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.     

Phosphorylation of rat liver glycogen synthase by phosphorylase kinase

Phosphorylation of rat liver glycogen synthase by rabbit skeletal muscle phosphorylase kinase results in the incorporation of approximately 0.8-1.2 mol of PO4/subunit. Analyses of the tryptic peptides by isoelectric focusing and thin layer chromatography reveal the presence of two major 32P-labeled peptides. Similar results were obtained when the synthase was phosphorylated by rat liver phosphorylase kinase. This extent of phosphorylation does not result in a significant change in the synthase activity ratio. In contrast, rabbit muscle glycogen synthase is readily inactivated by rabbit muscle phosphorylase kinase; this inactivation is further augmented by the addition of rabbit muscle cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1. Addition of cAMP-dependent protein kinase after initial phosphorylation of liver synthase with phosphorylase kinase, however, does not result in an inactivation or additional phosphorylation. The lack of additive phosphorylation under this condition appears to result from the phosphorylation of a common site by these two kinases. Partial inactivation of liver synthase can be achieved by sequential phosphorylation with phosphorylase kinase followed by synthase (casein) kinase-1. Under this assay condition, the phosphate incorporation into the synthase is additively increased and the synthase activity ratio (-glucose-6-P/+glucose-6-P) is reduced from 0.95 to 0.6. Nevertheless, if the order of the addition of these two kinases is reversed, neither additive phosphorylation nor inactivation of the synthase is observed. Prior phosphorylation of the synthase by phosphorylase kinase transforms the synthase such that it becomes a better substrate for synthase (casein) kinase-1 as evidenced by a 2- to 4-fold increase in the rate of phosphorylation. This increased rate of phosphorylation of the synthase appears to result from the rapid phosphorylation of a site neighboring that previously phosphorylated by phosphorylase kinase.     

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).     

Regulation of E. coli glycogen phosphorylase activity by HPr

Bacteria sense continuous changes in their environment and adapt metabolically to effectively compete with other organisms for limiting nutrients. One system which plays an important part in this adaptation response is the phosphoenol-pyruvate:sugar phosphotransferase system (PTS). Many proteins interact with and are regulated by PTS components in bacteria. Here we review the interaction with and allosteric regulation of Escherichia coli glycogen phosphorylase (GP) activity by the histidine phosphocarrier protein HPr, which acts as part of a phosphoryl shuttle between enzyme I and sugar-specific proteins of the PTS. HPr mediates crosstalk between PTS sugar uptake and glycogen breakdown. The evolution of the allosteric regulation of E. coli GP by HPr is compared to that of other phosphorylases.     

Stimulation of autophosphorylation of rabbit skeletal muscle phosphorylase kinase by glycogen synthase on glycogen particles

Glycogen synthase stimulated the autophosphorylation and autoactivation of phosphorylase kinase from rabbit skeletal muscle. This stimulation was additive to that by glycogen and the reaction was dependent on Ca2+. The effect by glycogen synthase was maximum within the activity ratio (the activity of enzyme without glucose-6-P divided by the activity with 10 mM glucose-6-P) of 0.3 and over 0.3 it was rather inhibitory. The results suggest that autophosphorylation of phosphorylase kinase in the presence of glycogen synthase on glycogen particles may be an important regulatory mechanism of glycogen metabolism in skeletal muscle.     

Amylin activates glycogen phosphorylase and inactivates glycogen synthase via a cAMP-independent mechanism.

Although the novel pancreatic peptide amylin has been shown to induce insulin resistance and decrease glucose uptake, the mechanism of amylin's actions is unknown. The following study evaluated the effect of amylin on glycogen metabolism in isolated soleus muscles in the presence and absence of insulin (200 microU/ml). Total glycogen, glycogen phosphorylase and glycogen synthases activities, and cAMP levels were measured. Total glycogen levels were significantly decreased by amylin (100 nM) in fed or fasted muscles under conditions of insulin stimulation. Amylin (100 nM) activated glycogen phosphorylase by as much as 100% and decreased glycogen synthase activity by over 60%, depending on the metabolic state of the muscles. These effects where comparable to those of the beta adrenergic agonist isoproterenol. A lower concentration of amylin (1 nM) did not significantly affect glycogen levels, glycogen phosphorylase, or glycogen synthase activity. Cyclic AMP levels were increased two-fold by isoproterenol but were unaffected by amylin. In conclusion, amylin induces glycogenolysis by decreasing glycogen synthesis and increasing breakdown. The effect of amylin on enzyme activity is consistent with a phosphorylation-dependent mechanism. It is likely that these events are mediated via a cAMP independent protein kinase.     

Regulation of glycogen metabolism in the liver and hepatic glycogenosis due to phosphorylase system deficiency]

Glycogen synthesis and breakdown in the liver are tightly controlled through different mechanisms. The purpose of this review is to describe some properties of the enzymes involved in the glycogen metabolism and the sequence of events by which glucose, allosteric effectors and hormones control this metabolism in the liver. Clinical, genetic and biological aspects of the phosphorylase and the phosphorylase kinase deficiencies are examined. The enzymatic analysis of the haemolysates from the patients allows discrimination of these two types of glycogenosis.     

Regulation of angiogenesis by glycogen synthase kinase-3beta

Glycogen synthase kinase-3beta (GSK3beta) plays important roles in metabolism, embryonic development, and tumorigenesis. Here we investigated the role of GSK3beta signaling in vascular biology by examining its function in endothelial cells (ECs). In EC, the regulatory phosphorylation of GSK3beta was found to be under the control of phosphoinositide 3-kinase-, MAPK-, and protein kinase A-dependent signaling pathways. The transduction of a nonphosphorylatable constitutively active mutant of GSKbeta promoted apoptosis under the conditions of prolonged serum deprivation or the disruption of cell-matrix attachments. Conversely, the transduction of catalytically inactive GSK3beta promoted EC survival under the conditions of cellular stress. Under normal cell culture conditions, the activation of GSK3beta signaling inhibited the migration of EC to vascular endothelial growth factor or basic fibroblast growth factor. Angiogenesis was inhibited by GSK3beta activation in an in vivo Matrigel plug assay, whereas the inhibition of GSK3beta signaling enhanced capillary formation. These data suggest that GSK3beta functions at the nodal point of converging signaling pathways in EC to regulate vessel growth through its control of vascular cell migration and survival.     

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.     

On the activities of glycogen phosphorylase and glycogen synthase in the liver of the rat

A procedure was developed for determination of glycogen synthase and phosphorylase activities in liver after various in vivo physiological treatments. Liver samples were obtained from anaesthetised rats by freeze-clamping in situ. Other procedures were shown to stimulate the activity of phosphorylase and depress the activity of glycogen in the liver. The direction of glycogen metabolism appears to be regulated by the relative proportions of the two enzymes, as shown by a strong positive correlation between total activities and active forms of phosphorylase and synthase. The enzyme activities responded as expected to stimuli such as insulin and glucose, which depressed phosphorylase and increased synthase activity, and glucagon, which increased phosphorylase and decreased synthase activity. In fasted animals approximately 50% of each enzyme was in the active form, which suggests the existence of a potential futile cycle for glycogen metabolism. The role for such a cycle in the regulation of glycogen synthesis and degradation is discussed.     

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.     

Regulation of the activity of rabbit skeletal muscle phosphorylase kinase by glycogen

The effects of glycogen on the non-activated and activated forms of phosphorylase kinase were studied. It was found that in the presence of glycogen the activity of non-activated kinase at pH 6.8 and 8.2 and that of the activated (in the course of phosphorylation) form are enhanced. The degree of activation depends on glycogen concentration. At saturating concentrations, this enzyme activity increases 2-3-fold; the enzyme affinity for the protein substrate, phosphorylase b, also shows an increase. The polysaccharide has no effect on the activity of phosphorylase kinase stimulated by limited proteolysis. In the presence of glycogen, the rate of autocatalytic phosphorylation of the enzyme is increased. Glycogen stabilizes the enzyme activity upon dilution. The experimental results suggest that the polysaccharide directly affects the phosphorylase kinase molecule. The maximal binding was shown to occur at the enzyme/polysaccharide ratio of 1:10 (w/w) in the presence of Ca2+ and Mg2+.