Juan E. Felíu

A phospho-oligosaccharide mimics insulin action on glycogen phosphorylase and pyruvate kinase activities in isolated rat hepatocytes.

A phospho-oligosaccharide which is the polar head group of a novel insulin-sensitive glycophospholipid has recently been involved in insulin action. We have investigated the insulin-like effects of this phospho-oligosaccharide on both glycogen phosphorylase a and pyruvate kinase activities of hepatocytes incubated in the presence of glucagon (0.1 nM). Similarly to insulin, the phospho-oligosaccharide antagonized glucagon-dependent activation of glycogen phosphorylase, as well as the inactivation of pyruvate kinase caused by this hormone. The antagonistic action of the phospho-oligosaccharide on glucagon effects was dose-dependent. Furthermore, it partially antagonized glucagon-stimulated cyclic AMP levels. These results support the hypothesis that this phospho-oligosaccharide mediates at least some insulin actions in hepatocytes.

Effect of glipizide on hepatic fructose 2,6-bisphosphate concentration and glucose metabolism

Glipizide raised, in a dose-dependent manner, the concentration of fructose 2,6-bisphosphate in hepatocytes isolated from 24-hour fasted rats and incubated in the presence of 10 mmol/L glucose. Simultaneously, the rate of L-lactate production, as well as the rate of 3H2O formation from (3-3H)glucose, increased markedly. The concentration of glipizide calculated as corresponding to the half-maximal effect in these metabolic parameters was 12 to 15 mumol/L. In hepatocytes isolated from fed rats, either normal or made diabetic by treatment with alloxan, glipizide inhibited the conversion of both (U-14C)pyruvate and (U-14C)lactate to (14C)glucose; an inverse correlation was established between hepatocyte fructose 2,6-bisphosphate levels and the rate of gluconeogenesis. The increase of fructose 2,6-bisphosphate concentration elicited by glipizide, which occurs without a significant modification of either 6-phospho-fructo 2-kinase activity or hepatocyte cyclic AMP levels, seems to be related to a significant accumulation of hexose 6-phosphates (glucose 6-phosphate and fructose 6-phosphate) in the hepatic cells.

Involvement of both phosphatidylinositol 3-kinase and p44/p42 mitogen-activated protein kinase pathways in the short-term regulation of pyruvate kinase L by insulin

Pyruvate kinase L (PK-L) is a key regulatory enzyme of the hepatic glycolytic/gluconeogenic pathway that can be dephosphorylated and activated in response to insulin. However, the signaling cascades involved in this insulin effect have not been established. In this work we have investigated the potential involvement of phosphatidylinositol 3-kinase (PI 3-K) and p44/p42 mitogen-activated protein kinase (MAPK) pathways in the short-term modulation of PK-L by insulin in primary cultures of rat hepatocytes. Wortmannin, at a concentration of 100 nm, caused a marked inhibition of the PI 3-K/protein kinase B pathway, which became complete at 500 nm wortmannin. Likewise, wortmannin at 100 and 500 nm, elicited partial and total inhibitions of insulin-mediated activation of PK-L, respectively. However, this PI 3-K inhibitor also reduced insulin-mediated phosphorylation of p44/p42 MAPK in cultured rat hepatocytes, indicating that both the PI 3-K and MAPK pathways could be involved in PK-L activation by insulin. Thre...

Effect of streptozotocin diabetes on the glycolytic flux and on fructose 2,6-bisphosphate levels in isolated rat enterocytes☆

In epithelial cells isolated from rat small intestine and incubated in the presence of 1 mM glucose, streptozotocin-induced diabetes reduced, by 46 and 29%, respectively, the rates of both glucose utilization and L-lactate formation. These effects were accompanied by a significant decrease of enterocyte fructose 2,6-bisphosphate concentration (about 50%) and of the glycolytic flux through the reaction catalyzed by 6-phosphofructo 1-kinase. The diminution of enterocyte fructose 2,6-bisphosphate levels caused by diabetes occurred in spite of an increase of hexose 6-phosphate concentration, and was associated with a reduction in the amount of active form of 6-phosphofructo 2-kinase; total activity of this enzyme was not significantly modified. Diabetes also caused an acceleration in the rate of 3-O-methyl-D-(14C) glucose uptake and increased hexokinase activity in enterocytes. Lactate dehydrogenase, pyruvate kinase and 6-phosphofructo 1-kinase activities were not found to be significantly different in epithelial cells isolated from control or diabetic animals. Our results indicate that a reduction of the glycolytic flux in enterocytes could collaborate to increase intestinal glucose absorption in the diabetic state.

Effect of sulfonylureas on hepatic glycogen metabolism: Activation of glycogen phosphorylase☆☆☆

In hepatocytes isolated from fed rats, both tolbutamide and glipizide caused a dose-dependent activation of glycogen phosphorylase, possibly by a Ca2+-mediated mechanism. Maximal effects (about twofold) were already obtained when drugs were used at 0.5 mmol/L, the calculated concentrations of tolbutamide and glipizide responsible for the half-maximal effects being 60 and 30 mumol/L, respectively. The activation of glycogen phosphorylase caused the mobilization of glycogen and increased the cellular concentration of hexose 6-phosphates (glucose 6-phosphate plus fructose 6-phosphate) and that of fructose 2,6-bisphosphate. Under the influence of sulfonylureas, glucose formation was slightly stimulated while the rate of L-lactate production was more markedly incremented, indicating that sulfonylureas canalize the metabolic flux coming from glycogen mainly to the glycolytic pathway. These results suggest that a glycogenolytic action of sulfonylureas could collaborate to raise hepatic fructose 2,6-bisphosphate concentration in the fed animal.

Sulfonylurea effects on acid and pepsinogen secretion in isolated rabbit gastric glands

The influence of different sulfonylureas on the rate of acid and pepsinogen secretion was studied in isolated rabbit gastric glands. Neither tolbutamide (10-500 microM), chlorpropamide (10-500 microM), glibenclamide (1-50 microM) nor glipizide (1-50 microM) exerted a secretory effect. In contrast, gliquidone caused a marked and dose-dependent stimulation of acid production in gastric glands incubated under basal conditions and potentiated the stimulatory effect of both histamine and carbachol. Gliquidone also increased the rate of pepsinogen release in gastric glands incubated either under basal conditions or in the presence of cholecystokinin-octapeptide or isoproterenol. The secretory effects of gliquidone were associated with a significant increase in the glandular content of cyclic AMP, caused by a competitive inhibition of low-Km cyclic AMP phosphodiesterase. Our results indicate that, among the assayed sulfonylureas, only gliquidone, in the micromolar range, stimulates acid and pepsinogen secretion through a cyclic AMP-dependent mechanism.

Interconversion phenomena between two kinetic forms of class a pyruvate kinase from Ehrlich ascites tumor cells

Summary1.The regulatory properties of two interconvertible kinetic forms of class A pyruvate kinase from Ehrlich ascites tumor cells have been studied with a partially purified enzyme preparation free of interfering enzymatic activities.2.The hyperbolic form shows Michaelis-Menten kinetics for P-pyruvate, with high affinity for this substrate and low affinity for the inhibitory amino acids alanine and phenylalanine. The sigmoidal form displays positive cooperativity respect to P-pyruvate (n = 1.4), with lower affinity for this substrate and higher affinity for the inhibitory amino acids.3.The equilibrium between the hyperbolic and the sigmoidal forms of the enzyme is affected by substrates and effectors. P-pyruvate, ADP and Fru-P2 shift the equilibrium to the hyperbolic form while ATP, alanine and phenylalanine stabilize the sigmoidal form.4.Effector metabolites affect the molecular weight of the protein, acting on an equilibrium between dimers and tetramers. P-pyruvate and ADP associate the enzyme to a tetramer while ATP, alanine and phenylalanine favor the occurrence as a dimer. The positive modifier Fru-P2 did not associate the enzyme to the tetramer, even at 1mm concentration.5.A tentative molecular model for pyruvate kinase A on the basis of the kinetic and aggregation interconversion is proposed.

Inhibition of gluconeogenesis by tolbutamide in isolated rat hepatocytes: Modulation of glucose-6-phosphate substrate cycle

In hepatocytes isolated from 24-hour fasted rats, the oral hypoglycemic agent tolbutamide (1 mmol/L) inhibited glucose formation from different concentrations (1 to 20 mmol/L) of galactose, dihydroxyacetone, glycerol, and a mixture of L-lactate:pyruvate (molar ratio, 10:1). Parallel to the reduction of gluconeogenesis, tolbutamide stimulated L-lactate formation when cells were incubated with either galactose, dihydroxyacetone, or glycerol. All these tolbutamide effects occurred without significant modification of hepatocyte fructose-2,6-bisphosphate (F-2,6-P2) levels. Only when glucose was included in the incubation medium was the inhibition of gluconeogenesis caused by the sulfonylurea accompanied by a significant increment of the cellular F-2,6-P2 concentration. Under these conditions, tolbutamide potentiated the effect of glucose in promoting the increase of this regulatory metabolite, as well as the stimulation of glycolysis; in addition, tolbutamide increased the cellular pool of hexose-6-phosphates and the rate of tritium release from (2-3H)glucose. These results support the hypothesis that tolbutamide regulates hepatic glucose metabolism, at least, by modulating the glucose-6-phosphate substrate cycle.

Glipentide and glucose metabolism in isolated rat hepatocytes

Glipentide, a second generation sulfonylurea, raised the cellular concentration of fructose 2,6-bisphosphate in isolated rat hepatocytes. Parallel to accumulating this regulatory metabolite, glipentide inhibited basal gluconeogenesis and increased the rate of L-lactate production, as well as the metabolic flux through the 6-phosphofructo 1-kinase reaction. Tolbutamide elicited similar metabolic effects to those reported for glipentide, although the latter sulfonylurea was about 10 times more potent. The biochemical mechanism by which sulfonylureas promote the accumulation of fructose 2,6-bisphosphate in hepatocytes seems to be related to a significant increase of the hexose 6-phosphate pool (glucose 6-phosphate plus fructose 6-phosphate), together with the activation of 6-phosphofructo 2-kinase and inactivation of fructose 2,6-bisphosphatase, enzyme activities responsible, respectively, for the synthesis and degradation of fructose 2,6-bisphosphate.

Modulation of Epinephrine-Stimulated Gluconeogenesis by Insulin in Hepatocytes Isolated from Genetically Obese (fa/fa) Zucker Rats

Genetically obese (fa/fa) Zucker rats present an impaired response of hepatic glucose production to the inhibition by insulin. In this work, we have investigated the modulation by this hormone of epinephrine-stimulated gluconeogenesis, in hepatocytes isolated from obese (fa/fa) rats and their lean (Fa/-) littermates. Epinephrine (1 μm) caused a maximal stimulation of [14C]lactate conversion to [14C]glucose in hepatocytes isolated from either obese or lean animals. The stimulation of gluconeogenesis by epinephrine was accompanied by a significant reduction of fructose 2,6-bisphosphate levels, an inactivation of both pyruvate kinase and 6-phosphofructo 2-kinase, and by a 2-fold increase in the cellular concentrations of cAMP. The presence of insulin in the incubation medium antagonized, in a concentration-dependent manner, the effects of epinephrine. In hepatocytes isolated from lean rats, the reversion caused by insulin was complete, the concentration required for half-maximal insulin action ranging from 0...