Insulin sensitivity of liver glycogen synthase b into a conversion

Summary Liver glycogen synthase b phosphatase, chromatographically separable from phosphorylase a phosphatase, is decreased in 48-hour alloxan diabetic rats. The phosphatase activities are measured in an in vitro system using exogenous isolated phospho-enzyme as substrates with added phosphatases. Synthase and phosphorylase phosphatases were shown to have differential catalytic properties by their reactivity in the presence of Pi, the heat-stable inhibitor of phosphorylase phosphatase and after incubation with added cAMP-dependent protein kinase.Supported by NIH Grants HD-07788, AM-21149 and, in part, by grants from the Greater St. Louis Diabetic Childrens Welfare Association and the American Diabetes Association, N.Y.     

Mechanism of palmityl coenzyme A inhibition of liver glycogen synthase.

Palmityl-CoA inhibits free liver glycogen synthase; the concentration required for half-maximum inhibition is 3 to 4 micrometer. Almost complete inhibition was observed at 50 micrometer. Palmityl-CoA inhibition is associated with dissociation of the tetrameric enzyme into monomers, and binding of palmityl-CoA to the monomers. Glycogen-bound enzyme is also inhibited by palmityl-CoA, resulting in dissociation of the enzyme into monomers and concomitant release of the enzyme from the primer glycogen. Palmityl-CoA inhibition of the enzyme is partially reversed by the glycogen synthase activator, glucose-6-P, whereas sodium lauryl sulfate-inhibited enzyme is not reactivated by glucose-6-P. Sodium lauryl sulfate inhibition results in the dissociation of the tetramer into the monomers. Bovine serum albumin and cyclodextrin can prevent palmityl-CoA inhibition only when they are added prior to palmityl-CoA addition. The possible physiological role of palmityl-CoA in glucose homeostasis is discussed.     

Presence of an intermediate synthase form during the conversion of glycogen synthase D into synthase I in rat liver extract

When synthase D was converted into synthase I in a liver extract, it progressed through a synthase form with activity characteristics that could not be explained by a mixture of the original synthase D and the final product, synthase I. This form was distinguished by an affinity for UDP-glucose, in the absence of glucose 6-phosphate, which was intermediate between those of the two known forms.     

Some properties of a liver protein that activates glycogen synthase b

A soluble protein has been identified in rat liver that increases the activity of glycogen synthase without causing synthase b to a conversion. The effect of the activator is to increase synthase b activity in the presence of saturating amounts of UDP-Glc and Glc-6-P. The activator is heat sensitive and does not have protease activity. The effect of the activator is linearly proportional to the amount assayed to a saturable level and its effect is not mimicked by other proteins associated with the control of glycogen metabolism (e.g., phosphorylase).     

The importance of nucleoside triphosphate inhibition of liver glycogen synthase phosphatase activity

The liver glycogen particle contains constitutive glycogen-synthase phosphatase activity which is inhibited by ATP-Mg in a concentration-dependent manner within the physiological range (I0.5 = 0.1 mM). Therefore, we determined whether other nucleoside triphosphate-magnesium complexes also inhibit synthase phosphatase activity. UTP-Mg, CTP-Mg and GTP-Mg were all found to be inhibitory. The maximum inhibition was 85-90% which was greater than that for ATP-Mg. The I0.5 for UTP-Mg was comparable to that of ATP-Mg but it was greater for CTP-Mg and for GTP-Mg. At in vivo physiological concentrations, both UTP and ATP are possible inhibitors of synthase phosphatase activity. In the presence of a saturating concentration of ATP-Mg, added UTP-Mg increased the inhibition suggesting the presence of at least two distinct nucleotide binding sites. Substitution of calcium for magnesium in an ATP complex had no effect on the I0.5, but increased the maximum inhibition. The present studies also suggest that in the multistep conversion of synthase D to synthase I, ATP-Mg inhibition occurs early in the sequence. Addition of glycogen, a known inhibitor of synthase phosphatase activity, to a reaction mixture containing 3 mM ATP-Mg did not further inhibit synthase phosphatase activity when added at concentrations up to 22 mg/ml. The latter data suggest that the presence of a nucleoside triphosphate may desensitize the phosphatase to glycogen inhibition. ATP-Mg and, to a lesser extent, UTP-Mg and CTP-Mg all stimulated phosphorylase phosphatase activity but GTP-Mg did not.     

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.     

High-affinity binding of glycogen-synthase phosphatase to glycogen particles in the liver. Role of glycogen in the inhibition of synthase phosphatase by phosphorylase a.

1. Post-mitochondrial supernatants were prepared from the livers of 24 h-fasted rats. Upon centrifugation at high speed, the major part of the glycogen-synthase phosphatase activity sedimented with the microsomal fraction. However, two approaches showed that the enzyme was associated with residual glycogen rather than with vesicles of the endoplasmic reticulum. Indeed, the activity was entirely solubilized when the remaining glycogen was degraded either by glucagon treatment in vivo or by alpha-amylolysis in vitro. No evidence could be found for an association of glycogen-synthase phosphatase with the smooth endoplasmic reticulum, as isolated with the use of discontinuous sucrose gradients. 2. After solubilization by glucagon treatment in vivo, synthase phosphatase could be transferred to glycogen particles with very high affinity. Half-maximal binding occurred at a glycogen concentration of about 0.25 mg/ml, whereas glycogen synthase and phosphorylase required 1.5-2 mg/ml. 3. In gel-filtered extracts prepared from glycogen-depleted livers, the activation of glycogen synthase was not inhibited at all by phosphorylase alpha. The inhibition was restored when the liver homogenates were prepared in a glycogen-containing buffer. The effect was half-maximal at a glycogen concentration of about 0.25 mg/ml, and virtually complete at 1 mg/ml. These findings explain long-standing observations that in fasted animals the liver contains appreciable amounts of both synthase and phosphorylase in the active form.     

Mechanism of activation of liver glycogen synthase by swelling.

The mechanism linking the stimulation of liver glycogen synthesis to swelling induced either by amino acids or hypotonicity was studied in hepatocytes, in gel-filtered liver extracts, and in purified preparations of particulate glycogen to which glycogen-metabolizing enzymes are bound. High concentrations of KCl, but not of potassium glutamate, were found to inhibit glycogen synthesis in permeabilized hepatocytes. Similarly, physiological concentrations (30-50 mM) of Cl- ions were also found to inhibit synthase phosphatase in vitro, whereas 10-20 mM Cl- ions, a concentration found in swollen hepatocytes, did not inhibit synthase phosphatase. Synthase phosphatase activity was more sensitive to inhibition by Cl- ions at low (0.1%) than at high (1%) concentrations of glycogen. By contrast, 10 mM glutamate and aspartate, a concentration observed in hepatocytes incubated with glutamine or proline, stimulated synthase phosphatase in vitro. Therefore, it is proposed that the fall in intracellular Cl- concentration as well as the increase in intracellular glutamate and aspartate concentrations, that are observed in swollen hepatocytes in the presence of amino acids, are responsible, at least in part, for the stimulation of synthase phosphatase and, hence, of glycogen synthesis.     

Control of glycogen synthase activity in developing rat liver.

Fasting newborn and growing young rats, though capable of synthesizing liver glycogen when fed, are, unlike adult fasted animals, insensitive to glucocorticoid stimulation of the rate of glucose and lactate incorporation into glycogen. Hormone resistance parallels a decreased liver capability for the synthase b to a conversion reaction up to 2 days after birth, after which the b to a transformation becomes adult type in nature. A comparison of the level of glucose 6-phosphate in liver to the effect of the activator on the synthase activity from newborn rat shows that the enzyme has a greater affinity toward the activator than comparable enzyme from the adult, suggesting the presence of an intermediate metabolite-regulated form of synthase in neonatal liver.     

Threonine phosphorylation of rat liver glycogen synthase

32P-labeled glycogen synthase specifically immunoprecipitated from 32P-phosphate incubated rat hepatocytes contains, in addition to [32P] phosphoserine, significant levels of [32P] phosphothreonine (7% of the total [32P] phosphoaminoacids). When the 32P-immunoprecipitate was cleaved with CNBr, the [32P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 "in vitro" (casein kinases I and II, cAMP-dependent protein kinase and glycogen synthase kinase-3). After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the "in vivo" phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase.     

Comparative characterization of human and rat liver glycogen synthase.

Glycogen synthase from human liver was studied, and its properties were compared with those of rat liver glycogen synthase. The rat and human liver glycogen synthases were similar in their pH profile, in their kinetic constants for the substrate UDP-glucose and the activator glucose 6-phosphate, and in their elution profiles from Q-Sepharose. The apparent molecular weight of the human synthase subunit was 80,000-85,000 by gel electrophoresis, which is similar to that of the rat enzyme. In addition, antibodies to rat liver glycogen synthase recognized human liver glycogen synthase, indicating that the enzymes of these two species share antigenic determinants. However, there were significant differences between the two enzymes. In particular, the total activity of the human enzyme was higher than that of the rat. The human enzyme, purified to near homogeneity, had a specific activity of 40 U/mg protein compared with 20 U/mg protein for the rat enzyme. The active forms of the rat enzyme had greater thermal stability than those of the human enzyme, but the human enzyme was more stable on storage in various buffers. Last, amino acid analysis indicated differences between the enzymes of the two species. Since glycogen synthase is an important enzyme in liver glycogen synthesis, the characterization of this enzyme in the human will help provide insight regarding human liver glycogen synthesis.     

Particulate glycogen of mammalian liver: specificity in binding phosphorylase and glycogen synthase.

The glycogen particle - glycogen metabolizing enzyme complex was investigated to gain some understanding of its physiological significance. Fractionations of populations of particles from mouse liver were carried out utilising open column and high performance liquid chromatography, and based either on the molecular weight of the particles or the hydrophobic interactions of the glycogen-associated proteins. The activities of glycogen phosphorylase and glycogen synthase were measured in these fractions. Fractionations were of tissue in different stages of glycogen deposition or mobilization. In animals fed ad libitum, glycogen synthase was associated with the whole spectrum of molecular weights, while the glycogen phosphorylase distribution was skewed in favour of the lower molecular weight species. Under conditions of glycogen mobilization, the phosphorylase distribution changed to include all molecular weights. The hydrophobic interaction separations demonstrated that glycogen synthase binds to a specific subpopulation of particles that is a minor proportion of the total. In general, there was a direct relationship of the total amount of phosphorylase and synthase bound during periods of mobilization and deposition, respectively. Two notable exceptions were the large amounts of glucose-6-P dependent synthase present during the early period of glycogen mobilization and the high amounts of active phosphorylase appearing shortly after food withdrawal, in spite of interim glycogen deposition from presumably already ingested food.     

Rabbit liver glycogen synthase kinases. Characterization of a protein kinase (PC0.7) able to phosphorylate glycogen synthase and phosvitin

A rabbit liver protein kinase (PC0.7), able to phosphorylate glycogen synthase and phosvitin, has been extensively purified. The enzyme had apparent Mr = 170,000-190,000 as judged by gel filtration and was associated with two major polypeptide species, alpha (Mr = 43,000) and beta (Mr = 25,000). Two other polypeptides, Mr = 38,000 and Mr = 35,000, were also detected. Treatment with trypsin led to an enzyme composed only of polypeptides of Mr = 35,000 and Mr = 25,000. The beta-polypeptide underwent autophosphorylation when incubated with Mg2+ and ATP or GTP. The protein kinase was effective in utilizing both ATP and GTP as the phosphoryl donor (apparent Km values 5-11 microM and 9-19 microM, respectively). The enzyme phosphorylated phosvitin, casein, and glycogen synthase but not histone or phosphorylase and was inhibited by heparin. Phosphorylation of glycogen synthase proceeded to approximately 0.5 phosphate/subunit with little inactivation of the glycogen synthase. The phosphorylation occurred predominantly in a 21,000-dalton CNBr fragment of glycogen synthase that had been previously shown to reside toward the COOH terminus of the molecule. The liver PC0.7 appeared very similar to an analogous enzyme isolated from rabbit muscle (DePaoli-Roach, A. A., Ahmad, Z., and Roach, P. J. (1981) J. Biol. Chem. 256, 8955-8962). The present work, therefore, provides a point of contact between the Ca2+ and cyclic nucleotide-independent glycogen synthase kinases of rabbit liver and muscle.     

Evidence for the coordinate control of activity of liver glycogen synthase and phosphorylase by a single protein phosphatase.

Homogeneous rabbit liver phosphorylase phosphatase (Brandt, H., Capulong, Z. L., and Lee, E. Y. C. (1975) J. Biol. Chem. 250, 8038-8044) also dephosphorylates glycogen synthase b. During purification, phosphorylase phosphatase and glycogen synthase phosphatase co-purified with a constant ratio of activities. The two activities co-migrated on disc gel electrophoresis. Both substrates competed with each other for the phosphatase, and both phosphatase activities were inhibited by lysine ethyl ester. It is concluded that liver phosphorylase phosphatase and glycogen synthase phosphatase have a common identity and that coordinate regulation of the phosphatase-catalyzed activation of glycogen synthase and inactivation of phosphorylase occurs in vivo. This provides a parallel and opposing mechanism to that mediated by adenosine 3':5'-monophosphate-dependent protein kinase, which coordinately inactivates glycogen synthase and, via phosphorylase kinase, activates phosphorylase. Maximal glycogen synthase phosphatase activity was observed near neutrality. Mg2+ and glucose-6-P activated the glycogen synthase phosphatase reaction and this activation was pH-dependent. The Km for glycogen synthase b was 0.12 muM.     

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.     

Phosphorylation of rat liver glycogen synthase bound to the glycogen particle

Rat liver glycogen synthase bound to the glycogen particle was partially purified by repeated high-speed centrifugation. This synthase preparation was labeled with ³²P by incubations with cAMP-dependent protein kinase and cAMP-independent synthase (casein) kinase-1 in the presence of [γ-³²P]ATP. The phosphorylated synthase was separated from other proteins in the glycogen pellet by immunoprecipitation with rabbit anti-rat liver glycogen synthase serum. Analysis of the immunoprecipitates by sodium dodecyl sulfate-gel electrophoresis showed that synthase subunits of M_r 85,000 and 80,000 were present in varying proportions. The ³²P-labeled synthase in the immunoprecipitate was digested with trypsin, and the resulting peptides were analyzed by isoelectric focusing. Synthase bound to the glycogen particle was phosphorylated by cAMP-dependent protein kinase at more sites and by cAMP-independent synthase (casein) kinase-1 at less sites than when the homogeneous synthase was incubated with these kinases. Phosphorylation of synthase in the glycogen pellet by either cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1 did not cause a significant inactivation as has been observed when the homogeneous synthase was incubated with these kinases. Inactivation of synthase in the glycogen pellet, however, can be achieved by the combination of both kinases. This inactivation appears to result from the phosphorylation of a new site by cAMP-independent synthase (casein) kinase-1 neighboring a site previously phosphorylated by cAMP-dependent protein kinase.     

Acute selective glycogen synthase kinase-3 inhibition enhances insulin signaling in prediabetic insulin-resistant rat skeletal muscle

Glycogen synthase kinase-3 (GSK3) has been implicated in the multifactorial etiology of skeletal muscle insulin resistance in animal models and in human type 2 diabetic subjects. However, the potential molecular mechanisms involved are not yet fully understood. Therefore, we determined if selective GSK3 inhibition in vitro leads to an improvement in insulin action on glucose transport activity in isolated skeletal muscle of insulin-resistant, prediabetic obese Zucker rats and if these effects of GSK3 inhibition are associated with enhanced insulin signaling. Type I soleus and type IIb epitrochlearis muscles from female obese Zucker rats were incubated in the absence or presence of a selective, small organic GSK3 inhibitor (1 microM CT118637, Ki < 10 nM for GSK3alpha and GSK3beta). Maximal insulin stimulation (5 mU/ml) of glucose transport activity, glycogen synthase activity, and selected insulin-signaling factors [tyrosine phosphorylation of insulin receptor (IR) and IRS-1, IRS-1 associated with p85 subunit of phosphatidylinositol 3-kinase, and serine phosphorylation of Akt and GSK3] were assessed. GSK3 inhibition enhanced (P <0.05) basal glycogen synthase activity and insulin-stimulated glucose transport in obese epitrochlearis (81 and 24%) and soleus (108 and 20%) muscles. GSK3 inhibition did not modify insulin-stimulated tyrosine phosphorylation of IR beta-subunit in either muscle type. However, in obese soleus, GSK3 inhibition enhanced (all P < 0.05) insulin-stimulated IRS-1 tyrosine phosphorylation (45%), IRS-1-associated p85 (72%), Akt1/2 serine phosphorylation (30%), and GSK3beta serine phosphorylation (39%). Substantially smaller GSK3 inhibitor-mediated enhancements of insulin action on these insulin signaling factors were observed in obese epitrochlearis. These results indicate that selective GSK3 inhibition enhances insulin action in insulin-resistant skeletal muscle of the prediabetic obese Zucker rat, at least in part by relieving the deleterious effects of GSK3 action on post-IR insulin signaling. These effects of GSK3 inhibition on insulin action are greater in type I muscle than in type IIb muscle from these insulin-resistant animals.     

Multiple phosphorylation sites of rat liver glycogen synthase

Rat liver glycogen synthase was purified to homogeneity by an improved procedure that yielded enzyme almost exclusively as a polypeptide of Mr 85,000. The phosphorylation of this enzyme by eight protein kinases was analyzed by cleavage of the enzyme subunit followed by mapping of the phosphopeptides using polyacrylamide gel electrophoresis in the presence of SDS, reverse-phase high-performance liquid chromatography and thin-layer electrophoresis. Cyclic AMP-dependent protein kinase, phosphorylase kinase, protein kinase C and the calmodulin-dependent protein kinase all phosphorylated the same small peptide (approx. 20 amino acids) located in a 14 kDa CNBr-fragment (CB-1). Calmodulin-dependent protein kinase and protein kinase C also modified second sites in CB-1. A larger CNBr-fragment (CB-2) of approx. 28 kDa was the dominant site of action for casein kinases I and II, FA/GSK-3 and the heparin-activated protein kinase. The sites modified were all localized in a 14 kDa species generated by trypsin digestion. Further proteolysis with V8 proteinase indicated that FA/GSK-3 and the heparin-activated enzyme recognized the same smaller peptide within CB-2, which may also be phosphorylated by casein kinase 1. Casein kinase 1 also modified a distinct peptide, as did casein kinase II. The results lead us to suggest homology to the muscle enzyme with regard to CB-1 phosphorylation and the region recognized by FA/GSK-3, which in rabbit muscle is characterized by a high density of proline and serine residues. A striking difference with the muscle isozyme is the apparent lack of phosphorylations corresponding to the muscle sites 1a and 1b. These results provide further evidence for the presence of liver- and muscle-specific glycogen synthase isozymes in the rat. That the isozymes differ subtly as to phosphorylation sites may provide a clue to the functional differences between the isozymes.