The effects of glucose and of potassium ions on the interconversion of the two forms of glycogen phosphorylase and of glycogen synthetase in isolated rat liver preparations.

In the isolated perfused rat liver, increasing glucose concentration from 5.5 to 55 mm in the perfusion medium caused a sequential inactivation of glycogen phosphorylase and activation of glycogen synthetase. The latter change was preceded by a lag period which corresponded to the time required to inactivate the major part of the phosphorylase. 2. The same sequence of events was observed in isolated rat hepatocytes incubated at 37C. In this preparation, the rate of phosphorylase inactivation was greatly increased by increasing the concentration of glucose and/or of K+ ions in the external medium. The same agents also caused the activation of glycogen synthetase, but this effect was secondary to the inactivation of phosphorylase. 3. In both types of preparations, the rate of synthetase activation was modulated by the residual amount of phosphorylase a that remained after the initial phase of rapid inactivation and was independent of glucose concentration. 4. In isolated hepatocytes, the rate of conversion of glucose into glycogen was propotional to the activity of synthetase a in the preparation. This conversion was preceded by a lag period which could be shortened by increasing either glucose or K+ concentration in the medium. The incorporation of labelled glucose into glycogen was simultaneous with a glycogenolytic process which could not be attributed to the activity of phosphorylase a.     

Interactions between insulin and alpha 1-adrenergic agents in the regulation of glycogen metabolism in isolated hepatocytes

Addition of insulin to liver cells from fed rats incubated in the absence of other hormones resulted in a 2-fold increase in glycogen synthase activity. This direct effect of insulin has been characterized and compared with the antagonism by insulin of alpha 1-adrenergic effects on glycogen metabolism. The activation of glycogen synthase by insulin developed slowly (20-25 min) and was most effective when the enzyme was partially preactivated by glucose. With glucose concentrations above 15 mM the effects of insulin and glucose were additive. In contrast to glucose, which caused inverse changes in phosphorylase and glycogen synthase activity, insulin activated glycogen synthase without affecting phosphorylase a. Treatment of hepatocytes with phenylephrine led to an activation of phosphorylase and inactivation of glycogen synthase, which could be partially blocked by insulin. This antagonistic effect of insulin was rapid (complete within 5 min of insulin addition) and showed an identical time course for both enzymes. The activation of glycogen synthase by insulin and inactivation by phenylephrine both resulted principally from alterations in the Vmax. Insulin added alone did not alter the basal cytosolic free Ca2+ concentration, which was 160 nM as measured with Quin 2 as an intracellular Ca2+ indicator. Both the magnitude and the initial rate of cytosolic free Ca2+ increase induced by phenylephrine were reduced by about 50% in cells pretreated with insulin. It is concluded that the direct activation of glycogen synthase by insulin is mediated by a glycogen synthase-specific kinase or phosphatase, whereas insulin antagonizes the effects of alpha 1-agonists by interfering with their ability to elevate cytosolic free Ca2+.     

Non-hormonal and hormonal control of glycogen metabolism in isolated sheep liver cells

1. Control of glycogen metabolism by various substrates and hormones was studied in ruminant liver using isolated hepatocytes from fed sheep. 2. In these cells glucose appeared uneffective to stimulate glycogen synthesis whereas fructose and propionate activated glycogen synthase owing to (i) a decrease in phosphorylase a activity and (ii) changes in the intracellular concentrations of glucose 6-phosphate and adenine nucleotides. 3. The activation of hepatic glycogenolysis by glucagon and alpha 1-adrenergic agents was associated with increased phosphorylase a and decreased glycogen synthase activities. 4. The simultaneous changes in these two enzyme activities suggest that in sheep liver, activation of phosphorylase a is not a prerequisite step for synthase inactivation. 5. In sheep hepatocytes, in the presence of propionate and after a lag period, insulin activated glycogen synthase without affecting phosphorylase a. 6. This latter result suggests that the direct activation of glycogen synthase by insulin is mediated by a glycogen synthase-specific kinase or phosphatase. Insulin also antagonized glucagon effect on glycogen synthesis by counteracting the rise of cAMP.     

Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells.

Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase...     

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.     

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.     

The glycogenic action of protein targeting to glycogen in hepatocytes involves multiple mechanisms including phosphorylase inactivation and glycogen synthase translocation

Expression of the glycogen-targeting protein PTG promotes glycogen synthase activation and glycogen storage in various cell types. In this study, we tested the contribution of phosphorylase inactivation to the glycogenic action of PTG in hepatocytes by using a selective inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a and sequential activation of glycogen synthase. Similar to CP-91194, graded expression of PTG caused a concentration-dependent inactivation of phosphorylase and activation of glycogen synthase. The latter was partially counter-acted by the expression of muscle phosphorylase and was not additive with the activation by CP-91149, indicating that it is in part secondary to the inactivation of phosphorylase. PTG expression caused greater stimulation of glycogen synthesis and translocation of glycogen synthase than CP-91149, and the translocation of synthase could not be explained by accumulation of glycogen, supporting an additional role for glycogen synthase translocation in the glycogenic action of PTG. The effects of PTG expression on glycogen synthase and glycogen synthesis were additive with the effects of glucokinase expression, confirming the complementary roles of depletion of phosphorylase a (a negative modulator) and elevated glucose 6-phosphate (a positive modulator) in potentiating the activation of glycogen synthase. PTG expression mimicked the inactivation of phosphorylase caused by high glucose and counteracted the activation caused by glucagon. The latter suggests a possible additional role for PTG on phosphorylase kinase inactivation.     

Regulation of Glycogen Metabolism in Primary and Transformed Astrocytes In Vitro

Glycogen metabolism was studied in primary and Herpesvirus-transformed cultures of neonatal rat brain astrocytes. A small fraction of the glucose consumed was conserved in glycogen in both the primary and the transformed astrocytic cell cultures. After addition of culture medium containing 5.5 mM glucose, glycogen increased to maximal levels within 2.5 h, the approximate time at which half of the medium glucose was consumed, and rapidly declined thereafter in both the primary and transformed astrocytic cultures. Maximum levels of glycogen were apparently related to the cell density of the Herpesvirus-transformed cultures, but primary cultures did not show this behavior. At any given cell density, maximal levels of glycogen were dependent on the concentration of extracellular glucose. Administration of glucose caused a transient activation of glycogen synthase alpha and a rapid inactivation of glycogen phosphorylase alpha.     

Effect of fructose 1-phosphate on the activation of liver glycogen synthase.

The activation (dephosphorylation) of glycogen synthase and the inactivation (dephosphorylation) of phosphorylase in rat liver extracts on the administration of fructose were examined. The lag in the conversion of synthase b into a was cancelled, owing to the accumulation of fructose 1-phosphate. A decrease in the rate of dephosphorylation of phosphorylase a was also observed. The latency re-appeared in gel-filtered liver extracts. Similar latency was demonstrated in extracts from glucagon-treated rats. Addition of fructose 1-phosphate to the extract was able to abolish the latency, and the activation of glycogen synthase and the inactivation of phosphorylase occurred simultaneously. Fructose 1-phosphate increased the activity of glycogen synthase b measured in the presence of 0.2-0.4 mM-glucose 6-phosphate. According to kinetic investigations, fructose 1-phosphate increased the affinity of synthase b for its substrate, UDP-glucose. The accumulation of fructose 1-phosphate resulted in glycogen synthesis in the liver by inducing the enzymic activity of glycogen synthase b in the presence of glucose 6-phosphate in vivo and by promoting the activation of glycogen synthase.     

Action of degradative enzymes on the light fraction of bovine septa protein polysaccharide

1. The administration of cortisol and of other glucocorticoid steroids to starved mice produced an increase in liver glycogen content, an elevation of glycogen-synthetase activity and a predominantly particulate localization of both phosphorylase and glycogen-synthetase enzymes. 2. Three daily doses of actinomycin D caused a marked glycogen depletion, a significant decrease in glycogen-synthetase activity, the solubilization of phosphorylase and glycogen synthetase and the following effects on the activities of various other enzymes: a decrease in UDP-glucose pyrophosphorylase and phosphoglucomutase, an increase in glucose 6-phosphate dehydrogenase and no change in glucose 6-phosphatase, 6-phosphogluconate dehydrogenase, pyruvate kinase and UDP-glucose dehydrogenase. 3. Glucose ingestion, but not cortisol administration, reversed the effects of actinomycin D on liver glycogen content and on the activities of phosphorylase and glycogen synthetase.     

Lithium's effects on rat liver glucose metabolism in vivo

Oral administration of lithium carbonate to fed-healthy rats strongly decreased liver glycogen content, despite the simultaneous activation of glycogen synthase and the inactivation of glycogen phosphorylase. The effect seemed to be related to a decrease in glucose 6-phosphate concentration and to a decrease in glucokinase activity. Moreover, in these animals lithium markedly decreased liver fructose 2,6-bisphosphate, which could be a consequence of the fall in glucose 6-phosphate and of the inactivation of 6-phosphofructo-2-kinase. Liver pyruvate kinase activity and blood insulin also decreased after lithium administration. Lower doses of lithium carbonate had less intense effects. Lithium administration to starved-healthy and fed-streptozotocin-diabetic rats caused a slight increase in blood insulin, which was simultaneous with increases in liver glycogen, glucose 6-phosphate, and fructose 2, 6-phosphate. Glucokinase, 6-phosphofructo-2-kinase, and pyruvate kinase activities also increased after lithium administration in starved-healthy and fed-diabetic rats. Lithium treatment activated glycogen synthase and inactivated glycogen phosphorylase in a manner similar to that observed in fed-healthy rats. Glycemia was not modified in any group of animals. These results indicate that lithium acts on liver glycogen metabolism in vivo in at least two different ways: one related to changes in insulinemia, and the other related to the direct action of lithium on the activity of some key enzymes of liver glucose metabolism.     

THE INCORPORATION OF GLUCOSE INTO BRAIN GLYCOGEN AND THE ACTIVITIES OF CEREBRAL GLYCOGEN PHOSPHORYLASE AND SYNTHETASE: SOME EFFECTS OF AMPHETAMINE

Abstract— The effects of amphetamine sulphate (5 mg/kg intraperitoneally) on the incorporation of radioactive carbon from [U-¹⁴C]glucose into the glycogen of mouse cerebral cortex, midbrain and hind-brain have been investigated. In all brain regions studied amphetamine induced a rapid decrease in glycogen followed by a slower return to control values. No significant alterations were observed in the steady state concentration of cerebral glucose. The initial fall in glycogen was associated with a fall in its specific radioactivity relative to that of cerebral glucose, whereas the resynthesis of the polysaccharide was associated with a marked increase in the relative specific radioactivity of glycogen. Other experiments demonstrated that amphetamine initially stimulates the breakdown of prelabelled glycogen and that the resulting molecule has fewer 1,4 linked glucose side chains.
Studies of the relative forms of the enzymes glycogen phosphorylase and glycogen synthetase suggested that rapid post mortem changes were less likely to occur if cerebral tissue was fixed by means of a freeze-blowing technique. Amphetamine administration resulted in a rapid though transient elevation of phosphorylase a activity in mouse forebrain. The level of glycogen synthetase I activity was unchanged initially but was markedly elevated during the period when there was a large increase in the rate of incorporation of glucose into glycogen. It is suggested that cerebral glycogen metabolism is controlled, at least in part, by the interconversion of the 'active' and 'inactive' forms of glycogen phosphorylase and synthetase.     

Cyclic AMP-mediated activation of hepatic glycogenolysis by fructose.

Isolated livers from fed and fasted rats were perfused for 30 min with recirculating blood-buffer medium containing no added substrate and then switched to a flow-through perfusion using the same medium for an additional 5, 10 and 30 min. Continuous infusion of fructose for the final 5, 10 or 30 min resulted in activation of glycogen phosphorylase, an increase in the activity of protein kinase, elevated levels of tissue adenosine 3', 5'-monophosphate (cyclic AMP), and no consistent effect on glycogen synthase. Infusion of glucose under the same conditions resulted in activation of glycogen synthase, inactivation of glycogen phosphorylase, no change in protein kinase, and no consistent change in tissue cyclic AMP. These results demonstrate that while glucose promotes hepatic glycogen synthesis, fructose promotes activation of the enzymatic cascade responsible for glycogen breakdown.     

Cyclic AMP-mediated activation of hepatic glycogenolysis by fructose

Isolated livers from fed and fasted rats were perfused for 30 min with recirculating blood-buffer medium containing no added substrate and then switched to a flow-through perfusion using the same medium for an additional 5, 10 and 30 min. Continous infusion of fructose for the final 5, 10 or 30 min resulted in activation of glycogen phosphorylase, an increase in the activity of protein kinase, elevated levels of tissue adenosine 3′,5′-monphosphate (cylic AMP), and no consistent effect on glycogen synthase. Infusion of glucose under the same conditions resulted in activation of glycogen synthase, inactivation of glycogen phosphorylase, no change in protein kinase, and no consistent change in tissue cyclic AMP. These results demonstrate that while glucose promotes hepatic glycogen synthesis, fructose promotes activation of the enzymatic cascade responsible for glycogen breakdown.     

Reciprocal regulation of glycogen phosphorylase and glycogen synthase by insulin involving phosphatidylinositol-3 kinase and protein phosphatase-1 in HepG2 cells

The effect of insulin on glycogen synthesis and key enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase, was studied in HepG2 cells. Insulin stimulated glycogen synthesis 1.83-3.30 fold depending on insulin concentration in the medium. Insulin caused a maximum of 65% decrease in glycogen phosphorylase 'a' and 110% increase in glycogen synthase activities in 5 min. Although significant changes in enzyme activities were observed with as low as 0.5 nM insulin level, the maximum effects were observed with 100 nM insulin. There was a significant inverse correlation between activities of glycogen phosphorylase 'a' and glycogen synthase 'a' (R² = 0.66, p < 0.001). Addition of 30 mM glucose caused a decrease in phosphorylase 'a' activity in the absence of insulin and this effect was additive with insulin up to 10 nM concentration. The inactivation of phosphorylase 'a' by insulin was prevented by wortmannin and rapamycin but not by PD98059. The activation of glycogen synthase by insulin was prevented by wortmannin but not by PD98059 or rapamycin. In fact, PD98059 slightly stimulated glycogen synthase activation by insulin. Under these experimental conditions, insulin decreased glycogen synthase kinase-3 activity by 30-50% and activated more than 4-fold particulate protein phosphatase-1 activity and 1.9-fold protein kinase B activity; changes in all of these enzyme activities were abolished by wortmannin. The inactivation of GSK-3 and activation of PKB by insulin were associated with their phosphorylation and this was also reversed by wortmannin. The addition of protein phosphatase-1 inhibitors, okadaic acid and calyculin A, completely abolished the effects of insulin on both enzymes. These data suggest that stimulation of glycogen synthase by insulin in HepG2 cells is mediated through the PI-3 kinase pathway by activating PKB and PP-1G and inactivating GSK-3. On the other hand, inactivation of phosphorylase by insulin is mediated through the PI-3 kinase pathway involving a rapamycin-sensitive p70^s6k and PP-1G. These experiments demonstrate that insulin regulates glycogen phosphorylase and glycogen synthase through (i) a common signaling pathway at least up to PI-3 kinase and bifurcates downstream and (ii) that PP-1 activity is essential for the effect of insulin.     

Hepatic glycogen synthesis is highly sensitive to phosphorylase activity: evidence from metabolic control analysis

We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mm glucose and by 2-fold at 20 mm glucose with a decrease in the concentration of glucose causing half-maximal rate (S(0.5)) from 26 to 19 mm. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states.     

Glucose 6-phosphate plays a central role in the regulation of glycogen synthesis in a glycogen-storing liver cell line

Two substrains of the epithelial liver cell line C1I, one storing large amounts of glycogen, the other one being very poor in glycogen were used as a model for studying glycogen synthesis. The glycogen content of glycogen-rich cells doubled during the proliferative phase and remained high in plateau phase although glycogen synthase I activity was not significantly altered during growth cycle and was too low to account for the increase in glycogen. However, the activity of the glucose 6-phosphate (Glc6-P)-dependent synthase rose continuously during growth cycle, and intracellular Glc6-P-concentration increased about 10-fold in log phase cells to 0.72 mumol g-1 wet weight. A0.5 of synthase for Glc6-P was 0.79 mM. It was also found that in contrast to the enzyme from normal liver, glycogen phosphorylase a from C1I cells was inhibited by Glc6-P, the apparent Ki being 0.45 mM. It was concluded that glycogen accumulation in C1I cells was due to stimulation of synthase and inhibition of phosphorylase by Glc6-P. Findings from the glycogen-poor cell line which revealed similar specific activities of synthase and phosphorylase but only low Glc6-P (0.056 mumol g-1 wet weight) supported this conclusion. Addition of glucose to starved cells resulted in a transient activation of synthase in both cell lines. Net glycogen synthesis, was, however, only observed in the cells with a high Glc6-P-content. Thus, modulation of synthase and phosphorylase by Glc6-P and not activation/inactivation of the enzymes seems to play a predominant role in glycogen accumulation in this cell line.     

Glycogen and glycogen enzymes in the liver and striated muscle of rats under altered thyroid states

Changes induced in liver and striated muscle glycogen and glycogen enzymes (glycogen synthetase, glycogen phosphorylase and alpha-amylase) by hypothyroidism and hyperthyroidism in rats have been determined. There were no changes in liver glycogen synthetase, phosphorylase and amylase activities in the hypothyroid group. Hyperthyroid rats showed lower liver glycogen synthetase, phosphorylase a and amylase activities. In muscle, hypothyroid rats had lower phosphorylase activity. In the hyperthyroid group glycogen synthetase was increased.--The results presented do not completely agree with the glycogen levels found in both tissues studied, and they are obviously more related to other factors such as glucose availability. It can be concluded that under the conditions studied, the glycogen enzyme levels could not alone explain the variations of glycogen levels.     

Stimulating role of potassium ions and ouabain on glycogen synthesis in adipose tissue.

1. Exposure of fat-pads to increasing concentrations of K+ in the presence of insulin stimulates the incorporation of labelled glucose into glycogen. In the absence of hormone, only a slight incorporation of glucose into glycogen and slight glucose oxidation were detectable. 2. Ouabain alone, up to 100 microM, had no effect on synthesis of glycogen. Ouabain reinforced the effect of insulin on the conversion of glucose into glycogen in a Na+ medium and in a equimolar Na+-K+ medium, but not in a K+ medium. In addition, ouabain modified the optimal K+/Na+ ratio for glycogen synthesis. 3. The proportion of glycogen synthase in the active form was increased in a K+ medium, and a faster rate of conversion of synthase b into a was observed under these conditions. No difference was detected in the rate of inactivation of phosphorylase in a K+ or a Na+ medium. 4. Even though these results, taken together, are consistent with the proposed role of phosphorylase a in the regulation of synthase activation, the molecular mechanism of action of K+ in adipose tissue in increasing synthesis of glycogen cannot be explained simply by a faster inactivation of phosphorylase a. It is concluded that some undetermined effector(s) or signal could itself be a primary determinant for the greater activation of synthase observed in a K+ medium.     

Effects of adrenalectomy on hormone action on hepatic glucose metabolism. Impaired glucagon activation of glycogen phosphorylase in hepatocytes from adrenalectomized rats.

The effects of adrenalectomy on glucagon activation of liver glycogen phosphorylase and glycogenolysis were studied in isolated hepatocytes. Adrenalectomy resulted in reduced responsiveness of glycogenolysis and phosphorylase to glucagon activation. Stimulation of cAMP accumulation and cAMP-dependent protein kinase activity by glucagon was unaltered in cells from adrenalectomized rats. Adrenalectomy did not alter the proportion of type I and type II protein kinase isozymes in liver, whereas this was changed by fasting. Activation of phosphorylase kinase by glucagon was reduced in hepatocytes from adrenalectomized rats, although the half-maximal effective concentration of glucagon was unchanged. No difference in phosphorylase phosphatase activity between liver cells from control and adrenalectomized rats was detected. Glucagon-activated phosphorylase declined rapidly in hepatocytes from adrenalectomized rats, whereas the time course of cAMP increase in response to glucagon was normal. Addition of glucose (15 mM) rapidly inactivated glucagon-stimulated phosphorylase in both adrenalectomized and control rat hepatocytes. The inactivation by glucose was reversed by increasing glucagon concentration in cells from control rats, but was accelerated in cells from adrenalectomized rats. It is concluded that impaired activation of phosphorylase kinase contributes to the reduced glucagon stimulation of hepatic glycogenolysis in adrenalectomized rats. The possible role of changes in phosphorylase phosphatase is discussed.