Chlorpropamide raises fructose-2,6-bisphosphate concentration and inhibits gluconeogenesis in isolated rat hepatocytes.

The addition of chlorpropamide to hepatocytes isolated from fed rats raised the cellular concentration of fructose-2,6-bisphosphate (F-2,6-P2), a regulatory metabolite that plays a relevant role in the control of hepatic glucose metabolism. The effect of chlorpropamide was dose dependent; a statistically significant increase was already seen at 0.2 mM of the sulfonylurea. The accumulation of F-2,6-P2 caused by chlorpropamide (1 mM) was parallel to the stimulation of L-lactate production (36.6 +/- 4.8 versus 26.1 +/- 2.6 mumol of lactate/g of cells X 20 min; N = 5, P less than 0.05) and to the inhibition of gluconeogenesis (0.57 +/- 0.1 versus 0.94 +/- 0.09 mumol of [U-14C]pyruvate converted to glucose/g of cells X 20 min; N = 5, P less than 0.05). In addition, chlorpropamide enhanced the inhibitory action evoked by insulin on glucagon-stimulated gluconeogenesis. This combined effect of chlorpropamide and insulin seems to be correlated with the synergistic accumulation of F-2,6-P2 provoked by the simultaneous action of these two agents on glucagon-treated hepatocytes. Finally, neither 6-phosphofructo-2-kinase activity nor hepatocyte cyclic AMP levels were significantly changed by the presence of the sulfonylurea in the incubation medium. Our results support the concept that chlorpropamide, by a cyclic AMP-independent mechanism, increases the hepatic content of F-2,6-P2 and, in this way, enhances the glycolytic flux and inhibits glucose output by the liver.

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 microM) 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 0.22 to 0.56 nM. In contrast, in obese rat hepatocytes, insulin only partially blocked epinephrine-mediated effects, and the sensitivity to insulin was 2- to 4-fold lower, as indicated by the corresponding half-maximal insulin action values. Furthermore, insulin (10 nM) almost completely blocked the increase in cAMP levels induced by epinephrine in lean rat hepatocytes, whereas it only provoked a small and nonsignificant reduction of epinephrine-stimulated levels of the cyclic nucleotide in hepatocytes obtained from obese rats.

Impairment of the modulation by glucose of hepatic gluconeogenesis in the genetically obese (fa/fa) Zucker rat.

Genetically obese (fa/fa) Zucker rats show oral glucose intolerance, an alteration that has been attributed at least in part to an impaired suppression of hepatic glucose output after the ingestion of glucose. In this work, we studied the influence of different concentrations of glucose (2.5-30 mM) on gluconeogenesis from a mixture of [14C]lactate-pyruvate as well as on fructose 2,6-bisphosphate levels, pyruvate kinase activity, and flux through the reaction catalyzed by 6-phosphofructo-1-kinase, in hepatocytes isolated from fed obese (fa/fa) or lean (Fa/-) rats. In hepatocytes isolated from lean rats, incubation with increasing concentrations of glucose caused a dose-dependent inhibition of gluconeogenesis (5.02 +/- 0.54 and 1.82 +/- 0.33 mumol lactate converted to glucose/g cells.20 min in hepatocytes incubated in the presence of 2.5 and 30 mM glucose, respectively; n = 4 experiments; P < 0.01) together with a significant elevation of the fructose 2,6-bisphosphate content and a stimulation of the flux through 6-phosphofructo-1-kinase reaction. Glucose also provoked a dose-dependent activation of pyruvate kinase in the absence of changes in the cellular concentration of cAMP. In liver cells from obese animals, gluconeogenesis was not significantly modified by raising the glucose concentration in the incubation medium (1.26 +/- 0.11 and 0.83 +/- 0.14 mumol lactate converted to glucose/g cells.20 min in hepatocytes incubated with 2.5 and 30 mM glucose, respectively; n = 4 experiments; P = 0.11) despite significant increases in both fructose 2,6-bisphosphate levels and flux through the 6-phosphofructo-1-kinase reaction. In these cells, pyruvate kinase was only slightly activated by high glucose concentrations. These results indicate that, unlike fructose 2,6-bisphosphate levels and flux through the 6-phosphofructo-1-kinase reaction, hepatic gluconeogenesis is unresponsive to high glucose concentrations in genetically obese (fa/fa) rats.

Impairment of glycosyl-phosphatidylinositol-dependent insulin signaling system in isolated rat hepatocytes by streptozotocin-induced diabetes.

The addition to different types of cells of an inositol-phosphate glycan, generated by the phospholipase C-catalyzed hydrolysis of a insulin-sensitive glycosyl-phosphatidylinositol (glycosyl-PI), mimics some of the biological effects of this hormone. Recently, a specific, time-, dose-, and energy-dependent transport system for this inositol-phosphate glycan has been identified in isolated rat hepatocytes. Here, we show that streptozotocin-induced diabetes mellitus reduced (by about 60%) the basal content of the insulin-sensitive glycosyl-PI in isolated rat hepatocytes. Moreover, streptozotocin-induced diabetes blocked the hydrolysis of the glycosyl-PI in response to insulin, diminished inositol phosphate-glycan uptake by the hepatocytes, and abolished the stimulatory effect of this compound on glycogen synthesis. All these metabolic changes caused by streptozotocin administration were reversed by treatment of the animals with insulin. Our results support the hypothesis that insulin resistance in streptozotocin-induced diabetic rats is related to the impairment of glycosyl-PI metabolism.

Insulin resistance in genetically obese (fa/fa) rats: changes in the glycosyl-phosphatidylinositol signaling system in isolated hepatocytes.

In different types of mammalian cells, insulin has been shown to promote the release of an inositol phosphate glycan (InsP-glycan) through the hydrolysis of a glycosyl-phosphatidylinositol (glycosyl-PtdIns). This InsP-glycan, which has been demonstrated to be taken up by intact cells, may mediate some of the biological effects of insulin. We have investigated how the insulin resistance expressed in genetically obese (fa/fa) rats affects the glycosyl-PtdIns signaling system in isolated hepatocytes compared to what occurs in hepatocytes isolated from lean (Fa/-) rats. The hepatocyte content of glycosyl-PtdIns was reduced by about 30% in obese rats, with respect to that measured in lean rats (2553 +/- 138 vs. 3334 +/- 115 dpm/mg protein; P < 0.01; n = 5). This reduction was accompanied by a marked blockade of the insulin-mediated glycosyl-PtdIns hydrolysis as well as a decrease (approximately 30%) in the rate of InsP-glycan uptake by the isolated liver cells. Obese Zucker rat hepatocytes also showed a significant decrease in the effects of both insulin and InsP-glycan on the stimulation of glycogen synthesis and the activation of glycogen synthase compared to hepatocytes isolated from lean rats. Our results demonstrate that genetic obesity in Zucker (fa/fa) rats is associated with an impairment of the glycosyl-PtdIns-dependent insulin signaling system.

Decreased responsiveness of basal gluconeogenesis to insulin action in hepatocytes isolated from genetically obese (fa/fa) Zucker rats.

In vivo studies have demonstrated that hepatic glucose production is poorly responsive to insulin in genetically obese Zucker rats. In this work, we have investigated the modulation by insulin of basal gluconeogenesis, fructose 2,6-bisphosphate levels, and pyruvate kinase and 6-phosphofructo 2-kinase activities in hepatocytes isolated from fed obese (fa/fa) or lean (Fa/-) rats. Gluconeogenesis was estimated by the conversion of a mixture of [14C]lactate-pyruvate to [14C]glucose. Basal gluconeogenesis was significantly reduced in hepatocytes isolated from obese rats compared to that measured in hepatocytes from lean animals (0.63 +/- 0.09 vs. 1.47 +/- 0.05 mumol lactate converted to glucose/g cells.20 min; n = 3-4; P < 0.001). In hepatocytes isolated from lean rats, insulin, without affecting the cellular cAMP concentration, caused a dose-dependent inhibition of the rate of gluconeogenesis, which was accompanied by a significant increase in fructose 2,6-bisphosphate levels and activation of both pyruvate kinase and 6-phosphofructo 2-kinase. In contrast, in hepatocytes isolated from obese (fa/fa) rats, neither basal gluconeogenesis nor any of the other metabolic parameters mentioned were significantly modified by insulin, even when assayed at high hormonal concentrations (10 nM). These results demonstrate a lack of responsiveness of hepatic gluconeogenesis to short term insulin action in genetically obese (fa/fa) rats.

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.

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.

Tolbutamide enhances insulin action on gluconeogenesis and on fructose 2,6-bisphosphate levels in isolated rat hepatocytes.

In hepatocytes isolated from fed rats and incubated either under basal conditions or in the presence of glucagon, tolbutamide reduced gluconeogenesis from (U-14C) pyruvate by increasing the cellular concentration of fructose 2,6-bisphosphate. Furthermore, this sulfonylurea enhanced the inhibitory action of insulin on glucagon-stimulated gluconeogenesis; this effect was accompanied by a more marked increase of the cellular concentration of fructose 2,6-bisphosphate than that elicited by either insulin or the sulfonylurea alone. In connection with this, tolbutamide--without significant modification of cellular cyclic AMP levels--raised the proportion of 6-phosphofructo 2-kinase in active form in hepatocytes incubated either under basal conditions or in the presence of glucagon, and reinforced the action of insulin in antagonizing the glucagon-mediated inactivation of this enzyme.

Modulation of gluconeogenesis by epinephrine in hepatocytes isolated from genetically obese (fa/fa) Zucker rats.

The obese (fa/fa) Zucker rat shows an impaired sympathetic tone which is accompanied by an altered thermogenesis and changes in both lipid and carbohydrate metabolism. In this work, we have investigated the regulatory effects of epinephrine on the rate of gluconeogenesis from a mixture of [(14)C]lactate/pyruvate, in hepatocytes isolated from obese (fa/fa) rats and their lean (Fa/-) littermates. Epinephrine caused a dose-dependent stimulation of the rate of [(14)C]glucose formation in both obese and lean rat hepatocytes, the maximal rates being five- and twofold higher than the corresponding basal values (0.50 +/- 0.06 and 1.96 +/- 0.15 micromol of lactate converted to glucose/g of cell x 20 min, respectively). No significant differences were found between the calculated half-maximal effective concentrations (EC(50)) for epinephrine in obese and lean rat liver cells. The stimulation of gluconeogenesis by epinephrine was accompanied by a decrease in the cellular concentration of fructose 2,6-bisphosphate, and an inactivation of both pyruvate kinase and 6-phosphofructo 2-kinase, to similar extents in both types of hepatocytes. Epinephrine also significantly raised the hepatocyte content of cyclic AMP, with about a twofold increase at a saturating concentration of the catecholamine (1 microM), in both lean and obese rat liver cells. However, at suboptimal concentrations of epinephrine, the rise in cyclic AMP levels was significantly less marked in obese than in lean rat hepatocytes. Nevertheless, no significant differences were found in either the affinity or the number of beta-adrenergic receptors, in radioligand binding studies carried out in liver plasma membranes obtained from obese and lean Zucker rats. In conclusion, compared to the corresponding basal values, the response of gluconeogenesis from lactate to the stimulatory effect of epinephrine is higher in obese (fa/fa) than in lean (Fa/-) Zucker rat hepatocytes, with no significant differences in the calculated EC(50) values for this hormone. This occurs in spite of an apparent decreased sensitivity of the adenylate cyclase system to the stimulatory effect of epinephrine in obese rat liver cells.

Impairment of H+-K+-ATPase-dependent proton transport and inhibition of gastric acid secretion by ethanol.

Ethanol (1-20% vol/vol) caused a dose-dependent reduction in the basal rate of acid formation in isolated rabbit gastric glands with a calculated EC(50) value of 4.5 +/- 0.2%. Ethanol also reduced ATP levels in isolated gastric glands and in cultured parietal cells (EC(50): 8.8 +/- 0.4% and 8.5 +/- 0.2%, respectively) and decreased both basal and forskolin-stimulated cAMP levels. In studies carried out in gastric gland microsomes, ethanol inhibited the hydrolytic activity of H+-K+-ATPase(EC(50): 8.5 +/- 0.6%), increased passive proton permeability (EC(50): 7.9%), and reduced H+-K+-ATPase-dependent proton transport (EC(50): 3%). Our results show that the inhibition of gastric acid secretion observed at low concentrations of ethanol (< or =5%) is mainly caused by the specific impairment of H+-K+-ATPase-dependent proton transport across cell membranes rather than inhibition of the hydrolytic activity of H+-K+-ATPase, reduction in the cellular content of ATP, or increase in the passive permeability of membranes to protons, although these changes, in combination, must be relevant at concentrations of ethanol > or =7%.

Changes in the insulin-sensitive glycosyl-phosphatidyl-inositol signalling system with aging in rat hepatocytes.

An inositol-phosphate glycan (InsP glycan), which is the polar head group of an insulin-sensitive glycosyl-phosphatidylinositol (glycosyl-PtdIns), has been reported to mimic some insulin actions when added to different types of cells. In connection with this, a specific, time-dependent and energy-dependent transport system for this InsP glycan has been identified in isolated rat hepatocytes [Alvarez, J. F., Sánchez-Arias, J. A., Guadaño, A., Estevez, F., Varela, I., Felíu, J. E. & Mato, J.M. (1991) Biochem. J. 274, 369-374]. Here we have investigated the glycosyl-PtdIns-dependent insulin-signalling system in hepatocytes isolated from either 3-month-old or 24-month-old rats. Aging reduced the stimulatory effect of insulin on [U-14C]glucose incorporation into glycogen, caused a significant decrease in basal glycosyl-PtdIns levels and blocked the insulin-mediated hydrolysis of this lipid. In 24-month-old rats, we also observed a diminution in the rate of hepatocyte InsP-glycan uptake and a marked reduction of the stimulatory effect of this compound on glycogen synthesis. These results support the hypothesis that insulin resistance associated with aging is accompanied by an impairment of the glycosyl-PtdIns-dependent cellular signalling system.