The r?le of mitochondrial pyruvate transport in the stimulation by glucagon and phenylephrine of gluconeogenesis from L-lactate in isolated rat hepatocytes.

The sensitivity of glucose production from L-lactate by isolated liver cells from starved rats to inhibition by alpha-cyano-4-hydroxycinnamate was studied. A small percentage of the maximal rate of gluconeogenesis was insensitive to inhibition by alpha-cyano-4-hydroxycinnamate, and evidence is presented to show that this is due to pyruvate entry into the mitochondria as alanine. After subtraction of this rate, Dixon plots of the reciprocal of the rate of gluconeogenesis against inhibitor concentration were linear both in the absence and presence of glucagon, phenylephrine or valinomycin, each of which stimulated gluconeogenesis by 30-50%. Pyruvate kinase activity was decreased by glucagon, but not by phenylephrine or valinomycin. Inhibition of gluconeogenesis by quinolinate (inhibitor of phosphoenolpyruvate carboxykinase) or monochloroacetate (probably inhibiting pyruvate carboxylation) caused a significant deviation from linearity of the Dixon plot obtained with alpha-cyano-4-hydroxycinnamate. Amytal, however, inhibited gluconeogenesis without affecting the linearity of this plot. These data, coupled with a computer simulation study, suggest that pyruvate transport may control gluconeogenesis from L-lactate and that hormones may stimulate this process through an effect on the respiratory chain. An additional role for pyruvate kinase and pyruvate carboxylase is quite compatible with the data presented.     

Control of glycogen synthase phosphorylation in isolated rat hepatocytes by epinephrine, vasopressin and glucagon

Isolated rat hepatocytes were incubated in a medium containing 0.1 mM [32P]phosphate (0.1 mCi/ml) before exposure to epinephrine, glucagon or vasopressin. 32P-labeled glycogen synthase was purified from extracts of control or hormone-treated cells by the use of specific antibodies raised to rabbit skeletal muscle glycogen synthase. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that a single 32P-labeled polypeptide, apparent Mr 88000, was removed specifically by the antibodies and corresponded to glycogen synthase. Similar electrophoretic analysis of CNBr fragments prepared from the immunoprecipitate revealed that 32P was distributed between two fragments, of apparent Mr 14000 (CB-1) and 28000 (CB-2). Epinephrine, vasopressin or glucagon increased the 32P content of the glycogen synthase subunit. CB-2 phosphorylation was increased by all three hormones while CB-1 was most affected by epinephrine and vasopressin. These effects correlated with a decrease in glycogen synthase activity. From studies using rat liver glycogen synthase, purified by conventional methods and phosphorylated in vitro by individual protein kinases, it was found that electrophoretically similar CNBr fragments could be obtained. However, neither cyclic-AMP-dependent protein kinase nor three different Ca2+-dependent enzymes (phosphorylase kinase, calmodulin-dependent protein kinase, and protein kinase C) were effective in phosphorylating CB-2. The protein kinases most effective towards CB-2 were the Ca2+ and cyclic-nucleotide-independent enzymes casein kinase II (PC0.7) and FA/GSK-3. The results demonstrate that rat liver glycogen synthase undergoes multiple phosphorylation in whole cells and that stimulation of cells by glycogenolytic hormones can modify the phosphorylation of at least two distinct sites in the enzyme. The specificity of the hormones, however, cannot be explained simply by the direct action of any known protein kinase dependent on cyclic nucleotide or Ca2+. Therefore, either control of other protein kinases, such as FA/GSK-3, is involved or phosphatase activity is regulated, or both.     

Control of gluconeogenesis in rat liver cells. I. Kinetics of the individual enzymes and the effect of glucagon

Control of gluconeogenesis from lactate was studied by titrating rat liver cells with lactate and pyruvate in a ratio of 10:1 in a perifusion system. At different steady states of glucose formation, the concentration of key gluconeogenic intermediates was measured and plotted against gluconeogenic flux (J glucose). Complete saturation was observed only in the plot relating J glucose to the extracellular pyruvate concentration. Measurement of pyruvate distribution in the cell showed that the mitochondrial pyruvate translocator operates close to equilibrium at high lactate and pyruvate concentrations. It can therefore be concluded that pyruvate carboxylase limits maximal gluconeogenic flux. Addition of glucagon did not cause a shift in the plots relating J glucose to glucose 6-phosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, and phosphoenolpyruvate. It can thus be concluded that glucagon does not affect the kinetic parameters of the enzymes involved in the conversion of phosphoenolpyruvate to glucose. Addition of glucagon led to a shift in the curves relating J glucose to the concentration of cytosolic oxalacetate and extracellular pyruvate. The shift in the curve relating J glucose to oxalacetate is due to glucagon-induced inhibition of pyruvate kinase. The stimulation of gluconeogenesis by glucagon can be accounted for almost completely by inhibition of pyruvate kinase. There was almost no stimulation by glucagon of pyruvate carboxylation. In the absence of glucagon, control on gluconeogenesis from lactate is distributed among different steps including pyruvate carboxylase and pyruvate kinase. Assuming that in the presence of glucagon all pyruvate kinase flux is inhibited, the control of gluconeogenesis in the presence of the hormone is confined exclusively to pyruvate carboxylase.     

Inability of glucagon to regulate glycogen metabolism in rat hepatocytes isolated after fasting and refeeding high-carbohydrate diets

Studies are described which demonstrate that the ability of glucagon, epinephrine, and dibutyryl-cAMP to stimulate glycogenolysis is impaired in rat hepatocytes isolated from animals starved for 24 h and then refed a sucrose-rich diet or refed standard rat chow. The impaired regulation of glycogenolysis by glucagon was observed within 24 h after refeeding and persisted for at least 3 days. The inability of glucagon to stimulate glycogen breakdown in the refed condition appeared to be due to a suppressed activation of glycogen phosphorylase and phosphorylase b kinase by the hormone. The capacity of glucagon to regulate pyruvate kinase and glycolysis was not altered by refeeding, suggesting that the defect lies beyond interaction of the hormone at its receptor. Prolonged incubation of hepatocytes from refed rats was accompanied by depletion of glycogen reserves and was accompanied by restoration of hormonal stimulation of glycogenolysis. Addition of glycogen to cell-free extracts was found to inhibit phosphorylase b kinase but not phosphorylase. The findings of this investigation are consistent with the interpretation that high levels of glycogen present of liver after refeeding may lead to a diminished activity of phosphorylase b kinase and its hormonal regulation.     

Interrelationship of insulin and glucagon ratios on carbohydrate metabolism in isolated hepatocytes containing high glycogen.

The effect of physiological concentrations of glucagon and insulin on glycogenolysis was studied in the presence and absence of substrates in isolated hepatocytes containing high glycogen. In the absence of substrates glucagon stimulated glycogenolysis at 10^?14M concentration, and addition of 100 μunits of insulin partially inhibited glucagon stimulated glycogenolysis (10^?14M to 10^?11M). However, in the presence of substrates, insulin completely inhibited glucagon stimulated glycogenolysis (10^?14M to 10^?11M), indicating that molar glucagon and insulin ratios control carbohydrate metabolism in liver. Additional studies showed incorporation of amino acid into protein was linear for only 3 to 4 hr in cells containing low glycogen, whereas in cells containing high glycogen, incorporation was linear for 8 to 10 hr.     

Studies on the differential response to glucagon concentration on gluconeogenesis in isolated hepatocytes containing high and low glycogen

Rat liver parenchymal cells were isolated with (a) collagenase alone and (b) with both collagenase and hyaluronidase. Addition of hyaluronidase significantly decreased intracellular glycogen content of cells from fed rats. Effects of various concentrations of glucagon on gluconeogenesis were also studied in isolated hepatocytes from fed and fasted rats. Glucagon at the concentration of 10^?12M to 10^?10M stimulated gluconeogenesis in fed rats. Higher concentrations (10^?8M) had no further stimulating effect. In fasted rats, glucagon at the concentrationsof 10^?12M had no effect whereas at 10^?10M to 10^?8M concentrations, it stimulated gluconeogenesis by 2 fold. These studies suggest that glucagon functions in gluconeogenesis both in the fed and fasted state.     

Regulation by insulin of gluconeogenesis in isolated rat hepatocytes.

Insulin (10nM) completely suppressed the stimulation of gluconeogenesis from 2 mM lactate by low concentrations of glucagon (less than or equal to 0.1 nM) or cyclic AMP (less than or equal to 10 muM), but it had no effect on the basal rate of gluconeogenesis in hepatocyctes from fed rats. The effectiveness of insulin diminished as the concentration of these agonists increased, but insulin was able to suppress by 40% the stimulation by a maximally effective concentration of epinephrine (1 muM). The response to glucagon, epinephrine, or insulin was not dependent upon protein synthesis as cycloheximide did not alter their effects. Insulin also suppressed the stimulation by isoproterenol of cyclic GMP. These data are the first demonstration of insulin antagonism to the stimulation of gluconeogenesis by catecholamines. Insulin reduced cyclic AMP levels which had been elevated by low concentrations of glucagon or by 1 muM epinephrine. This supports the hypothesis that the action of insulin to inhibit gluconeogenesis is mediated by the lowering of cyclic AMP levels. However, evidence is presented which indicates that insulin is able to suppress the stimulation of gluconeogenesis by glucagon or epinephrine under conditions where either the agonists or insulin had no measurable effect on cyclic AMP levels. Insulin reduced the glucagon stimulation of gluconeogenesis whether or not extracellular Ca2+ were present, even though insulin only lowered cyclic AMP levels in their presence. Insulin also reduced the stimulation by epinephrine plus propranolol where no significant changes in cyclic AMP were observed without or with insulin. In addition, insulin suppressed gluconeogenesis in cells that had been preincubated with epinephrine for 20 min, even though the cyclic AMP levels had returned to near basal values and were unaffected by insulin. Thus insulin may not need to lower cyclic AMP levels in order to suppress gluconeogenesis.     

Stimulatory effect of the intestinal peptide PHI on glycogenolysis and gluconeogenesis in isolated rat hepatocytes.

The newly isolated peptide PHI provoked a dose-dependent stimulation of glycogenolysis and gluconeogenesis in isolated rat hepatocytes; at 1 microM-PHI, both processes were increased 1.6-fold as compared with basal values. These PHI-mediated effects were accompanied by the activation of glycogen phosphorylase and the inactivation of pyruvate kinase. PHI (1 microM) also caused a 2-fold increase in hepatocyte cyclic AMP.     

Role of fructose 2,6-bisphosphate in the control by glucagon of gluconeogenesis from various precursors in isolated rat hepatocytes.

Hepatocytes from overnight-starved rats were incubated with 1-20 mM-fructose, -dihydroxyacetone, -glycerol, -alanine or -lactate and -pyruvate with or without 0.1 microM-glucagon. The production of glucose and lactate was measured, as was the content of fructose 2,6-bisphosphate. The concentrations of fructose (below 5 mM) and dihydroxyacetone (above 1 mM) that gave rise to an increase in fructose 2,6-bisphosphate were those at which a glucagon effect on the production of glucose and lactate could be observed. Glycerol had no effect on fructose 2,6-bisphosphate content or on production of lactate, and glucagon did not stimulate the production of glucose from this precursor. With alanine or lactate/pyruvate as substrates, glucagon stimulated glucose production whether the concentration of fructose 2,6-bisphosphate was increased or not. The extent of inactivation of pyruvate kinase by glucagon was not affected by the presence of the various gluconeogenic precursors. The role of fructose 2,6-bisphosphate in the effect of glucagon on gluconeogenesis from precursors entering the pathway at the level of triose phosphates or pyruvate is discussed.     

Iron deficiency decreases gluconeogenesis in isolated rat hepatocytes

Dietary iron deficiency in rats results in increased blood glucose turnover and recycling. We measured the rates of glucose production in isolated hepatocytes from iron-sufficient (Fe+) and iron-deficient (Fe-) rats to assess the intrinsic capacity of the Fe- liver to carry out gluconeogenesis. Low-iron and control diets were given to 21-day-old female rats. After 4-5 wk, hemoglobin concentrations averaged 4.1 g/dl in the Fe- and 14.3 g/dl in the Fe+ animals. In the hepatocytes from Fe- rats, there was a 35% decrease in the rate of glucose production from 1 mM pyruvate + 10 mM lactate, a 48% decrease from 0.1 mM pyruvate + 1 mM lactate, a 39% decrease from 1 mM alanine, and a 48% decrease from 1 mM glycerol. The addition of 5 microM norepinephrine or 0.5 microM glucagon to the incubation media produced stimulatory effects on hepatocytes from both Fe- and Fe+ rats, resulting in the maintenance of an average difference of 38% in the rates of gluconeogenesis between the two groups. Studies on isolated liver mitochondria and cytosol revealed alpha-glycerophosphate-cytochrome c reductase and phospho(enol)pyruvate carboxykinase activities to be decreased by 27% in Fe- rats. We conclude that because severe dietary iron deficiency decreases gluconeogenesis in isolated rat hepatocytes, the increased gluconeogenesis demonstrated by Fe- rats in vivo is attributable to increased availability of gluconeogenic substrates and upregulation of the pathway.     

Control of glycogen metabolism by gluconeogenic and ketogenic substrates in isolated hepatocytes from fed rats.

1. This study was conducted to examine the effects of gluconeogenic and ketogenic substrates on the activities of the glycogen-metabolizing enzymes and on glycogenolysis in isolated hepatocytes from fed rats. 2. Gluconeogenic substrates like fructose, dihydroxyacetone or lactate turned out to stimulate the glucose-induced activation of glycogen synthase and this effect may be linked, to some extent, to the increase of the cellular glucose 6-phosphate concentration. 3. The effect of fructose was accompanied by the onset of glycogen synthesis. 4. Energetic substrates like fatty acids were also potent activators of glycogen synthase, especially in the presence of glucose. 5. When fatty acids were added alone or together with a physiological concentration of glucose, they induced or potentiated the inhibition of glycogen phosphorylase-a. 6. This inhibitory effect was mediated by a decrease of lactate release. 7. The stimulatory effect of amino acids on glycogen synthase seemed to be direct, non mediated by an inhibition of the phosphorylase-a activity although hepatic glycogenolysis markedly decreased. 8. Moreover, the amino acid action could be linked to their capacities to induce cell swelling and/or to limit proteolysis.     

A histochemical study of liver enzymes involved in glycogen metabolism in the X-irradiated rat

Summary The liver enzymes responsible for the breakdown and synthesis of glycogen from glucose have been investigated cytochemically in rats exposed to 1200 rads of x-irradiation. It was found that significant changes occur in their activities and that amylophosphorylase and amylo-1,6-glucosidase (debranching enzyme), both of which are responsible for the conversion of glycogen to glucose, are markedly inhibited by radiation. A significant inhibition of the activity of 1,41,6 transglucosidase (branching enzyme) was also observed. In contrast, the activity of UDPG-glycogen transglucosylase, which is responsible for the in vivo synthesis of 1,4-polysaccharides, was found to be stimulated.     

Distinctive effects of glucagon on gluconeogenesis and ketogenesis in hepatocytes isolated from normal and biotin-deficient rats.

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.     

Regulation of phosphofructokinase activity by glucagon in isolated rat hepatocytes.

Glucagon addition to isolated hepatocytes from fed rats resulted in an inhibition of the activity of phosphofructokinase measured in extracts of the cells. Glucagon caused a shift in the fructose 6-phosphate concentration curve to the right resulting in an increase in the K_0.5 for F6P from 0.09 mM to 0.31 mM. No effect of glucagon was seen when the enzyme was assayed with saturating concentrations of fructose 6-phosphate or in the presence of 1 mM AMP. The effect of glucagon was seen within minutes and the concentration of hormone giving half-maximal inhibition was 0.2 nM. This effect of glucagon on phosphofructokinase activity may contribute to the effect of glucagon on substrate cycling at the fructose 6-phosphate-fructose bisphosphate level.     

Effects of glucagon and alpha- and beta-agonists on glycogenolysis and gluconeogenesis in isolated ovine hepatocytes

(1) The effects of glucagon, dibutyryl cyclic AMP, vasopressin, phenylephrine, and isoproterenol on glycogenolysis and gluconeogenesis were investigated using isolated ovine hepatocytes. (2) Glycogenolysis was stimulated by all effectors except vasopressin. The response to alpha-agonists was greater than that of beta-agonists in older animals. Stimulation by beta-agonists increased after 30 h primary culture. (3) Gluconeogenesis from propionate or L-lactate plus pyruvate was stimulated to a small extent by dibutyryl cyclic AMP, glucagon and isoproterenol but not by vasopressin or phenylephrine. (4) No effects of lactation were observed. (5) Data are compared to results obtained in other species and the physiological significance of the results in relation to the ruminant is discussed.     

Vanadate counteracts glucagon effects in isolated rat hepatocytes

The incubation of isolated rat hepatocytes with vanadate increased the concentration of fructose 2,6-bisphosphate without modifying 6-phosphofructo-2-kinase activity. Vanadate also reverted and prevented the decrease of fructose 2,6-bisphosphate levels, of the "active" form of the 6-phosphofructo 2-kinase and of the pyruvate kinase activity ratio produced by glucagon, by probably counteracting the increase in cyclic AMP concentration.     

Effect of methylamalonic acid on gluconeogenesis in isolated rat and guinea-pig hepatocytes.

Gluconeogenesis from pyruvate, alanine, lactate and propionate was inhibited by methylmalonate in both rat and guinea-pig hepatocytes. The effect was dose-dependent. Gluconeogenesis from glycerol and xylitol was not affected.     

Inhibition of gluconeogenesis by 2,5-anhydro-D-mannitol in isolated rat hepatocytes

2,5-Anhydro-D-mannitol inhibited glucose synthesis, increased the pyruvate/phosphoenolpyruvate ratio and altered adenine nucleotide concentrations in hepatocytes isolated from fasted rats. The accumulations of 2,5-anhydro-D-mannitol 1,6-diphosphate, an allosteric activator of pyruvate kinase, and of ADP in treated hepatocytes can account for the increase in pyruvate/phosphoenolpyruvate ratio and the inhibition of glucose synthesis from lactate.