Glycogen Synthase Phosphatase Research Articles

Alteration of hepatic glycogen synthase phosphatase activity by insulin deficiency

Perfused livers from normal and alloxan-diabetic rats were studied to determine whether the diabetes-related decrease in glycogen synthase phosphatase was due to an alteration of the synthase molecule, an increase in synthase phosphatase activity inhibition by phosphorylase a, or generation of inhibitor of the phosphatase. With purified rat liver synthase as substrate for the phosphatase, the diabetic tissue remained 90-95% deficient in the ability to catalyze synthase D to I conversion, showing that the defect cannot be solely due to an altered substrate. When synthase phosphatase assays were carried out in the presence of rat liver glycogen phosphorylase antiserum, phosphatase activity remained 70-75% deficient in diabetic tissue. Therefore, the defect cannot be attributed to increased inhibition of synthase phosphatase by increased amounts of phosphorylase a. When synthase phosphatase assays were run by mixing extracts from normal and diabetic livers, phosphatase activity was additive, indicating that a phosphatase inhibitor was probably not involved in the phosphatase deficiency in the diabetic. These data are consistent with the hypothesis that the diabetes-related defect in glucose regulation of hepatic glycogen synthase is due to a molecular alteration or a deficiency of a specific glycogen synthase phosphatase.

Hormonal regulation of hepatic glycogen synthase phosphatase.

Perfusion of livers from fed rats with medium containing glucagon (2 x 10(-10) or 1 x 10(-8) M) resulted in both time- and concentration-dependent inactivation of glycogen synthase phosphatase. Expected changes occurred in cAMP, cAMP-dependent protein kinase, glycogen synthase, and glycogen phosphorylase. The effect of glucagon on synthase phosphatase was partially reversed by simultaneous addition of insulin (4 x 10(-8) M), an effect paralleled by a decrease in cAMP. Addition of arginine vasopressin (10 milliunits/ml) resulted in a similar inactivation of synthase phosphatase and activation of phosphorylase, but independent of any changes in cAMP or its kinase. Phosphorylase phosphatase activity was unaffected by any of these hormones. Synthase phosphatase activity, measured as the ability of a crude homogenate to catalyze the conversion of purified rat liver synthase D to the I form, was no longer inhibited by glucagon or vasopressin when phosphorylase antiserum was added to the phosphatase assay mixture in sufficient quantity to inhibit 90-95% of the phosphorylase a activity. These data support the following conclusions: 1) hepatic glycogen synthase phosphatase activity is acutely modulated by hormones, 2) hepatic glycogen synthase phosphatase and phosphorylase phosphatase are regulated differently, 3) the hormone-mediated changes in synthase phosphatase cannot be explained by an alteration of the synthase D molecule affecting its behavior as a substrate, and 4) glycogen synthase phosphatase activity is at least partially controlled by the level of phosphorylase a.

The role of glycogen synthase phosphatase in the glucocorticoid-induced deposition of glycogen in foetal rat liver.

1. The mechanism that underlies the induction of glycogen synthesis in the foetal rat liver by glucocorticoids was reinvestigated in conditions where the accumulation of glycogen is either precociously induced with dexamethasone or inhibited by steroid deprivation. It appears that glucocorticoids act as the physiological trigger for glycogen synthesis by inducing both glycogen synthase (a known effect) and its activating enzyme, glycogen synthase phosphatase. 2. The activity of glycogen synthase phosphatase in adult liver stems from the interaction of two protein components [Doperé, Vanstapel & Stalmans (1980) Eur. J. Biochem. 104, 137--146]. Two independent experimental approaches indicate that the cytosolic 'S-component' is already well developed in the foetal liver before the onset of glycogen synthesis. The manifold glucocorticoid-dependent increase in synthase phosphatase activity during late gestation must be attributed to the specific development of the glycogen-bound 'G-component'.

Effects of insulin treatment on the activities of phosphoprotein phosphatase and its inhibitors.

An increase in glycogen synthase phosphatase (phosphoprotein phosphatase) activity was observed in the rat skeletal muscle extract following insulin administration. The phosphoprotein phosphatase activity present in the muscle extract from insulin treated rats was observed to remain elevated after the extract had been subjected to a molecular sieve chromatography. These results indicate that the stimulatory effects of insulin is due to modification of phosphatase itself or some macromolecular weight modifiers. The heat-stable protein inhibitors of the phosphoprotein phosphatase were isolated from skeletal muscle of insulin treated and control rats and their inhibitory potencies were compared over a wide range of protein concentrations. The inhibitory potency in the insulin treated rat skeletal muscle was found to be significantly less than that in the control muscle. Since type-1 inhibitor is well-known to be active only after being phosphorylated by cyclic AMP-dependent protein kinase, we suggest that the observed change in phosphoprotein phosphatase inhibitor potency is most likely mediated by an alteration in the phosphorylation state of type-1 inhibitor.

Influence of fructose on the glycogen synthase and phosphorylase systems in rat liver

Fructose and glucose, when administered as a single, large intravenous dose (500 mg/kg) produced opposite effects on key regulatory enzymes of glycogen metabolism in intact normal fed animals. Glucose rapidly stimulated glycogen synthase phosphatase activity and increased the proportion of glycogen synthase in the active (I) form as expected; fructose reduced synthase phosphatase activity and the proportion of synthase in the I form. Glucose also stimulated a reduction in the proportion of phosphorylase in the active ( a) form, whereas fructose stimulated an increase in the proportion of phosphorylase in the a form. The effect of fructose was not mediated by an increase in cyclic adenylate (cAMP) concentration nor by a conversion of phosphorylase kinase b to phosphorylase kinase a. As expected, the concentration of ATP decreased significantly. The increase in proportion of phosphorylase in the a form may be due to stimulation of phosphorylase kinase b activity by a decrease in the intracellular ATP:Mg ++ ratio or by an increase in intracellular Ca ++ concentration. The mechanism of the fructose-induced change in synthase phosphatase activity and in synthase I activity is unknown.

Studies on the Insulin Mediator: II. Separation of Two Antagonistic Biologically Active Materials from Fraction II

Insulin treatment significantly altered the elution profile of deproteinized muscle extracts chromatographed on Sephadex G-25 columns, particularly in fraction II, which contains the insulin mediator. Further purification of fraction II by high-voltage paper electrophoresis at pH 1.9 and 3.5 resulted in two active fractions. Fraction 1 leads to 4 stimulated the cyclic AMP-dependent protein kinase and inhibited glycogen synthase phosphoprotein phosphatase, and may be a novel substance. Fractions 1 leads to 6 and 3 leads to 6 inhibited the cyclic AMP-dependent protein kinase and stimulated glycogen synthase phosphatase. It is proposed that the insulin mediator is present in fractions 1 leads to 6 and 3 leads to 6.

Liver glycogen synthase phosphatase and phosphorylase phosphatase activities in vitro following glucose and glucagon administration

Correlation of the changes in phosphorylase a concentration with the synthase phosphatase velocity in a glycogen particle preparation in the presence of EDTA revealed that the initial synthase phosphatase rate was greatest in extracts from glucose-treated rats and least in extracts from glucagon-treated rats. In all cases the velocity increased with time and with a decrease in phosphorylase a. However, a threshold release of phosphatase activity when phosphorylase a reached a critical level was not observed. The data are compatible with either an independent regulation of synthase phosphatase by glucose and glucagon or regulation of the activity by phosphorylase over a range of phosphorylase a concentrations.

Liver glycogen synthase in rats with a glycogen-storage disorder. The role of glycogen in the regulation of glycogen synthase.

1. A strain of rats with a genetically‐determined liver glycogen‐storage disorder (gsd/gsd) caused by a deficiency of liver phosphorylase kinase has a very high concentration of glycogen in the liver with a total glycogen synthase activity higher than in liver of fed normal animals, but only a small amount of the enzyme in the active form. In the livers of both starved and fed normal rats and gsd/gsd rats, there is a positive correlation between glycogen content and total glycogen synthase (r= 0.79) and an inverse correlation between glycogen content and active glycogen synthase (r= 0.86).2. Homogenates of livers from the gsd/gsd rats have lower glycogen‐synthase phosphatase activities than homogenates from fed normal rats which in turn have lower activities than those from starved rats. The addition of glycogen to homogenates of liver from starved rats reduced the glycogen‐synthase phosphatase activity.3. Dilution of homogenates of gsd/gsd rat liver produced an increase in glycogen‐synthase phosphatase activity which could be reversed by adding glycogen.4. The gsd/gsd rats were shown to have liver glycogen‐synthase phosphatase activity comparable to normal rats when the enzyme was measured in dilute homogenates from both groups with the same glycogen concentration.5. It is suggested that inhibition of glycogen‐synthase phosphatase by high glycogen concentrations is responsible for the low levels of activation of glycogen synthase in the gsd/gsd rats and that glycogen itself is probably the major factor controlling glycogen synthesis in gsd/gsd and fed normal rats.

Stimulation of muscle glycogen synthase phosphatase by polyamines

The naturally-occurring polyamines were found to stimulate glycogen synthase phosphatase from rat skeletal muscle. The sequence of effectiveness in the stimulation was spermine >. spermidine > putrescine. It was shown that the spermine-sensitive phosphatase was present primarily in the soluble fraction of the muscle extract. In the presence of spermine, the phosphatase activity can be further stimulated by Mn 2+; however, a lower Mn 2+ concentration is required for the activation of the enzyme in comparison with that in the absence of spermine. Kinetic studies indicated that activation of glycogen synthase phosphatase by spermine was achieved by an increase in its V without significant alteration in the K m, suggesting that spermine directly stimulated the catalytic efficiency of the phosphatase enzyme.

Dephosphorylation and activation of exogenous glycogen synthase by adipose-tissue phosphatase

Exogenous purified rabbit skeletal-muscle glycogen synthase was used as a substrate for adipose-tissue phosphoprotein phosphatase from fed and starved rats in order to (1) compare the relationship between phosphate released from, and the kinetic changes imparted to, the substrate and (2) ascertain if decreases in adipose-tissue phosphatase activity account for the apparent decreased activation of endogenous glycogen synthase from starved as compared with fed rats. Muscle glycogen synthase was phosphorylated with [gamma-(32)P]ATP and cyclic AMP-dependent protein kinase alone, or in combination with a cyclic AMP-independent protein kinase, to 1.7 or 3mol of phosphate per subunit. Adipose-tissue phosphatase activity determined with phosphorylated skeletal-muscle glycogen synthase as substrate was decreased by 35-60% as a consequence of starvation. This decrease in phosphatase activity had little effect on the capacity of adipose-tissue extracts to activate exogenous glycogen synthase (i.e. to increase the glucose 6-phosphate-independent enzyme activity), although there were marked differences in the activation profiles for the two exogenous substrates. Glycogen synthase phosphorylated to 1.7mol of phosphate per subunit was activated rapidly by adipose-tissue extracts from either fed or starved rats, and activation paralleled enzyme dephosphorylation. Glycogen synthase phosphorylated to 3mol of phosphate per subunit was activated more slowly and after a lag period, since release of the first mol of phosphate did not increase the glucose 6-phosphate-independent activity of the enzyme. These patterns of enzyme activation were similar to those observed for the endogenous adipose-tissue glycogen synthase(s): the glucose 6-phosphate-independent activity of the endogenous enzyme from fed rats increased rapidly during incubation, whereas that of starved rats, like that of the more highly phosphorylated muscle enzyme, increased only very slowly after a lag period. The observations made here suggest that (1) changes in glucose 6-phosphate-independent glycogen synthase activity are at best only a qualitative measure of phosphoprotein phosphatase activity and (2) the decrease in glycogen synthase phosphatase activity during starvation is not sufficient to explain the differential glycogen synthase activation in adipose tissue from fed and starved rats. However, alterations in the phosphorylation state of glycogen synthase combined with decreased activity of phosphoprotein phosphatase, both as a consequence of starvation, could explain the apparent markedly decreased enzyme activation.

Impaired glycogen synthase activating system in human diabetic polymorphonuclear leukocytes.

The effects of diabetes mellitus on glycogen synthase and its activating system (synthase phosphatase) were studied using human polymorphonuclear leukocytes (PMN). PMN were obtained from control subjects and diabetic patients by a gradient sedimentation technique. Enzyme activities of endogenous synthase-l and total synthase were not statistically different in diabetic and control cells. For measurement of endogenous synthase phosphatase, cells were sonicated in 50 mM Tris buffer (pH 7.5) and incubated at 30 degrees C. Conversion of synthase-D to -l and the maximum percent synthase-l attained were decreased in homogenates of diabetic cells. There was no correlation between the plasma glucose concentration and the rate of conversion of synthase-D to -l. Synthase phosphatase activities were also measured using a purified synthase-D substrate. Under these experimental conditions, glycogen synthase phosphatase activities did not differ in control and diabetic cells. These results are consistent with a diabetes-induced defect in conversion of endogenous synthase-D to -l at the level of the synthase enzyme rather than at that of the activating phosphatase.

Glycogen-synthase phosphatase activity in rat liver. Two protein components and their requirement for the activation of different types of substrate.

Three subfractions of glycogen synthase b (termed b1, b2, b3) have been isolated from the glycogen fraction of dog liver on the basis of a different affinity for DEAE-cellulose. Their kinetic properties and chromatographic behaviour are compatible with the presence of an increasing number of phosphorylated sites from synthase b1 towards b3. Synthase phosphatase activity in rat liver stems from two heat-labile and trypsin-labile proteins. These components are conveniently prepared from the cytosolic fraction of glycogen-depleted liver; the 'G-component' of the phosphatase co-sediments with added particulate glycogen, whereas the 'S-component' remains in the supernatant. The G-component alone did not convert any available synthase b to the a form. The synthase phosphatase activity of the S-component was variable according to the actual type of substrate. When acting on synthase b2 and b3, the S-component had a low phosphatase activity that was increased 7-fold and 11-fold, respectively, upon addition of the G-component. Synthase b1, however, was efficiently activated by the S-component, and only 35% faster in the presence of both components. When the cytosolic fraction of glycogen-depleted livers was analysed by sucrose-gradient centrifugation a single peak of phosphatase activity (S20, W = 10.2 S; provisional Mr = 254000) was detected with synthase b2 as substrate. In addition to this peak, presumably an S-G complex, synthase b1 also identified free S-component of lower and heterogeneous molecular weight. Our results illustrate in general the influence of the type of synthase b on the detection of synthase phosphatase activity, and specifically may provide an explanation for some discrepant reports on the subcellular distribution of the enzyme.

Insulin action in intact mouse diaphragm I.

The incubation of intact mouse diaphragms with insulin caused a dose and time dependent increase in the independent activity of glycogen synthase in tissue extracts. 2-deoxyglucose (2-10 mM) alone markedly stimulated the conversion of glycogen synthase to the independent activity under conditions in which tissue ATP concentrations were not affected. The incubation of diaphragms with both insulin and 2-deoxyglucose resulted in a greater than additive effect. Insulin stimulated the uptake of 2-deoxyglucose into mouse diaphragms, accumulating as 2-deoxyglucose-6-phosphate. The accumulation of 2-deoxyglucose-6-phosphate correlated well with the increase in the independent activity of glycogen synthase and with the activation of glycogen synthase phosphatase in tissue extracts. The uptake of 3-0 methyl glucose was also markedly stimulated by insulin, without affecting the activity of glycogen synthase. Both glucose-6-phosphate and 2-deoxyglucose-6-phosphate stimulated the activation of endogenous glycogen synthase phosphatase activity in muscle homogenates. We conclude that insulin, in addition to its effects in the absence of exogenous sugars, increases the independent activity of glycogen synthase through increased sugar transport resulting in increased concentrations of sugar-phosphates which promote the activity of glycogen synthase phosphatase.

The divalent cation dependence of liver glycogen synthase phosphatase activity.

In a previous report it was shown that EDTA inhibition of liver glycogen synthase phosphatase activity in preparations from normal, fed rats could be increased upon glucagon or cAMP treatment. This occurred without a change in the half-maximum inhibitory concentration of EDTA. Glucose administration to animals resulted in decreased EDTA inhibition. The inhibitory action of EDTA has been further characterized by comparing its action with that of other chelators (CDTA and EGTA) and examining the effects of various divalent cations on chelator inhibition. Both CDTA and EDTA which differ structurally were inhibitory at 5 mm concentrations whereas EGTA which is structurally similar to EDTA was not inhibitory at concentrations up to 10 mm. The lack of inhibition by EGTA could be explained by its weak affinity for Mg++ in the preparation. A comparison of CDTA and EDTA revealed that CDTA was a more potent inhibitor than EDTA (I0.5, 0.15 mm vs 0.3 mm). Glucagon and glucose treatment of rats resulted in changes in CDTA inhibition which closely paralleled those of EDTA. A large group of divalent cations were tested but only Mg++, Ca++, and Mn++ both prevented and reversed CDTA or EDTA inhibition. Fifty percent reversal using either chelator occurred at calculated free-metal ion concentrations of approximately 2 µm, 0.08 µm and 0.0004 µm, respectively. Thus, it is clear that EDTA inhibition is due to its chelation effect and is not due to a nonspecific anionic effect.

The effects of food deprivation and re-feeding on bovine adipose-tissue glycogen synthase.

Bovine adipose-tissue glycogen metabolism was studied during food deprivation and re-feeding. Changes in the specific activity of adipose-tissue glycogen synthase paralleled changes in tissue glycogen content: both parameters increased during food deprivation and remained so during the first 10 days of re-feeding. The values for the A0.5 (activation constant) for glucose 6-phosphate of the freshly isolated enzyme from adipose tissue from fed and starved steers were 2.9 +/- 0.1 mM and 0.90 +/- 0.05 mM respectively. Additionally, whereas incubation of adipose-tissue extracts from fed steers did not activate endogenous glycogen synthase (through a presumed phosphoprotein phosphatase mechanism), the enzyme from starved or re-fed (up to 3 days re-feeding) steers was reversibly activated as measured by changes in the value for the A0.5 for glucose 6-phosphate. Thus activation of bovine adipose-tissue glycogen synthase during food deprivation appears to be related to expression of glycogen synthase phosphatase activity. These effects of food deprivation on bovine glycogen metabolism contrast markedly with the effects observed in rat adipose tissue.

Association of glycogen synthase phosphatase and phosphorylase phosphatase activities with membranes of hepatic smooth endoplasmic reticulum.

A detailed investigation was conducted to determine the precise subcellular localization of the rate-limiting enzymes of hepatic glycogen metabolism (glycogen synthase and phosphorylase) and their regulatory enzymes (synthase phosphatase and phosphorylase phosphatase). Rat liver was homogenized and fractionated to produce soluble, rough and smooth microsomal fractions. Enzyme assays of the fractions were performed, and the results showed that glycogen synthase and phosphorylase were located in the soluble fraction of the livers. Synthase phosphatase and phosphorylase phosphatase activities were also present in soluble fractions, but were clearly identified in both rough and smooth microsomal fractions. It is suggested that the location of smooth endoplasmic reticulum (SER) within the cytosome forms a microenvironment within hepatocytes that establishes conditions necessary for glycogen synthesis (and degradation). Thus the location of SER in the cell determines regions of the hepatocyte that are rich in glycogen particles. Furthermore, the demonstration of the association of synthase phosphatase and phosphorylase phosphatase with membranes of SER may account for the close morphological association of SER with glycogen particles (i.e., disposition of SER membranes brings the membrane-bound regulatory enzymes in close contact with their substrates).

Insulin activates glycogen synthase in phosphorylase kinase deficient mice

Glycogen synthase, the rate limiting enzyme in glycogen synthesis, is regulated by a phosphorylationdephospho~lation mechanism. The enzyme can exist as either a high activity dephospho~lated a-form, whose activity is enhanced only slightly by glucose&phosphate, or as low activity phosphorylated b-forms, which can be activated considerably by glucosed-phosphate. An important action of insulin in skeletal muscle is its ability to activate glycogen synthase and thereby stimulate the deposition of glycogen. The effect is observed in diaphragm [ 1 ] and soieus muscle [2] in the absence of glucose in the incubation medium, in~cating that it is not a consequence of the increased transport of glucose into muscle which is also stimulated by insulin [3,4]. Insulin increases the activity ratio of glycogen synthase (defined as the activity in the absence of glucose8-phosphate divided by the activity in the presence of glucose-&phosphate). It therefore produces kinetic changes that are equiv‘alent to a net decrease in the extent of phosphorylation of the enzyme, and such a decrease in the phosphate content of rabbit skeletal muscle glycogen synthase has been demonstrated following the administration of the hormone in vivo [S]. These experiments indicate that insulin either decreases the activity of glycogen synthase kinase or increases the activity of glycogen synthase phosphatase. However, this apparently simple picture is complicated

Glycogen synthase: The existence of a single demonstrable form in bovine adipose tissue

Glycogen synthase from bovine adipose tissue has been kinetically characterized. Glucose 6-phosphate increased enzyme activity 50-fold with an activation constant ( A 0.5) of 2.6 m m. Mg 2+ reversibly decreased this A 0.5 to 0.75 m m without changing the amount of stimulation by glucose 6-phosphate. Mg 2+ did not alter the apparent K m for UDP-glucose (0.13 m m). The pH optimum was broad and centered at pH 7.6. The glucose 6-phosphate activation of the enzyme was reversible and competitively inhibited by ATP ( K i = 0.6 mm) and P i( K i = 2.0 mm). The use of exogenous sources of glycogen synthase and glycogen synthase phosphatase suggests that (i) adipose tissue glycogen synthase phosphatase activity in fed mature steers is low or undetectable, and (ii) endogenous bovine adipose tissue glycogen synthase can be activated to other glucose 6-phosphate-dependent forms by addition of adipose tissue extracts from fasted steers or fed rats.

A novel glycogen synthase phosphatase from canine heart

Summary Two molecular forms (M r =49,300 and 26,000) of a divalent cation-dependent (Mg 2+ =Mn 2+ >Co 2+ ) protein phosphatase, which represent the major glycogen synthase phosphatase activity in canine heart extracts, have been partially purified and characterized. Although a general protein phosphatase of M r =35,000 is also active toward synthase D, it represents the major phosphorylase phosphatase activity in heart muscle. The present findings indicate that the dephosphorylation of synthase D and phosphorylase a may be regulated by two distinct phosphatases rather than by a single nonspecific enzyme.

Glycogen Synthase, Synthase Phosphatase, and Phosphorylase Response to Glucose in Somatostatin-Pretreated Intact Rats

The response of the glycogen synthase and phosphorylase systems of the liver to intravenous glucose in the presence and absence of short-term somatostatin blockade of insulin secretion was determined in fed and 20-h fasted rats. These enzyme systems regulate glycogen synthesis and degradation, respectively. In the presence of somatostatin, intravenous glucose (1.0 g/kg), promptly (5 min) increased the proportion of synthase in the I (active) form, and the increase was similar to that in animals that had not received somatostatin. In the same animals, phosphorylase a also was decreased, and the decrease was similar in all groups. When a smaller dose of glucose (250 mg/kg) was used that only modestly increased plasma glucose (139 mg/dl) and produced a less than maximal synthase response, insulin (1 U/kg) did not potentiate glucose activation of synthase either in the presence or absence of somatostatin. Phosphorylase a did not change significantly in any group. Glucose, in both the presence and absence of somatostatin, also rapidly (2 min) converted synthase phosphatase from a form inhibited by EDTA to a form not inhibited by EDTA. These data indicate that the synthase and phosphorylase systems in vivo respond primarily to a rise in plasma glucose and not to a simultaneous elevation in plasma insulin. Thus, glucose is regulating its own storage as glycogen in the liver. The effect of glucose on the synthase may be mediated through a conversion of the synthase phosphatase to a form that is not dependent on divalent cation for activity. This form presumably is more active in vivo.