Optimization and Validation of an In Vitro Standardized Glycogen Phosphorylase Activity Assay

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway and a potential therapeutic target in the management of type 2 diabetes. It catalyzes a reversible reaction: the release of the terminal glucosyl residue from glycogen as glucose 1-phosphate; or the transfer of glucose from glucose 1-phosphate to glycogen. A colorimetric method to follow in vitro the activity of GP with usefulness in structure-activity relationship studies and high-throughput screening capability is herein described. The obtained results allowed the choice of the optimal concentration of enzyme of 0.38 U/mL, 0.25 mM glucose 1-phosphate, 0.25 mg/mL glycogen, and temperature of 37 °C. Three known GP inhibitors, CP-91149, a synthetic inhibitor, caffeine, an alkaloid, and ellagic acid, a polyphenol, were used to validate the method, CP-91149 being the most active inhibitor. The effect of glucose on the IC50 value of CP-91149 was also investigated, which decreased when the concentration of glucose increased. The assay parameters for a high-throughput screening method for discovery of new potential GP inhibitors were optimized and standardized, which is desirable for the reproducibility and comparison of results in the literature. The optimized method can be applied to the study of a panel of synthetic and/or natural compounds, such as polyphenols.

Chronic overeating impairs hepatic glucose uptake and disposition

Dogs consuming a hypercaloric high-fat and -fructose diet (52 and 17% of total energy, respectively) or a diet high in either fructose or fat for 4 wk exhibited blunted net hepatic glucose uptake (NHGU) and glycogen deposition in response to hyperinsulinemia, hyperglycemia, and portal glucose delivery. The effect of a hypercaloric diet containing neither fructose nor excessive fat has not been examined. Dogs with an initial weight of ≈25 kg consumed a chow and meat diet (31% protein, 44% carbohydrate, and 26% fat) in weight-maintaining (CTR; n = 6) or excessive (Hkcal; n = 7) amounts for 4 wk (cumulative weight gain 0.0 ± 0.3 and 1.5 ± 0.5 kg, respectively, P < 0.05). They then underwent clamp studies with infusions of somatostatin and intraportal insulin (4× basal) and glucagon (basal). The hepatic glucose load was doubled with peripheral (Pe) glucose infusion for 90 min (P1) and intraportal glucose at 4 mg·kg−1·min−1 plus Pe glucose for the final 90 min (P2). NHGU was blunted ( P < 0.05) in Hkcal during both periods (mg·kg−1·min−1; P1: 1.7 ± 0.2 vs. 0.3 ± 0.4; P2: 3.6 ± 0.3 vs. 2.3 ± 0.4, CTR vs. Hkcal, respectively). Terminal hepatic glucokinase catalytic activity was reduced nearly 50% in Hkcal vs. CTR ( P < 0.05), although glucokinase protein did not differ between groups. In Hkcal vs. CTR, liver glycogen was reduced 27% ( P < 0.05), with a 91% increase in glycogen phosphorylase activity ( P < 0.05) but no significant difference in glycogen synthase activity. Thus, Hkcal impaired NHGU and glycogen synthesis compared with CTR, indicating that excessive energy intake, even if the diet is balanced and nutritious, negatively impacts hepatic glucose metabolism.

Glycogen catabolism enzymes and protein fractions in the third and fourth larval stages ofAnisakis simplex

Extracts ofAnisakis simplexthird (L3) and fourth (L4) larval stages were assayed for protein content and activity and properties of α-amylase, glucoamylase and glycogen phosphorylase. Protein content in L4 was twice that in L3. SDS–PAGE applied to both larval stages revealed 22 protein fractions in each, including five stage-specific fractions in each larval stage. The L3 extracts contained three amylase isoenzymes: α1, α2 and α3; their molecular weights were 64, 29 and 21 kDa, respectively. Only one amylase isoenzyme (64 kDa) was found in the L4 extracts. Glycogen in L3 was found to be broken down mostly by hydrolysis because of low glycogen phosphorylase activity. The α-amylase activity in L4 was higher than that in L3 by half and the glycogen phosphorylase activity was ten times higher. In addition, the same enzymes isolated from L3 and L4 were found to differ in their properties. These differences could be manifestations of metabolic adaptations ofA. simplexlarvae to host switch from fish (L3) to mammals (L4), i.e. adaptations to a new habitat.

Glycogen Phosphorylase, the Product of the glgP Gene, Catalyzes Glycogen Breakdown by Removing Glucose Units from the Nonreducing Ends in Escherichia coli

ABSTRACT To understand the biological function of bacterial glycogen phosphorylase (GlgP), we have produced and characterized Escherichia coli cells with null or altered glgP expression. glgP deletion mutants (ΔglgP) totally lacked glycogen phosphorylase activity, indicating that all the enzymatic activity is dependent upon the glgP product. Moderate increases of glycogen phosphorylase activity were accompanied by marked reductions of the intracellular glycogen levels in cells cultured in the presence of glucose. In turn, both glycogen content and rates of glycogen accumulation in ΔglgP cells were severalfold higher than those of wild-type cells. These defects correlated with the presence of longer external chains in the polysaccharide accumulated by ΔglgP cells. The overall results thus show that GlgP catalyzes glycogen breakdown and affects glycogen structure by removing glucose units from the polysaccharide outer chains in E. coli.

Muscle-Specific Deletion of the Glut4 Glucose Transporter Alters Multiple Regulatory Steps in Glycogen Metabolism

ABSTRACT Mice with muscle-specific knockout of the Glut4 glucose transporter (muscle-G4KO) are insulin resistant and mildly diabetic. Here we show that despite markedly reduced glucose transport in muscle, muscle glycogen content in the fasted state is increased. We sought to determine the mechanism(s). Basal glycogen synthase activity is increased by 34% and glycogen phosphorylase activity is decreased by 17% (P < 0.05) in muscle of muscle-G4KO mice. Contraction-induced glycogen breakdown is normal. The increased glycogen synthase activity occurs in spite of decreased signaling through the insulin receptor substrate 1 (IRS-1)-phosphoinositide (PI) 3-kinase-Akt pathway and increased glycogen synthase kinase 3β (GSK3β) activity in the basal state. Hexokinase II is increased, leading to an approximately twofold increase in glucose-6-phosphate levels. In addition, the levels of two scaffolding proteins that are glycogen-targeting subunits of protein phosphatase 1 (PP1), the muscle-specific regulatory subunit (RGL) and the protein targeting to glycogen (PTG), are strikingly increased by 3.2- to 4.2-fold in muscle of muscle-G4KO mice compared to wild-type mice. The catalytic activity of PP1, which dephosphorylates and activates glycogen synthase, is also increased. This dominates over the GSK3 effects, since glycogen synthase phosphorylation on the GSK3-regulated site is decreased. Thus, the markedly reduced glucose transport in muscle results in increased glycogen synthase activity due to increased hexokinase II, glucose-6-phosphate, and RGL and PTG levels and enhanced PP1 activity. This, combined with decreased glycogen phosphorylase activity, results in increased glycogen content in muscle in the fasted state when glucose transport is reduced.