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+.     

Regulation of glycogen synthase activity in the isolated mouse soleus muscle effect of insulin,epinephrine, glucose and anti-insulin receptor antibodies

The effects of insulin, epinephrine, glucose and anti-insulin receptor antibodies on enzymes involved in the regulation of glycogen synthesis were investigared in the isolated mouse soleus muscle. Insulin maximally increased the percentage of glycogen synthase active form after 15 min in the absence of glucose in the extracellular medium; half-maximal and maximal effects were obtained with 1.5 and 33 nM insulin, respectively. The basal percentage of glycogen phosphorylase active form was not altered by insulin. Antibodies to the insulin receptor had similar effects to those of insulin on both enzymes. The percentage of glycogen synthase active form was maximally decreased and that of phosphorylase maximally increased after a 2 min exposure to epinephrine in the absence of extracellular glucose. Glucose alone had no effect on muscle glycogen synthase. When muscles were incubated with insulin (33 nM) plus glucose (20 mM) for 5–10 min, the increase in the percentage of glycogen synthase active form was greater than with insulin alone. This enhancing effect of glucose on insulin activation of glycogen synthase disappeared after 20 min. The results suggest the existence of two mechanisms whereby insulin activates muscle glycogen synthase. The main effect is operative in the absence of extracellular glucose and occurs at insulin concentrations close to the physiological range. The other effect requires glucose and may result from the stimulation by insulin of glucose transport and/or metabolism.     

Regulation of glycogen synthase activity in the isolated mouse soleus muscle. Effect of insulin, epinephrine, glucose and anti-insulin receptor antibodies

The effects of insulin, epinephrine, glucose and anti-insulin receptor antibodies on enzymes involved in the regulation of glycogen synthesis were investigated in the isolated mouse soleus muscle. Insulin maximally increased the percentage of glycogen synthase active form after 15 min in the absence of glucose in the extracellular medium; half-maximal and maximal effects were obtained with 1.5 and 33 mM insulin, respectively. The basal percentage of glycogen phosphorylase active form was not altered by insulin. Antibodies to the insulin receptor had similar effects to those of insulin on both enzymes. The percentage of glycogen synthase active form was maximally decreased and that of phosphorylase maximally increased after a 2 min exposure to epinephrine in the absence of extracellular glucose. Glucose alone had no effect on muscle glycogen synthase. When muscles were incubated with insulin (33 nM) plus glucose (20 mM) for 5-10 min, the increase in the percentage of glycogen synthase active form was greater than with insulin alone. This enhancing effect of glucose on insulin activation of glycogen synthase disappeared after 20 min. The results suggest the existence of two mechanisms whereby insulin activates muscle glycogen synthase. The main effect is operative in the absence of extracellular glucose and occurs at insulin concentrations close to the physiological range. The other effect requires glucose and may result from the stimulation by insulin of glucose transport and/or metabolism.     

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.     

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.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of insulin and glucose in cells from diabetic rats

Defects in the deposition of glycogen and the regulation of glycogen synthesis in the livers of severely insulin-deficient rats can be reversed, in vivo, within hours of insulin administration. Using primary cultures of hepatocytes isolated from normal and diabetic rats in a serum-free chemically defined medium, the present study addresses the chronic action of insulin to facilitate the direct effects of insulin and glucose on the short term regulation of the enzymes controlling glycogen metabolism. Primary cultures were maintained in the presence of insulin, triiodothyronine, and cortisol for 1-3 days. On day 1 in alloxan diabetic cultures, 10(-7) M insulin did not acutely activate glycogen synthase over a period of 15 min or 1 h, whereas insulin acutely activated synthase in cultures of normal hepatocytes. By day 3 in hepatocytes isolated from alloxan diabetic rats, insulin effected an approximate 30% increase in per cent synthase I within 15 min as was also the case for normal cells. The acute effect of insulin on synthase activation was independent of changes in phosphorylase alpha. Whereas glycogen synthase phosphatase activity could not be shown to be acutely affected by insulin, the total activity in diabetic cells was restored to normal control values over the 3-day culture period. The acute effect of 30 mM glucose to activate glycogen synthase in cultured hepatocytes from normal rats after 1 day of culture was missing in hepatocytes isolated from either alloxan or spontaneously diabetic (BB/W) rats. After 3 days in culture, glucose produced a 50% increase in glycogen synthase activity during a 10-min period under the same conditions. These studies clearly demonstrate that insulin acts in a chronic manner in concert with thyroid hormones and steroids to facilitate acute regulation of hepatic glycogen synthesis by both insulin and glucose.     

The effects of fasting and refeeding on liver glycogen synthase and phosphorylase in obese and lean mice.

The responses of hepatic glycogen synthase and phosphorylase to fasting and refeeding were assessed as part of an investigation into possible sites of insulin resistance in gold thioglucose (GTG) obese mice. The active forms glycogen synthase and phosphorylase (synthase I and phosphorylase a) and the total activity of these enzymes were estimated in lean and GTG mice over 48 h of food deprivation, and for 120 min after glucose gavage (1 g/kg wt). In lean mice there was a maximal reduction in hepatic glycogen content after 12 h of starvation and the activity of phosphorylase a decreased from 23.8 +/- 1.9 to 6.8 +/- 0.7 mumol/g protein/min. These changes were accompanied by an increase in the activity of synthase I (from 0.14 +/- 0.01 to 0.46 +/- 0.04 mumol/g protein/min). In obese mice, similar changes in enzyme activity occurred after 48 h of starvation. These changes were accompanied by a significant reduction in the hyperinsulinemia and hyperglycemia of the GTG mice. After glucose gavage in both lean and obese mice, the activity of synthase I further increased over the first 30 min and declined thereafter. The activity of phosphorylase a increased progressively after refeeding. Results from this study suggest that despite increased hepatic glycogen deposition, the responses of glycogen synthase and phosphorylase, in livers of obese mice, to fasting and refeeding are similar to those of control mice even in the presence of insulin resistance.     

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...     

Activation of glucose transport in muscle by prolonged exposure to insulin. Effects of glucose and insulin concentrations

Glucose transport activity was found to increase over 5 h in rat epitrochlearis muscle in response to a moderate concentration (50-100 microunits/ml) of insulin. This process was examined using 3-methylglucose. The increase in permeability to 3-methylglucose was 2- to 4-fold greater after 5 h than after 1 h in muscles incubated with 50 microunits/ml of insulin and 1 or 8 mM glucose. The increase in permeability to 3-methylglucose during the period between 1 and 5 h of exposure to 50 microunits/ml of insulin and 1 mM glucose was due to an increase in the apparent Vmax of sugar transport. There were two components to this activation of glucose transport. One, which was not influenced by inhibition of protein synthesis, resulted in activation of sugar transport to the same extent by 50 microunits/ml as by 20,000 microunits/ml of insulin; however, this activation took approximately 20 times longer with 50 microunits/ml insulin. The other, which was blocked by cycloheximide, resulted in a further activation of sugar transport to a level higher than that attained in response to 20,000 microunits/ml of insulin. Glucose had no effect on activation of sugar transport during the first hour, but a high concentration (20-36 mM) of glucose prevented the further activation of glucose transport during prolonged treatment with 50 microunits/ml of insulin. It appears from these results that prolonged exposure to a moderate concentration of insulin has previously unrecognized effects that include: a progressive activation of glucose transport over a long time that eventually results in as great a response as a "supramaximal" insulin concentration, and in the presence of low glucose concentration, further activation of glucose transport by an additional, protein synthesis-dependent mechanism. The results also show that a high concentration of glucose can, under some conditions, inhibit stimulation of its own transport.     

Control of glycogen synthase and phosphorylase by insulin in rat adipocytes which is unrelated to the concentration of cyclic AMP

Incubation of adipocytes in glucose-free medium with adrenocorticotrophic hormone, epinephrine, isoproterenol, or norepinephrine increased the concentration of cyclic AMP and the percentage of phosphorylase a activity, and decreased the percentage of glycogen synthase I activity. Glucose was essentially without effect on glycogen synthase or phosphorylase in either the presence or absence of epinephrine. Although glucose potentiated the action of insulin to activate glycogen synthase, the hexose did not enhance the effectiveness of insulin in the presence of epinephrine. Likewise, glucose did not increase the ability of insulin to oppose the activation of phosphorylase by epinephrine.The activation of glycogen synthase by insulin was not associated with a decrease in the concentration of cyclic AMP. Insulin partially blocked the rise in cyclic AMP due to isoproterenol, adrenocorticotrophic hormone, and norepinephrine. The maximum effects of isoproterenol on glycogen synthase and phosphorylase were observed when the concentration of cyclic AMP was increased twofold. However, insulin clearly opposed the changes in enzyme activity produced by isoproterenol (and also adrenocorticotrophic hormone, epinephrine and norepinephrine) even though concentrations of cyclic AMP were still increased three- to fourfold. Nicotinic acid opposed the increases in cyclic AMP due to adrenocorticotrophic hormone, isoproterenol and norepinephrine to the same extent as insulin; however, nicotinic acid was ineffective in opposing the activation of phosphorylase and inactivation of glycogen synthase produced by these agents. Thus, it is unlikely that the effects of insulin on glycogen synthase and phosphorylase result from an action of the hormone to decrease the concentration of cyclic AMP.     

Adaptations of glycogen metabolism in rat epididymal adipose tissue during fasting and refeeding.

It is well documented that adipose tissue glycogen content decreases during fasting and increases above control during refeeding. We now present evidence that these fluctuations result from adaptations intrinsic to adipose tissue glycogen metabolism that persist in vitro: in response to insulin (1 milliunit/ml), [3H]glucose incorporation into rat fat pad glycogen was reduced to 10% of control after a 3-day fast; incorporation increased 6-fold over fed control on the 4th day of refeeding following a 3-day fast. We have characterized this adaptation with regard to alterations in glycogen synthase and phosphorylase activity. In addition, we found that incubation of fat pads from fasted rats with insulin (1 milliunit/ml) increased glucose-6-P content, indicating that glucose transport was not the rate-limiting step for glucose incorporation into glycogen in the presence of insulin. In contrast, feeding a fat-free diet resulted in dramatic increases in glycogen content of fat pads without a concomitant increase in glucose incorporation into glycogen in response to insulin (1 milliunit/ml). Thus, fasting and refeeding appeared to alter insulin action on adipose tissue glycogen metabolism more than this dietary manipulation.     

NMR measurements of in vivo myocardial glycogen metabolism

Using 13C and 1H NMR we measured the rate of glycogen synthesis (0.23 +/- 0.10 mumol/min gram wet weight tissue (gww) in rat heart in vivo during an intravenous infusion of D-[1-13C]glucose and insulin. Glycogen was observed within 10 min of starting and increased linearly throughout a 50-min infusion. This compared closely with the average activity of glycogen synthase I (0.22 +/- 0.03 mumol/min gww) measured at physiologic concentrations of UDP-glucose (92 microM) and glucose-6-phosphate (110 microM). When unlabeled glycogen replaced D-[1-13C]glucose in the infusate after 50 min the D-[1-13C]glycogen signal remained stable for another 60 min, indicating that no turnover of the newly synthesized glycogen had occurred. Despite this phosphorylase a activity in heart extracts from rats given a 1 h glucose and insulin infusion (3.8 +/- 2.4 mumol/min gww) greatly exceeded the total synthase activity and if active in vivo should promote glycogenolysis. We conclude that during glucose and insulin infusion in the rat: (a) the absolute rate of myocardial glycogen synthesis can be measured in vivo by NMR; (b) glycogen synthase I can account for the observed rates of heart glycogen synthesis; (c) there is no futile cycling of glucose in and out of heart glycogen; and (d) the activity of phosphorylase a measured in tissue extracts is not reflected in vivo. These studies raise the question whether significant regulation of phosphorylase a activity in vivo is mediated by factors in addition to its phosphorylation state.     

Insulin controls key steps of carbohydrate metabolism in cultured HT29 colon cancer cells.

Effects of insulin on key steps of carbohydrate metabolism were investigated in cultured HT29 colon cancer cells by two different approaches, i.e. incubation of the cells either in the absence or in the presence of glucose in the medium. In glucose-deprived cells, insulin decreased glycogen breakdown, but did not affect polysaccharide levels when glucose was present. Glycogen synthase became activated after insulin treatment in both conditions, even though the activation was more evident when glucose was omitted. No effect on glycogen phosphorylase activity was evident under our experimental conditions. In cells incubated with glucose, the hormone stimulated in a dose-dependent manner the rates of glucose uptake and lactate release. Concomitantly with the increase in glycolytic rate, insulin caused a strong increase in fructose 2,6-bisphosphate. This effect was not observed in the absence of glucose. It is concluded that the carbohydrate metabolism of cultured HT29 cells responds to insulin, making this biological model suitable for investigations in vitro on the mechanism of insulin action.     

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.     

Synergistic activation of rat hepatocyte glycogen phosphorylase by A23187 and phorbol ester

The combination of 1.6 microM 4 beta phorbol, 12 beta myristate, 13 alpha acetate (PMA) and 1 microM A23187 produced a five-fold greater stimulation of rat hepatocyte glycogen phosphorylase activity than was seen with PMA alone. Vasopressin activation of glycogen phosphorylase was comparable to that seen with PMA plus A23187. Glycogen phosphorylase activity due to PMA plus A23187 was increased significantly after 30 sec, maximal at 120 and sustained at elevated levels for 240 sec. In contrast, activation due to vasopressin was maximal at 30 sec followed by a decrease. The addition of PMA 5 min prior to the A23187 abolished the synergism between these two agents. These data are compatible with the hypothesis that diacylglycerol and Ca2+ synergistically increase glycogen phosphorylase activity in rat hepatocytes.     

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.     

Synthesis of glycogen from fructose in the presence of elevated levels of glycogen phosphorylase a in rat hepatocytes

Incubation of hepatocytes with glucose promoted the increase in the glycogen synthase (-glucose 6-phosphate/+glucose 6-phosphate) activity ratio, the decrease in the levels of phosphorylase a and a marked increase in the intracellular glycogen level. Incubation with fructose alone promoted the simultaneous activation of glycogen synthase and increase in the levels of phosphorylase a. Strikingly, glycogen deposition occurred in spite of the elevated levels of phosphorylase a. When glucose and fructose were added to the media the activation of glycogen synthase was always higher than when the hexoses were added separately. On the other hand the effects on glycogen phosphorylase were a function of the relative concentrations of both sugars. Inactivation of glycogen phosphorylase occurred when the fructose to glucose ratio was low while activation took place when the ratio was high. The simultaneous presence of glucose and fructose resulted, in all cases, in an enhancement in the deposition of glycogen. The effects described were not limited to fructose as D-glyceraldehyde, dihydroxyacetone, L-sorbose, D-tagatose and sorbitol, compounds metabolically related to fructose, provoked the same behaviour.     

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.