Effect of starvation and insulin treatment on glycogen synthase D and synthase D phosphatase activity in rat heart

Summary We have previously shown that synthase phosphatase activity was decreased in starved animals and was rapidly restored by insulin administration (1). In order to determine whether the decreased phosphatase activity was due to a decrease in phosphatase enzyme per se or to a change in the substrate, synthase D, phosphatase activity has been determined using purified synthase D substrate. Using purified heart or liver synthase D, phosphatase activity was lower in extracts from starved animals than in fed animals. Insulin administration rapidly increased phosphatase activity in extracts from the starved animals. The total amount of endogenous synthase D which was convertible to synthase I was lower in extracts from starve animals, but this was rapidly increased within 15 minutes following insulin administration. These data suggest that starvation and insulin have a direct effect on the phosphatase enzyme activity per se and probably on the substrate suitability of synthase D as well.     

Insulin regulation of glycogen synthase phosphatase in primary cultures of hepatocytes

Activation of glycogen synthase in the perfused rat liver is defective in severely diabetic rats. In the present study, activation of glycogen synthase by glucose and increased incorporation of [14C]glucose into glycogen by insulin are defective in hepatocytes isolated from alloxan diabetic rats. Acute activation of glycogen synthase in hepatocytes isolated from diabetic rats was restored by treatment of the rats with insulin in vivo. Restoration of synthase activation was not achieved by incubation of hepatocytes in the presence of insulin in vitro for up to 12 h. When isolated hepatocytes from diabetic rats were placed in primary culture in a serum-free defined medium over a 3-day period, glycogen synthesis was partially restored by cortisol and triiodothyronine and dramatically increased by insulin. Concomitant with restoration of [14C]glycogen synthesis was an insulin-mediated increase in glycogen synthase I and synthase phosphatase activity. Restoration of regulation of glycogen synthesis in primary cultures of hepatocytes from diabetic rats by insulin required the presence of cortisol and triiodothyronine. Primary cultures of hepatocytes from normal rats did not require triiodothyronine for insulin to effect glycogenesis over a 3-day period. These data demonstrate that insulin acts in a chronic manner in concert with other hormones to control synthase phosphatase activity, an effect which may be influencing acute control of hepatic glycogen synthesis.     

Direct glucose stimulation of glycogen synthase phosphatase activity in a liver glycogen particle preparation

In glycogen particle suspensions prepared from fed rats given either glucagon or glucose in order to increase or decrease the phosphorylase a concentration, respectively, glucose stimulation of synthase phosphatase activity was observed. In preparations from glucagon-treated rats, addition of glucose stimulated synthase and phosphorylase phosphatase simultaneously and not sequentially. Synthase phosphatase stimulation was glucose concentration dependent even when phosphorylase a had been rapidly reduced to a low level. The estimated A0.5 for glucose stimulation of synthase phosphatase activity was 27 mM. An A0.5 for glucose stimulation of phosphorylase phosphatase activity could not be estimated since activity was still increasing with concentrations of glucose as high as 200 mM. In preparations from glucose-treated rats which contain virtually no phosphorylase a, glucose stimulation was still apparent but the A0.5 was increased modestly (36 mM). Stimulation of synthase phosphatase activity was specific for glucose. Several other monosaccharides and the polyhydric alcohol sorbitol were ineffective.     

Phosphohistone and glycogen synthase D phosphatase activities in a liver glycogen pellet preparation.

A liver glycogen pellet preparation previously found to contain synthase D phosphatase activity was shown to contain also phosphohistone phosphatase activity. Pellet phosphohistone phosphatase and synthase D phosphatase competed for the same substrates and appeared to be the same enzyme. ATP, a potent inhibitor, and G-6-P, a potent activator of the synthase phosphatase reaction, had little effect on the phosphohistone phosphatase reaction. These observations suggest that the ATP and G-6-P effects are relatively specific and are probably caused by binding to the synthase D substrate. The observed effects of NaCl and KCl were more complex. They stimulated phosphohistone phosphatase activity but strikingly inhibited synthase phosphatase activity. Sodium fluoride inhibited both reactions.     

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.     

Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A.

A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae. The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis. This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C. The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer. On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration. The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid. Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines. Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50%. The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A. An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not. These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.     

Phosphorylation of bovine neurofilament proteins by protein kinase FA (glycogen synthase kinase 3).

A highly purified preparation of protein kinase FA (where FA is the activating factor for phosphatase 1)/glycogen synthase kinase 3 from rabbit muscle readily phosphorylated bovine neurofilaments. All three neurofilament proteins, the high, middle, and low molecular proteins (NF-H, NF-M, and NF-L), were phosphorylated when intact filaments were incubated with the kinase. Experiments with individual proteins showed that NF-M was the best substrate. At protein concentrations of 0.13 mg/ml, the initial rate of NF-M phosphorylation was 30% of that observed for glycogen synthase. Km values were 0.24 mg/ml (7 x 10(-7) M tetramer) for glycogen synthase and 0.10 mg/ml (5 x 10(-7) M dimer) for NF-M. Vmax values were 0.36 mumol/min/mg for glycogen synthase and 0.035 mumol/min/mg for NF-M. Dephosphorylated NF-M was phosphorylated only half as much as native NF-M; this is consistent with the known substrate specificity of the kinase. The possible involvement of FA/GSK-3 in the phosphorylation of neurofilaments in vivo is discussed.     

Dephosphorylation of glycogen synthase in rat heart extracts by E. coli alkaline phosphatase

Regulation of the dephosphorylation of glycogen synthase in extracts from rat heart has been studied by adding exogenous phosphatase to the extract. These experiments were possible only because the endogenous protein phosphatase activity of the extract could be inhibited by KF under conditions where alkaline phosphatase activity was not. The concentration of substrate (glycogen synthase from the heart extract) and catalyst (purified E. coli alkaline phosphatase) could be varied independently, by adding known amounts of alkaline phosphatase to the KF-containing heart extracts. Alkaline phosphatase could completely dephosphorylate glycogen synthase while phosphorylase was unchanged. The rate of dephosphorylation was proportional to both the concentration of alkaline phosphatase added to the tissue extract and the amount of glycogen synthase in the extract. The Km for glycogen synthase was close to the concentration found in heart tissue. The Km and the maximum rate of dephosphorylation were both dependent on the phosphorylation state of the glycogen synthase. Less phosphorylated enzyme forms were dephosphorylated faster. These results indicate the necessity for precise control of many variables in studying the rate of glycogen synthase dephosphorylation. Alkaline phosphatase-catalyzed dephosphorylation could be inhibited by physiological concentrations of glycogen. Glycogen synthase dephosphorylation in extracts from fasted-refed rats was less sensitive to glycogen inhibition than in extracts from normal animals. The phosphorylation state of the glycogen synthase in these animals was assessed by kinetic studies to show that differences in phosphorylation state probably could not account for the observations. Fasting led to a decreased rate of dephosphorylation of glycogen synthase due to both an apparent change in kinetic properties of glycogen synthase as a substrate for alkaline phosphatase, and an increased inhibitory effect of glycogen. Stable modifications of glycogen synthase caused by altered nutritional states in the animals are thought to produce these effects.     

Purification of bovine heart glycogen synthase

Glycogen synthase was purified to apparent homogeneity from bovine heart muscle by a procedure involving precipitation of the enzyme in the presence of added glycogen by polyethylene glycol, chromatography on DEAE-Sephacel, and high-speed centrifugation through a sucrose-containing buffer. The enzyme was maintained in the presence of glycogen during the isolation procedure. Glycogen synthase I and D preparations were obtained having specific activities of 21-25 and 30-35 units/mg protein at pH 7.8 and 30 degrees C and having activity ratios of 0.5-0.6 and 0.05-0.10, respectively, when assayed in the absence and in the presence of glucose 6-P.     

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.     

Insulin resistance of glycogen synthase mediated by o-linked N-acetylglucosamine

We have investigated the mechanism by which high concentrations of glucose inhibit insulin stimulation of glycogen synthase. In NIH-3T3-L1 adipocytes cultured in low glucose (LG; 2.5 mm), the half-maximal activation concentration (A(0.5)) of glucose 6-phosphate was 162 +/- 15 microm. Exposure to either high glucose (HG; 20 mm) or glucosamine (GlcN; 10 mm) increased the A(0.5) to 558 +/- 61 or 612 +/- 34 microm. Insulin treatment with LG reduced the A(0.5) to 96 +/- 10 microm, but cells cultured with HG or GlcN were insulin-resistant (A(0.5) = 287 +/- 27 or 561 +/- 77 microm). Insulin resistance was not explained by increased phosphorylation of synthase. In fact, culture with GlcN decreased phosphorylation to 61% of the levels seen in cells cultured in LG. Hexosamine flux and subsequent enzymatic protein O-glycosylation have been postulated to mediate nutrient sensing and insulin resistance. Glycogen synthase is modified by O-linked N-acetylglucosamine, and the level of glycosylation increased in cells treated with HG or GlcN. Treatment of synthase in vitro with protein phosphatase 1 increased basal synthase activity from cells cultured in LG to 54% of total activity but was less effective with synthase from cells cultured in HG or GlcN, increasing basal activity to only 13 or 16%. After enzymatic removal of O-GlcNAc, however, subsequent digestion with phosphatase increased basal activity to over 73% for LG, HG, and GlcN. We conclude that O-GlcNAc modification of glycogen synthase results in the retention of the enzyme in a glucose 6-phosphate-dependent state and contributes to the reduced activation of the enzyme in insulin resistance.     

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 liver glycogen synthase phosphatase activity by ATP-Mg

The kinetics of a synthase phosphatase reaction inhibited by ATP-Mg in a liver glycogen particle preparation were complex. In the presence of a physiological concentration of ATP-Mg, synthase phosphatase activity in the glycogen particle follows a biphasic course. Initially, the reaction was inhibited but later the reaction rate accelerated. The reaction was inhibited but the rate was constant in the presence of ATP-Mg with the addition of a physiological concentration of glucose 6-phosphate (Glc 6-P). Therefore, in most subsequent experiments Glc 6-P was added. The concentration of ATP-Mg at which 50% maximal inhibition (I0.5) occurred was approximately 0.1 mM in preparations obtained from rats given glucagon prior to being killed. In preparations from animals given glucose, the I0.5 was increased to 2.0 mM. The maximum inhibition was little changed in preparations from glucose- or glucagon-treated animals. Thus, administration of glucose in vivo reduced the sensitivity of the synthase phosphatase to ATP-Mg inhibition. Complexes of ATP with paramagnetic ions such as Co2+ and Mn2+ were less inhibitory than complexes with diamagnetic ions, including Ca2+ and Mg2+. Magnesium complexes of adenosine tetraphosphate and 5'-adenylimidodiphosphate also were inhibitory. Inhibition was independent of phosphorylase a and not a nonspecific, polyvalent anion effect. The best explanation for the distinctive effects of ATP-Mg in preparations from glucagon- and glucose-treated animals is that the respective treatments promote and stabilize different forms of synthase D or possibly synthase phosphatase with different affinities for ATP-Mg. These forms are interconvertible, as previously suggested, in studies employing EDTA (20).     

Dephosphorylation of glycogen synthase in rat heart extracts by E. coli alkaline phosphatase

Summary Regulation of the dephosphorylation of glycogen synthase in extracts from rat heart has been studied by adding exogenous phosphatase to the extract. These experiments were possible only because the endogenous protein phosphatase activity of the extract could be inhibited by KF under conditions where alkaline phosphatase activity was not. The concentration of substrate (glycogen synthase from the heart extract) and catalyst (purified E. coli alkaline phosphatase) could be varied independently, by adding known amounts of alkaline phosphatase to the KF-containing heart extracts. Alkaline phosphatase could completely dephosphorylate glycogen synthase while phosphorylase was unchanged. The rate of dephosphorylation was proportional to both the concentration of alkaline phosphatase added to the tissue extract and the amount of glycogen synthase in the extract. The K_m for glycogen synthase was close to the concentration found in heart tissue. The K_m and the maximum rate of dephosphorylation were both dependent on the phosphorylation state of the glycogen synthase. Less phosphorylated enzyme forms were dephosphorylated faster. These results indicate the necessity for precise control of many variables in studying the rate of glycogen synthase dephosphorylation.Alkaline phosphatase-catalyzed dephosphorylation could be inhibited by physiological concentrations of glycogen. Glycogen synthase dephosphorylation in extracts from fasted-refed rats was less sensitive to glycogen inhibition than in extracts from normal animals. The phosphorylation state of the glycogen synthase in these animals was assessed by kinetic studies to show that differences in phosphorylation state probably could not account for the observations. Fasting led to a decreased rate of dephosphorylation of glycogen synthase due to both an apparent change in kinetic properties of glycogen synthase as a substrate for alkaline phosphatase, and an increased inhibitory effect of glycogen. Stable modifications of glycogen synthase caused by altered nutritional states in the animals are thought to produce these effects.%GSI represents the percentage of glycogen synthase activity that is active without glucose 6-P.     

Substrate specificity of glycogen synthase phosphatase from bovine heart: action on phosphorylase a and histone

Glycogen synthase phosphatase has been purified from bovine heart. This preparation catalyzes conversion of synthase D into I and phosphorylase $\text{a}$ into $\text{b}$ and is able to dephosphorylate synthase D, phosphorylase $\text{a}$, active phosphorylase kinase, and phosphorylated histone and casein. The activity of phosphatase was assayed with synthase D, phosphorylase $\text{a}$, and histone as substrates after chromatography on Sephadex G-100, after sucrose gradient centrifugation, and after isoelectric focusing in a sucrose gradient. In all cases no separation of enzyme activity was observed with the above substrates. The phosphatase activity on all substrates was lost at the same rate by heat denaturation. These results indicate that this enzyme preparation contains a single phosphoprotein phosphatase which is responsible for the activity observed on the above substrates.