Alpha 1-Adrenergic stimulation of Ca2+ mobilization without phosphorylase activation in hepatocytes from phosphorylase b kinase-deficient gsd/gsd rats.

Phenylephrine, vasopressin and the bivalent cation ionophore A23187 mobilized Ca2+ normally, but failed to activate phosphorylase, in hepatocytes from gsd/gsd rats with a deficiency of liver phosphorylase b kinase. These data provide strong evidence that phosphorylase b kinase is the site of action of the Ca2+ mobilized intracellularly during alpha 1-adrenergic activation of phosphorylase in liver cells.     

Age-related augmentation of phosphorylase b kinase in hepatic tissue from the glycogen-storage-disease (gsd/gsd) rat.

The effects of food deprivation on body weight, liver weight, hepatic glycogen content, glycogenolytic enzymes and blood metabolites were compared in young and old phosphorylase b kinase-deficient (gsd/gsd) rats. Although the concentration of glycogen in liver from 9-week-old female gsd/gsd rats (730 mumol of glucose equivalents/g wet wt.) was increased by 7-8% during starvation, total hepatic glycogen was decreased by 12% after 24 h without food. In 12-month-old male gsd/gsd rats the concentration of liver glycogen (585 mumol of glucose equiv./g wet wt.) was decreased by 16% and total hepatic glycogen by nearly 40% after food deprivation for 24 h. Phosphorylase b kinase and phosphorylase a were present at approx. 10% of the control activities in 9-week-old gsd/gsd rats, but both enzyme activities were increased more than 3-fold in 12-month-old affected rodents. It is concluded that the age-related ability to mobilize hepatic glycogen appears to result from the augmentation of phosphorylase b kinase during maturation of the gsd/gsd rat.     

The activation of glycogen synthase in hepatocytes from rats with a glycogen storage disorder (gsd/gsd)

A rapid high resolution method of purification of the Trp-containing S100 proteins (S100a, S100a′) and of the S100b protein has been developed. The principle of this method is based on the fact that S100b protein becomes highly hydrophobic upon Zn²⁺ binding, whereas S100a and S100a′ are not affected. On an affinity chromatography of phenyl—Sepharose column, S100b is selectively bound in presence of zinc, whereas the Trp-containing S100 patients are quickly eluted. The S100b protein is further eluted with a buffer containing EDTA.     

Effects in vivo of food deprivation and 3-mercaptopicolinate in the glycogen-storage-disease (gsd/gsd) rat.

Intraperitoneal injection of 3-mercaptopicolinate into 24 h-food-deprived 27-week-old female control (GSD/GSD) rats lowered the concentration of circulating glucose by 66%, but glycerol and lactate concentrations were increased up to 3- and 4-fold respectively. In phosphorylase b kinase-deficient (gsd/gsd) rats the corresponding changes for blood glucose, lactate and glycerol were half those observed in the controls. Although the concentration of liver glycogen (approx. 12%, w/w) in the gsd/gsd rats was not altered during food deprivation, total hepatic glycogen was decreased by 17%. It is suggested that the gradual breakdown of the extensive hepatic glycogen stores during starvation assists in the maintenance of normoglycaemia in the gsd/gsd rat.     

Skeletal muscle glycogen content, structure, and metabolism are normal in rats with hepatic glycogen phosphorylase kinase deficiency

Skeletal muscle glycogen content and structure, and the activities of several enzymes of glycogen metabolism are reported for the hepatic glycogen phosphorylase b kinase deficient (gsd/gsd) rat. The skeletal muscle glycogen content of the fed gsd/gsd rat is 0.50 +/- 0.11% tissue wet weight, and after 40 hours of starvation this value is lowered 40% to 0.30 +/- 0.05% tissue wet weight. In contrast the gsd/gsd rat liver has an elevated glycogen content which remains high after starvation. The skeletal muscle phosphorylase b kinase, glycogen phosphorylase, glycogen synthase and acid alpha-glucosidase activities are 17.2 +/- 2.9 units/g tissue, 119.9 +/- 6.4 units/g tissue, 12.2 +/- 0.4 units/g tissue and 1.4 +/- 0.4 milliunits/g tissue, respectively, with approx. 20% of phosphorylase and approx. 24% of synthase in the active form (at rest). These enzyme activities resemble those of Wistar skeletal muscle, and again this contrasts with the situation in the liver where there are marked differences between the Wistar and the gsd/gsd rat. Fine structural analysis of the purified glycogen showed resemblance to other glycogens in branching pattern. Analysis of the molecular weight distribution of the purified glycogen indicated polydispersity with approx. 66% of the glycogen having a molecular weight of less than 250 X 10(6) daltons and approx. 25% greater than 500 X 10(6) daltons. This molecular weight distribution resembles those of purified Wistar liver and skeletal muscle glycogens and differs from that of the gsd/gsd liver glycogen which has an increased proportion of the low molecular weight material.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of hepatic glycogen phosphorylase and glycogen synthase by calcium and diacylglycerol

Incubation of rat hepatocytes with angiotensin II (1 nM) produced a time-dependent accumulation of 1, 2-diacylglycerol and inactivation of glycogen synthase with maximum effects at 10 min. The level of diacylglycerol then gradually declined and the activity of glycogen synthase I returned to control values at 30 min. In contrast, angiotensin II caused an increase in cytosolic Ca2+ and an activation of glycogen phosphorylase which were rapid and transient, reaching maximum values in less than 2 min and then returning to control levels at 15 min. There were excellent correlations between the changes in glycogen synthase I and diacylglycerol levels and between the changes in phosphorylase alpha and cytosolic Ca2+ in these time-course studies. However, there was no correlation between the changes in diacylglycerol and phosphorylase alpha or between the changes in cytosolic Ca2+ and glycogen synthase I. Norepinephrine also caused a slow increase in diacylglycerol and inactivation of glycogen synthase, and a rapid increase in cytosolic free Ca2+ and activation of glycogen phosphorylase. Addition of an alpha1-adrenergic blocker (prazosin or phentolamine) caused rapid decreases in cytosolic free Ca2+ and phosphorylase alpha, but only slowly reversed the inactivation of synthase and accumulation of diacylglycerol. The dose-response curves for norepinephrine and prazosin on glycogen synthase were well correlated with those on diacylglycerol. It is proposed that in liver cells, Ca2+-mobilizing hormones regulate phosphorylase a through a Ca2+-dependent mechanism and inactivate glycogen synthase through the generation of diacylglycerol, at least in part. The data provide additional support for the view that protein kinase C may be important in the regulation of glycogen synthase in liver.     

Insulin regulation of hepatic glycogen synthase and phosphorylase.

The relative roles of insulin and glucose in the regulation of hepatic glycogen synthase and phosphorylase were studied in hepatocytes from fed rats. Elevation of extra-cellular glucose led to a rapid decrease in phosphorylase a activity followed by a slower increase in glycogen synthase I activity. A reciprocal and coordinate relationship between phosphorylase inactivation and synthase activation in response to glucose was observed; following initial glucose-induced inactivation of phosphorylase, there was a highly significant linear inverse relationship between residual phosphorylase activity and glycogen synthase activation. Insulin led to a further decrease in phosphorylase activity and a 30-50% additional increase in glycogen synthase activity over that caused by glucose. The effects of insulin required the presence of glucose and served to augment acute glucose stimulation of glycogen synthase and inhibition of phosphorylase. Insulin did not perturb the reciprocal and coordinate relationship between phosphorylase inactivation and synthase activation in response to glucose. The results suggest that the ability of insulin to activate hepatic glycogen synthase can be entirely accounted for by its ability to inactivate phosphorylase.     

Expression and regulation of glycogen phosphorylase in preneoplastic and neoplastic hepatic lesions in rats

Glycogen phosphorylase (PHO) was demonstrated immunocytochemically and enzyme histochemically in cryostat sections of liver from rats treated for 7 weeks with N-nitrosomorpholine (120 mg/l and 200 mg/l drinking water) and from untreated controls. The activity and distribution of PHO protein were studied in normal liver and correlated with morphologically defined stages of hepatic tumour development. In normal liver the amount of enzyme protein, as visualized by the immunoperoxidase method using antibodies against phosphorylase, showed some heterogeneity within the liver lobule. The intralobular and intracellular distribution of PHO protein was the same as that of glycogen, namely coarse and granular in periportal hepatocytes and very fine in perivenular cells. In glycogen storage foci the amount of PHO protein was increased. In contrast, PHO activity was generally decreased. In other preneoplastic and neoplastic lesions such as mixed cell foci, neoplastic nodules and hepatocellular carcinomas, PHO protein was increased in all glycogen-loaded cells while PHO activity was reduced. In all glycogen-poor and basophilic cells, both PHO protein and PHO activity were decreased or absent. It was concluded that the decrease in PHO activity in glycogen storage foci was not the direct consequence of genetic changes leading to a loss in enzyme protein but was due to a defect in the cascade of phosphorylation processes resulting in active PHO. Alteration in gene expression leading to a loss of PHO protein was a late event in the process of hepatocarcinogenesis.     

Hepatic carbon flux after re-feeding in the glycogen-storage-disease (gsd/gsd) rat.

In this study we utilized the phosphorylase b kinase-deficient (gsd/gsd) rat as a model of hepatic substrate utilization where there is a constraint on glycogenesis imposed by the maintenance of high glycogen concentrations. Glucose re-feeding of 48 h-starved gsd/gsd rats led to suppression of hepatic glucose output. In contrast with the situation in normal rats, activation of the pyruvate dehydrogenase complex and lipogenesis was observed. It is suggested that impeding glycogenic flux may divert substrate into lipogenesis, possibly via activation of the pyruvate dehydrogenase complex.     

The action of anoxia and cyanide on glycogen breakdown in the liver of the gsd/gsd rat

Perfusion of normal rat livers under anoxic conditions or the addition of KCN to aerobic perfusions activated phosphorylase and stimulated glycogen breakdown and glucose output. Livers from rats with a deficiency of liver phosphorylase kinase (gsd/gsd) showed a much smaller activation of phosphorylase with anoxia or KCN and produced glucose at about half the rate of normal livers. The increase in phosphorylase a in gsd/gsd livers was insufficient to account for the increase in glucose output. The addition of KCN to normal hepatocytes, activated phosphorylase and stimulated glucose output almost as effectively as glucagon. Hepatocytes from gsd/gsd rats showed only a very small increase in phosphorylase a on the addition of KCN, and glucose output did not increase. We conclude that in the perfused liver, anoxia and KCN stimulate glycogen breakdown and glucose output, at least in part, by a mechanism that does not involve conversion of phosphorylase b to phosphorylase a. In isolated hepatocytes KCN stimulates glucose output only by increasing the content of phosphorylase a.     

The behavior of hepatic phosphorylase b kinase, phosphorylase a and b after administration of glucagon to patients with glycogen storage disease type VIa

In the patients with glycogen storage disease (GSD) type VIa and different serum glucose response to glucagon, the activities of hepatic phosphorylase b kinase, phosphorylase a and b were estimated before and after the intravenous administration of glucagon. 3 min after the administration of glucagon an increase in the activities of phosphorylase b kinase and phosphorylase a was found in liver tissue of all patients except one. These enzymatic activities, however, did not exceed the values of these enzymes in the control liver biopsies without glucagon loading. After the intravenous administration of glucagon an unsuspected increase of phosphorylase b activity was observed in the control liver tissues and in patients with GSD type VIa, except one. In vitro investigations revealed that an increase of hepatic phosphorylase b activity occurs during its conversion to phosphorylase a. We suppose that this phosphorylase b represents a partially phosphorylated form of this enzyme (an intermediate form) that is due to the action of the active phosphorylase b kinase. The correlations between the activities of phosphorylase b kinase, phosphorylase a and an intermediate form of phosphorylase b and hepatic glycogen degradation after administration of glucagon has been discussed.