Hormonal regulation of glycogen synthase: Insulin decreases protein kinase sensitivity to cyclic AMP

Rat hemidiaphragms incubated with epinephrine exhibited increases in cyclic AMP content and protein kinase activity which were proportional to the logarithm of the hormone concentration from 0.1–2 μM. The fraction of glycogen synthase made independent of glucose-6-P for activity (%I) decreased concomitantly, but correlated only with epinephrine concentrations up to 0.2 μM. Insulin (0–100 mU/ml) increased glycogen synthase %I in a dose-dependent manner with no change in cyclic AMP concentration. Protein kinase activity increased slightly at the lowest insulin concentration, then decreased slightly as glycogen synthase %I increased. Insulin was without effect when administered with a supramaximal dose of epinephrine. In the presence of submaximal epinephrine, insulin produced a dose-dependent increase in glycogen synthase %I which correlated with a decrease in protein kinase activity, without changing cyclic AMP. Insulin had no effect on the increases in cyclic AMP produced by varying levels of epinephrine. However, the activation of protein kinase activity by endogenous cyclic AMP was inhibited in the presence of insulin. The glycogen synthase %I response to epinephrine also was less sensitive in the presence of insulin. Insulin antagonizes the activation of cyclic AMP-dependent protein kinase by epinephrine without altering cyclic AMP levels.     

Activation of protein kinase and glycogen phosphorylase in isolated rat liver cells by glucagon and catecholamines.

In liver cells isolated from fed female rats, glucagon (290nM) increased adenosine 3':5'-monophosphate (cyclic AMP) content and decreased cyclic AMP binding 30 s after addition of hormones. Both returned to control values after 10 min. Glucagon also stimulated cyclic AMP-independent protein kinase activity at 30 s and decreased protein kinase activity assayed in the presence of 2 muM cyclic AMP at 1 min. Glucagon increased the levels of glycogen phosphorylase a, but there was no change in total glycogen phosphorylase activity. Glucagon increased glycogen phosphorylase a at concentrations considerably less than those required to affect cyclic AMP and protein kinase. The phosphodiesterase inhibitor, 1-methyl-3-isobutyl xanthine, potentiated the action of glucagon on all variables, but did not increase the maximuM activation of glycogen phosphorylase. Epinephrine (1muM) decreased cyclic AMP binding and increased glycogen phosphorylase a after a 1-min incubation with cells. Although 0.1 muM epinephrine stimulated phosphorylase a, a concentration of 10 muM was required to increase protein kinase activity. 1-Methyl-3-isobutyl xanthine (0.1 mM) potentiated the action of epinephrine on cyclic AMP and protein kinase. (-)-Propranolol (10muM) completely abolished the changes in cyclic AMP binding and protein kinase due to epinephrine (1muM) in the presence of 0.1mM 1-methyl-3-isobutyl xanthine, yet inhibited the increase in phosphorylase a by only 14 per cent. Phenylephrine (0.1muM) increased glycogen phosphorylase a, although concentrations as great as 10 muM failed to affect cyclic AMP binding or protein kinase in the absence of phosphodiesterase inhibitor. Isoproterenol (0.1muM) stimulated phosphorylase and decreased cyclic AMP binding, but only a concentration of 10muM increased protein kinase. 1-Methyl-3-isobutyl xanthine potentiated the action of isoproterenol on cyclic AMP binding and protein kinase, and propranolol reduced the augmentation of glucose release and glycogen phosphorylase activity due to isoproterenol. These data indicate that both alpha- and beta-adrenergic agents are capable of stimulating glycogenolysis and glycogen phosphorylase a in isolated rat liver cells. Low concentrations of glucagon and beta-adrenergic agonists stimulate glycogen phosphorylase without any detectable increase in cyclic AMP or protein kinase activity. The effects of alpha-adrenergic agents appear to be completely independent of changes in cyclic AMP protein kinase activity.     

Activation of rat adipocyte glycogen synthase by insulins.

Incubation of fat cells with insulin increased glycogen synthase I activity without changing total synthase activity. This effect of insulin was dependent upon the particular lot of albumin present in the medium and was abolished by incubating cells with trypsin. Half-maximal activation of glycogen synthase was obtained with 8 microunits/ml of insulin, a concentration very similar to that which half-maximally stimulated 3-O-methylglucose uptake. The basal percentage of phosphorylase a activity was not detectably altered by insulin, although it was decreased by incubating cells with 5 mM glucose. Insulin (50 microunits/ml) markedly opposed actions of epinephrine (0.05 to 10 muM) to increase phosphorylase a activity and decrease glycogen synthase I activity, effects which were observed without glucose. Partial activation of glycogen synthase by insulin was seen after 1 min and complete activation after 4 min. Glucose alone produced a transient increase in synthase I activity. When cells were incubated with insulin plus glucose for 4 min, the increase in the percent synthase I activity was much greater than the additive effects of insulin and glucose alone. This potentiation of the effect of insulin on glucogen synthase I activity depended on the time of incubation with glucose and on the concentration of the hexose. If cells were incubated with cytochalasin B before insulin plus glucose, the effect of glucose was abolished. These results suggest that there are at least two mechanisms by which insulin can increase fat cell glycogen synthase I activity. One requires glucose and activation occurs secondary to an increase in glucose transport; where another mechanism(s) is operative even in the absence of glucose.     

The control of glycogen metabolism in yeast. 1. Interconversion in vivo of glycogen synthase and glycogen phosphorylase induced by glucose, a nitrogen source or uncouplers

The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6-monophosphate and of fructose 2,6-bisphosphate. The activity of glycogen synthase increased about 4-fold and that of glycogen phosphorylase decreased 3-5-fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6-phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26 degrees C, but were much more pronounced at the nonpermissive temperature of 35 degrees C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate-cyclase-deficient mutant (cyr1), able to incorporate exogenous cyclic AMP. When a nitrogen source or uncouplers were added to the incubation medium after glucose, they had effects on glycogen metabolism and on the activity of glycogen synthase and glycogen phosphorylase which were directly opposite to those of glucose. By contrast, like glucose, these agents also caused, under most experimental conditions, a detectable rise in cyclic AMP concentration and a series of cyclic-AMP-dependent effects such as an activation of phosphofructokinase 2 and of trehalase and an increase in the concentration of fructose 2,6-bisphosphate and in the rate of glycolysis. Under all experimental conditions, the rate of glycolysis was proportional to the concentration of fructose 2,6-bisphosphate. Uncouplers, but not a nitrogen source, also induced an activation of glycogen phosphorylase and an inactivation of glycogen synthase when added to the cdc35 mutant incubated at the restrictive temperature of 35 degrees C without affecting cyclic AMP concentration.     

Glucose activation of liver glycogen synthase. Insulin-mediated restoration of glucose effect in diabetic rate is blocked by protein synthesis inhibitor

The loss of glucose regulation of glycogen synthase in perfused livers from diabetic rats was associated with a substantial reduction in synthase phosphatase activity. Treatment of diabetic rats with insulin alone resulted in total restoration of the glucose effect and synthase phosphatase activity, while simultaneous treatment with cycloheximide severely reduced the hormonal effect. Although treatment of normal rats with cycloheximide had no effect on glucose activation of synthase, it did result in severe depletion of liver glycogen increased liver glycogen phosphorylase activity, and elevation of liver adenosine 3′,5′-monosphosphate (cyclic AMP), but without elevation of liver protein kinase activity. Simultaneous treatment of alloxan-diabetic rats with insulin and cycloheximide resulted in reduction of total liver glycogen, increased phosphorylase activity, a reduction in the ability of insulin to lower hepatic cyclic AMP, and a further reduction of protein kinase activity.In summary, the effect of insulin treatment of diabetic rats to restore glucose regulation of hepatic glycogen synthase probably involves synthesis of new protein, and the data remain consistent with the hypothesis that the defect may be due to a diabetes-related deficiency in a specific synthase phosphatase and/or alteration of the synthase molecule itself.     

Glucose activation of liver glycogen synthase. Insulin-mediated restoration of glucose effect in diabetic rats is blocked by protein synthesis inhibitor.

The loss of glucose regulation of glycogen synthase in perfused livers from diabetic rats was associated with a substantial reduction in synthase phosphatase activity. Treatment of diabetic rats with insulin alone resulted in total restoration of the glucose effect and synthase phosphatase activity, while simultaneous treatment with cycloheximide severely reduced the hormonal effect. Although treatment of normal rats with cycloheximide had no effect on glucose activation of synthase, it did result in severe depletion of liver glycogen, increased liver glycogen phosphorylase activity, and elevation of liver adenosine 3',5'-monophosphate (cyclic AMP), but without elevation of liver protein kinase activity. Simultaneous treatment of alloxan-diabetic rats with insulin and cycloheximide resulted in reduction of total liver glycogen, increased phosphorylase activity, a reduction in the ability of insulin to lower hepatic cyclic AMP, and a further reduction of protein kinase activity. In summary, the effect of insulin treatment of diabetic rats to restore glucose regulation of hepatic glycogen synthase probably involves synthesis of new protein, and the data remain consistent with the hypothesis that the defect may be due to a diabetes-related deficiency in a specific synthase phosphatase and/or alteration of the synthase molecule itself.     

Role of AMP on the activation of glycogen synthase and phosphorylase by adenosine, fructose, and glutamine in rat hepatocytes

The mechanism for glycogen synthesis stimulation produced by adenosine, fructose, and glutamine has been investigated. We have analyzed the relationship between adenine nucleotides and glycogen metabolism rate-limiting enzymes upon hepatocyte incubation with these three compounds. In isolated hepatocytes, inhibition of AMP deaminase with erythro-9-(2-hydroxyl-3nonyl)adenine further increases the accumulation of AMP and the activation of glycogen synthase and phosphorylase by fructose. This ketose does not increase cyclic AMP or the activity of cyclic AMP-dependent protein kinase. Adenosine raises AMP and ATP concentration. This nucleotide also activates glycogen synthase and phosphorylase by covalent modification. The correlation coefficient between AMP and glycogen synthase activity is 0.974. Nitrobenzylthioinosine, a transport inhibitor of adenosine, blocks (by 50%) the effect of the nucleoside on AMP formation and glycogen synthase but not on phosphorylase. 2-Chloroadenosine and N6-phenylisopropyladenosine, nonmetabolizable analogues of adenosine, activate phosphorylase (6-fold) without increasing the concentration of adenine nucleotides or the activity of glycogen synthase. Cyclic AMP is not increased by adenosine in hepatocytes from starved rats but is in cells from fed animals. [Ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA) blocks by 60% the activation of phosphorylase by adenosine but not that of glycogen synthase. Glutamine also increases AMP concentration and glycogen synthase and phosphorylase activities, and these effects are blocked by 6-mercaptopurine, a purine synthesis inhibitor. Neither adenosine nor glutamine increases glucose 6-phosphate. It is proposed that the observed efficient glycogen synthesis from fructose, adenosine, and glutamine is due to the generation of AMP that activates glycogen synthase probably through increases in synthase phosphatase activity. It is also concluded that the activation of phosphorylase by the above-mentioned compounds can be triggered by metabolic changes.     

Interaction between alpha and beta adrenergic receptors and cholinergic receptors in isolated perfused rat heart: effects on cAMP-protein kinase and phosphorylase

The ability of acetylcholine to antagonize catecholamine-induced activation of myocardial cyclic AMP dependent protein kinase and glycogen phosphorylase activity was assessed using isolated perfused rat hearts. Perfused hearts were treated with either saline, epinephrine, epinephrine plus phentolamine or isoproterenol. After 1 minute of infusion of the indicated drug a second infusion containing acetylcholine was started. After an additional minute hearts were frozen and analyzed for cyclic nucleotide content and enzyme activity. In the presence of the alpha receptor blocking agent, phentolamine, epinephrine is a more effective activator of protein kinase than in its absence. Under these conditions the antagonistic action of acetylcholine on protein kinase activation is more pronounced. In the presence of epinephrine plus phentolamine or in the presence of isoproterenol the antagonistic action of acetylcholine on phosphorylase activity can be accounted for by a reduction in cyclic AMP-protein kinase. This same action of acetylcholine on epinephrine-stimulated phosphorylase in the aabsence of phentolamine, however, cannot be totally accounted for by a reduction in cyclic AMP content or in protein kinase activity.     

The concentrations of glucose 1,6-bisphosphate and other regulatory metabolites, and the activities of enzymes of the glycogen metabolism in the perfused rabbit psoas muscle

The following parameters were determined in the rabbit psoas muscle after perfusion in the presence of either insulin, propranolol, or isoproterenol: Concentrations of cyclic AMP, glucose 1,6-bisphosphate, fructose 2,6-bisphosphate, glucose-1-phosphate, glucose 6-phosphate, and fructose-1,6-bisphosphate. Maximum and "regulatory" activities of the enzymes glycogen phosphorylase, glycogen synthase, phosphofructokinase, and histone-phosphorylating protein kinase.     

Uterine cyclic AMP formation: biphasic effect of estrogen on catecholamine sensitivity.

Female, ovariectomized rats were treated with estradiol and then, after various time periods, given an intravenous injection of isoproterenol or epinephrine. 30 seconds later uteri were frozen $\text{in}$$\text{situ}$ and assayed for cyclic AMP and glycogen phosphorylase. The cyclic AMP response to catecholamines was significantly depressed as early as 30 minutes after estrogen and at 6, 12 and 24 hours was 50% of that in non-estrogen-treated controls. Catecholamine-induced glycogen phosphorylase activation was unchanged until 24 hours after estrogen when it was significantly increased over controls. At 48 hours of estrogen both the cyclic AMP and phosphorylase responses to catecholamines were greater than controls. Estrogen regulates uterine β-adrenergic sensitivity but the time courses of estrogen effects on the cyclic AMP and glycogen phosphorylase response changes are different. Catecholamine-induced uterine cyclic AMP formation is biphasic: suppression during the first 24 hours of estrogen followed by recovery and finally augmentation by 48 hours. Catecholamine-induced glycogen phosphorylase activation shows only augmentation after 24–48 hours of estrogen. It is concluded that estrogen has independent effects on the β-adrenergic-glycogen phosphorylase activation pathway at two different points; one prior to cyclic AMP formation and another after cyclic AMP formation.     

Effect of acetylcholine on glycogen phosphorylase activity and cyclic nucleotide content in isolated perfused rat hearts.

Acetylcholine (1muM) increased cyclid GMP content in paced perfused rat hearts within 15 sec., with peak content occurring at 1 min. No effect of acetylcholine on cyclic AMP content, phosphorylase activity or glycogen synthase was observed. Epinephrine (1muM) infusion increased both cyclic AMP content and phosphorylase, but did not alter cyclic GMP content or glycogen synthase activity. When acetylcholine was infused during the second min. of a 2 min. infusion of epinephrine, the cholinergic agent increased cyclic GMP and reduced the stimulated phosphorylase activity and elevated cyclic AMP.     

Attenuation of epinephrine-induced increase in liver cyclic AMP by endogeneous insulin in vivo.

1. Epinephrine-induced increase in rat liver cyclic AMP in vivo was potentiated when the circulating insulin was suppressed by injection of anti-insulin serum or by induction of diabetes. Consequently, phosphorylase was activated, glycogen synthetase was inactivated and glycogen accumulation induced by glucose load was prevented by epinephrine in the insulin-deficient rats to a much larger extent than in normal rats. 2. Insulin lack was effective in potentiating epinephrine-induced increase in liver and muscule cyclic AMP even after the treatment of rats with theophylline; the potentiation could not be solely accounted for by the inhibition of cyclic AMP phosphodiesterase. Thus, it is likely that insulin lack enhaces epinephrine activation of adenylate cyclase. 3. Unlike epinephrine, glucagon increased liver cyclic AMP to essentially the same extent whether the rat was treated with anti-insulin serum or not. 4. Based on the difference in dose-response curves between normal and insulin-deficient rats, a possibility is discussed that there are two adenylate cylase in the liver with higher and lower affinities for epinephrine and that circulating insulin blocks the high affinity enzyme selectively.     

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 glycogen metabolism in astrocytoma and neuroblastoma cells in culture.

The regulation of glycogen metabolism in C-6 astrocytoma and C-1300 neuroblastoma cells in culture has been investigated. Two modes of control of glycogen metabolism appear to be operative. The regulation of intracellular glycogen concentrations and the predominant forms of glycogen phosphorylase and glycogen synthase vary with (a) the available energy supply, and (b) altered intracellular concentration of cyclic adenosine 3':5'-monophosphate (cyclic AMP). Both cell lines respond to glucose in the medium; when glucose levels are high, glycogen is synthesized, glycogen phosphorylase a decreases, and glycogen synthase a increases. When glucose in the medium decreases to a critical level, the phosphorylase a increases and glycogen concentrations in the cells decrease in aprallel with the medium glucose. The critical glucose concentration is 2.5 mM for the astrocytoma cells and 4 mM for the neuroblastoma cells. Insulin promotes the conversion of phosphorylase to the b form and synthase to the a form in both cell lines. All of these changes occur without alteration in the intracellular cyclic AMP concentrations. When cyclic AMP concentrations are increased in either cell line, phosphorylase a is increased, synthase a is decreased, and glycogen concentrations decrease. Isobutyl methylxanthine is effective in promoting glycogenolysis in both cell lines. Norepinephrine is effective with the astrocytoma cells, and prostaglandin E1 is effective with the neuroblastoma cells.     

Hormonal stimulation of cyclic AMP accumulation and glycogen phosphorylase activity in calcium-depleted hepatocytes from euthyroid and hypothyroid rats.

Activation of glycogen phosphorylase by hormones was examined in hepatocytes isolated from euthyroid and hypothyroid female rats and incubated by Ca2+-free buffer containing 1 mM-EGTA. Basal glycogen phosphorylase activity was decreased in Ca2+-free buffer. However, the activation of hepatocyte glycogen phosphorylase, in the absence of extracellular Ca2+, in response to adrenaline, glucagon or phenylephrine was slightly lower, whereas that by vasopressin was abolished. The activation of glycogen phosphorylase by phenylephrine, adrenaline or isoproterenol (isoprenaline) in hepatocytes from euthyroid rats incubated in the absence of Ca2+ was not accompanied by any detectable increase in total cyclic AMP. The log-dose/response curves for activation of phosphorylase by phenylephrine or low concentrations of adrenaline were the same in hepatocytes from hypothyroid as compared wit euthyroid rats, whereas the response to isoproterenol was greater in hepatocytes from hypothyroid rats. However, the increases in total cyclic AMP accumulation caused by adrenaline or isoproterenol were greater in hepatocytes from hypothyroid rats than in hepatocytes from euthyroid rats. The increases in cyclic AMP accumulation caused by adrenaline or isoproterenol in Ca2+-depleted hepatocytes from hypothyroid rats were blocked by propranolol, a beta-adrenergic antagonist. In contrast, propranolol was only partially effective asan inhibitor of the activation of glycogen phosphorylase by phenylephrine or adrenaline in hepatocytes from hypothyroid rats and ineffective on phosphorylase activation in cells from euthyroid rats. These data indicate that the alpha-adrenergic activation of glycogen phosphorylase is not affected by the absence of extracellular Ca2+, and the extent to which total cyclic AMP was increased by adrenergic amines did not correlate with glycogen phosphorylase activation.     

‘Insulin-like’ effects of lithium ion on isolated rat adipocytes II. Specific activation of glycogen synthase

Lithium ion, like insulin, activated adipocyte glycogen synthase with or without glucose in the medium. However, the effect of lithium ion was much greater than that of insulin under both conditions. The lithium-activated glycogen synthase was stable to both Sephadex chromatography and ethanol precipitation of the enzyme, indicating that the effect of lithium ion on glycogen synthase was through covalent modification of the enzyme. Glycogen synthase was significantly activated by lithium ion under conditions where concentrations of cellular ATP were unaffected. The effect of lithium ion on glycogen synthase was rapid and observed at concentrations as low as 1 to 3 mM, reaching a maximum at the concentration of 40 mM. It was thus the most sensitive of all the effects studied (see previous paper). Insulin further stimulated glycogen synthase at low concentrations but not at maximal concentration of lithium ion. Lithium-activated glycogen synthase was inhibited by both epinephrine and dibutyryl cyclic AMP, but was not affected by the removal of extracellular Ca++. Interestingly, lithium ion had no detectable effect on basal pyruvate dehydrogenase as well as on epinephrine-stimulated phosphorylase. The failure of lithium ion to thus mimic insulin actions on pyruvate dehydrogenase and on phosphorylase suggests that the action of lithium ion on glycogen synthase is quite specific and may be mediated by stimulating a phosphatase or by inhibiting a protein kinase acting specifically on glycogen synthase.     

Amylin activates glycogen phosphorylase and inactivates glycogen synthase via a cAMP-independent mechanism.

Although the novel pancreatic peptide amylin has been shown to induce insulin resistance and decrease glucose uptake, the mechanism of amylin's actions is unknown. The following study evaluated the effect of amylin on glycogen metabolism in isolated soleus muscles in the presence and absence of insulin (200 microU/ml). Total glycogen, glycogen phosphorylase and glycogen synthases activities, and cAMP levels were measured. Total glycogen levels were significantly decreased by amylin (100 nM) in fed or fasted muscles under conditions of insulin stimulation. Amylin (100 nM) activated glycogen phosphorylase by as much as 100% and decreased glycogen synthase activity by over 60%, depending on the metabolic state of the muscles. These effects where comparable to those of the beta adrenergic agonist isoproterenol. A lower concentration of amylin (1 nM) did not significantly affect glycogen levels, glycogen phosphorylase, or glycogen synthase activity. Cyclic AMP levels were increased two-fold by isoproterenol but were unaffected by amylin. In conclusion, amylin induces glycogenolysis by decreasing glycogen synthesis and increasing breakdown. The effect of amylin on enzyme activity is consistent with a phosphorylation-dependent mechanism. It is likely that these events are mediated via a cAMP independent protein kinase.     

The effect of insulin on the glycogenolytic cascade and on the activity of glycogen synthase in the liver of anaesthetized rabbits.

1. The administration of insulin to anaesthetized rabbits caused the inactivation of liver phosphorylase and phosphorylase kinase, but did not change either the hepatic concentration of cyclic AMP or the activity of cyclic AMP-dependent histone kinase. All measured parameters were increased by the subsequent administration of glucagon. 2. Activation of glycogen synthase by insulin was only observed when phosphorylase had been strongly inactivated.     

Altered liver glycogen metabolism in fed genetically obese mice.

The cyclic AMP and glycogen concentrations and the activities of phosphorylase kinase, phosphorylase a and glycogen synthase a were not different in livers from lean or ob/ob mice despite increased plasma glucose and insulin in the obese group. The liver water content was decreased by 10% in the obese mice. In hepatocytes isolated from lean mice and incubated with increasing glucose concentrations (14-112 mM), a sequential inactivation of phosphorylase and activation of glycogen synthase was observed. In hepatocytes from obese mice the inactivation of phosphorylase was not followed by an activation of synthase. The inactivation of phosphorylase occurred more rapidly and was followed by an activation of synthase in hepatocytes isolated from both groups of mice when in the incubation medium Na+ was replaced by K+ or when Ca2+ was omitted and 2.5 mM-EGTA included. The inactivation of phosphorylase and activation of synthase were not different in broken-liver-cell preparations from lean and obese animals. The re-activation of phosphorylase in liver filtrates in the presence of 0.1 microM-cyclic AMP and MgATP was inhibited by about 70% by EGTA and stimulated by Ca2+ and was always greater in preparations from ob/ob mice. The apparent paradox between the impairment of glycogen metabolism in isolated liver preparations and the situation in vivo in obese mice is discussed.     

Rat liver glycogen metabolism in the perinatal period

The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen sythase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximun on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose of AMP on the respective phosphatases was considered. It was found that the Km of phosphatase for glucose in the prenatal period was 5–7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6–15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time.Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver.It is suggested that the rapid changes observed immediately after birth are due to an effect of hypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.