Regulation of glycogen synthetase and phosphorylase phosphatase activities in rat adipose tissue.

Incubation of a rat adipose tissue homogenate causes a time and temperature dependent activation of glycogen synthetase (UDP glucose:glycogen 4-alpha-glucosyltransferase) and simultaneous inactivation of phosphorylase (1,4-alpha-D-glucan: orthophosphate alpha-glucosyltransferase, EC 2.4.1.1). Activation of glycogen synthetase at 15 and 23 degrees C was preceded by a lag period. The duration of the lag period could not be correlated with significant changes in phosphorylase activity. Addition of glucose and methylxanthines caused an increase in the rates of glycogen synthetase activation and phosphorylase inactivation. The effect on glycogen synthetase activation was mainly on the linear phase. Addition of AMP inhibited phosphorylase inactivation and accelerated glycogen synthetase activation. Addition of muscle phosphorylase alpha caused a prolongation of the lag period which lasted until phosphorylase alpha activity had decreased to the level originally present in the preparation. It is concluded that in adipose tissue activation of glycogen synthetase is not dependent on prior inactivation of phosphorylase and that other factors should be looked for to explain the lag period preceding glycogen synthetase activation.     

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.     

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.     

Stabilization of glycogen stores and stimulation of glycogen synthesis in hepatocytes by phenacyl imidazolium compounds

LY177507 is representative of a series of phenacyl imidazolium compounds that cause marked lowering of blood glucose levels in animal models of noninsulin-dependent diabetes mellitus. In studies conducted with isolated rat hepatocytes, LY177507 inhibited net glucose production from a variety of substrates, inhibited glycolysis from exogenous glucose and endogenous glycogen, inhibited glycogenolysis, and stimulated glycogenesis. These effects of LY177507 appear to be the consequence of activation of glycogen synthase and inactivation of glycogen phosphorylase. In vivo studies with normal fed rats demonstrated a decrease in blood glucose, an increase in hepatic glycogen stores, and an inactivation of glycogen phosphorylase. Phenacyl imidazolium compounds appear to lower blood glucose levels and affect hepatic carbohydrate metabolism by a mechanism unlike other known hypoglycemic compounds.     

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.     

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 glucose on phosphorylase and glycogen synthase in hepatocytes from diabetic rats.

The effects of glucose on phosphorylase and glycogen synthase were investigated in hepatocytes isolated from acutely (40 h) and chronically (90 h) alloxan-diabetic rats. The glucose-induced inactivation of phosphorylase proceeded normally in all conditions. The ensuing activation of glycogen synthase was slightly blunted in acute diabetes, but became virtually absent in 72 h diabetes of similar severity. In hepatocytes from rats with various degrees of chronic diabetes, the maximal activation of glycogen synthase (at 60 mM-glucose) was inversely correlated with the plasma glucose concentration.     

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.     

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.     

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.     

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.     

The activity of glycogen synthase phosphatase limits hepatic glycogen deposition in the adrenalectomized starved rat.

Hepatocytes from adrenalectomized 48 h-starved rats responded to increasing glucose concentrations with a progressively more complete inactivation of phosphorylase. Yet no activation of glycogen synthase occurred, even in a K+-rich medium. Protein phosphatase activities in crude liver preparations were assayed with purified substrates. Adrenalectomy plus starvation decreased synthase phosphatase activity by about 90%, but hardly affected phosphorylase phosphatase activity. Synthase b present in liver extracts from adrenalectomized starved rats was rapidly and completely converted into the a form on addition of liver extract from a normal fed rat. Glycogen synthesis can be slowly re-induced by administration of either glucose or cortisol to the deficient rats. In these conditions there was a close correspondence between the initial recovery of synthase phosphatase activity and the amount of synthase a present in the liver. The latter parameter was strictly correlated with the measured rate of glycogen synthesis in vivo. The decreased activity of synthase phosphatase emerges thus as the single factor that limits hepatic glycogen deposition in the adrenalectomized starved rat.     

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.     

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+.     

Activation of hepatocyte glycogen synthase by metabolic inhibitors

Incubation of isolated rat hepatocytes with metabolic inhibitors causes an increase in the -glucose 6-P/+glucose 6-P activity ratio of glycogen synthase after decreasing ATP and increasing AMP levels. Concomitantly, the activity of phosphorylase is increased six-fold by the same treatment. This activation of both enzymes remains after gel filtration of the hepatocyte extracts. Addition of metabolic inhibitors to cells pretreated with an inhibitor of AMP-deaminase results in an accumulation of AMP and, simultaneously, in a further increase in the activation state of glycogen synthase. The correlation coefficient between the intracellular concentration of AMP and glycogen synthase activity is r = 0.93. It is proposed that the covalent activation of glycogen synthase by metabolic inhibitors can be triggered by changes in the level of the intracellular concentrations of adenine nucleotides.     

Glucagon and glucose as major regulators of glycogen metabolism in primary cultured rat hepatocytes

The short-term controls of glycogen synthase [EC 2.4.1.11] and glycogen phosphorylase [EC 2.4.1.1] by major regulators, such as insulin, glucose, catecholamine, and glucagon, were compared in a simple, yet organized experimental system, i.e., adult rat hepatocytes in primary culture. Glycogen synthase was activated by glucose markedly and dose-dependently (5-40 mM), but insulin alone (1 X 10(-8) M) activated this enzyme only two-fold. Therefore, activation of the enzyme by the two regulators together was mostly due to activation by glucose. Glucagon at a concentration of 5 X 10(-10) M suppressed this activation almost completely. Glucagon at this concentration activated phosphorylase considerably and this activation was slightly inhibited by insulin. Phenylephrine also activated phosphorylase, and this activation was inhibited by phenoxybenzamine or prazosin, suggesting that activation by catecholamine is through the alpha 1-adrenergic receptor. Similarly a high concentration of glucose diminished the effects of glucagon and phenylephrine. These results suggest that in rat liver, glycogen metabolism is controlled mainly by glucagon, catecholamine, and glucose; the former two activate phosphorylase and inactivate synthase, while glucose activates synthase strongly and inactivates phosphorylase partially. Insulin plays a minor role in both reactions. Thus, the liver is primarily an organ for glucose production, which is regulated by hormones, not for glycogen storage, which is increased only by a high glucose concentration in the portal blood.     

Effects of hormones and serum on glycogen metabolism in adult rat liver parenchymal cell primary cultures.

Parenchymal cells from adult rat liver, isolated by a collagenase perfusion technique, have been maintained in primary culture and a detailed study on carbohydrate metabolism carried out over the initial 48-hour culture period. The glucose concentration of the medium exerts a major influence on glycogen accumulation by the cells. Insulin, particularly at high glucose concentrations, stimulates glycogen biosynthesis, whereas glucagon prevents glycogen accumulation. Dexamethasone was without effect on glycogen metabolism. Glucose appears to stimulate glycogen accumulation by activation of glycogen synthetase enzyme. However, there is a gradual loss of synthetase activity throughout the culture period. Similar decreases in activity were noted for pyruvate kinase, aldolase and hexokinase. Glucose, insulin and dexamethasone were unable to prevent these decreases in enzyme activity. Foetal bovine serum contains fructose and this hexose appears to be the factor in serum which is responsible for the activation of glycogen accumulation in the presence of physiological glucose concentrations. The lactic acid content of the serum may also stimulate glycogen accumulation. In general, there is a gradual loss of the pattern of carbohydrate metabolism typical of differentiated hepatocytes during the culture period.     

Synergistic inhibition of hepatic glycogenolysis in the presence of insulin and a cAMP antagonist

The effects of insulin on the ability of the specific intracellular cAMP-dependent protein kinase antagonist, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate, to inhibit glycogenolysis induced by the Sp diastereomer was studied in hepatocytes isolated from fed rats. Addition of the cAMP agonist, (Sp)-cAMPS, to hepatocytes resulted in a concentration-dependent increase in glycogenolytic glucose production concomitant with the cAMP-dependent activation of phosphorylase and inhibition of glycogen synthase. Activity curves were shifted to the right in the presence of the cAMP antagonist, (Rp)-cAMPS. Preincubation of the hepatocytes with a maximally effective concentration of insulin did not affect the concentration of (Sp)-cAMPS required for half-maximal activation of phosphorylase but did result in a 10-fold shift in the concentration of (Sp)-cAMPS required for half-maximal inactivation of glycogen synthase. Preincubation of hepatocytes with a combination of the cAMP antagonist, (Rp)-cAMPS, and insulin resulted in synergistic inhibition of (Sp)-cAMPS-induced phosphorylase activation, glycogen synthase inactivation, and glycogenolytic glucose production. Since neither phosphorothioate diastereomer was hydrolyzed significantly during the course of the experiments, the synergistic effects of insulin are postulated to be working through a mechanism subsequent to the phosphodiesterase activation step.     

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.