic flux. The rate of lactate + pymvate production increased whereas there was no effect on glycerone utilization. This effect was caused by an activation of pyruvate kinase. Our data indicate that this enzyme is an extremely sensitive sensor of the cytosolic ATP/ADP ratio. Hence, under our experimental conditions, the cytosolic ATP/ADP ratio decrease affects only the balance between glucose and lactate + pyruvate productions, and not the phosphorylation of glycerone, the first and controlling step of this pathway.Almitrine is a drug which is used for the treatment of patients suffering from hypoxia due to chronic lung diseases [l , 21. The increase in arterial blood oxygen pressure in patients treated with this drug is mainly explained by a specific effect on chemoreceptors [3-61 and/or by changes in the ventilatiodperfusion ratio [7, 81. Moreover some effects on coupling between oxidation and phosphorylation in brain mitochondria isolated from senescent rats have been reported: an increase at low almitrine concentrations and a decrease at high almitrine concentrations [9].Recently we have described the effects of almitrine in mitochondria isolated from yeast, bovine heart and rat liver [lo, 111. Briefly, almitrine (a) inhibited ATPase activity, (b) decreased the ATP/O ratio without any change in the magnitude of the protonmotive force and (c) had no direct effect on respiratory chain activity. Moreover the number of H' ions transferred by ATPase/mole hydrolyzed ATP increased. Taken together, these properties led us to propose that almitrine acts by changing the mechanistic stoichiometry of mitochondria] ATPase/ATP synthase.The purpose of the present work was to investigate the effects of almitrine in intact rat hepatocytes. As expected from our previous results in isolated rat liver mitochondria, almitrine decreases cytosolic and mitochondrial ATP/ADP ratios without changing the respiratory rate. This unique property of almitrine (i.e. changing the oxidative ATP proCorrespo...

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“…As described elsewhere (Argaud et al, 1991) the perfusion fluid was Krebs-bicarbonate buffer containing 1.3 mM Ca", 0.1 % bovine serum albumin and 0.1 mM oleate. Concentrations of dihydroxyacetone were added to obtain different steady states of Glc and lactate (Lac) production.…”

Section: Perifusion Of Hepatocytesmentioning

confidence: 99%

Metformin decreases gluconeogenesis by enhancing the pyruvate kinase flux in isolated rat hepatocytes

Argaud

Roth

Wiernsperger

et al. 1993

European Journal of Biochemistry

144

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Metformin (dimethylbiguanide) has been used for more than 30 years as an antihyperglycemic agent in the treatment of diabetes mellitus, but its effect on gluconeogenesis is still controversial. In isolated hepatocytes from fasted rats, a significant inhibition of glucose production from lactate/ pyruvate (10: 1, mol/mol), fructose, alanine or glutamine, following metformin addition, is observed. Moreover, in hepatocytes perifused with dihydroxyacetone as the gluconeogenic substrate and treated with 0.5 mM metformin, an inhibition of the glucose flux and a simultaneous stimulation of the lactate/pyruvate flux were observed. This enhancement of lactate/pyruvate formation appears to be due to an effect on the pyruvate-kinase enzyme. A direct effect of metformin on pyruvate kinase cannot explain this result, since pyruvate-kinase activity was not affected by metformin at this concentration. In contrast, the addition of metformin caused a significant decrease in the cellular ATP concentration, a known allosteric inhibitor of this enzyme. This could explain the stimulation of pyruvate-kinase activity following metformin addition and thus the inhibition of gluconeogenesis.

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

Cited by 43 publications

References 36 publications

Glucose 6-Phosphate Hydrolysis Is Activated by Glucagon in a Low Temperature-sensitive Manner

Glucose 6-Phosphate Hydrolysis Is Activated by Glucagon in a Low Temperature-sensitive Manner

Decrease in Cytosolic ATP/ADP Ratio and Activation of Pyruvate Kinase after in vitro Addition of Almitrine in Hepatocytes Isolated from Fasted Rats

Metformin decreases gluconeogenesis by enhancing the pyruvate kinase flux in isolated rat hepatocytes

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