Abstract
We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mm glucose and by 2-fold at 20 mm glucose with a decrease in the concentration of glucose causing half-maximal rate (S(0.5)) from 26 to 19 mm. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states.
Highlights
We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus
CP-91149 Causes Inactivation of Phosphorylase and Sequential Activation of Synthase and Increases the Affinity of Glycogen Synthesis for Glucose—We used a potent inhibitor of liver phosphorylase a, [R-(R,S)-5-chloro-N-[3-(dimethylamino)-2-hydroxy-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide (CP-91149) [5], to determine the relation between phosphorylase activity and glycogen synthesis
Glucose caused a concentration-dependent inactivation of phosphorylase, and the inactivation by CP-91149 was additive with the effect of glucose (Fig. 2A)
Summary
Materials—CP-91149 [5] was a kind gift from Pfizer Global Research & Development, Groton Laboratories. After cell attachment (2–3 h), they were incubated for 2 h in serum-free minimal essential medium containing recombinant adenovirus encoding either muscle glycogen phosphorylase (AdCMV-MGP [15]) or liver glucokinase (AdCMV-GK [17]). Enzyme Activity Determination—For determination of glycogen synthase and phosphorylase, hepatocyte monolayers were extracted as described previously [7]. To determine the catalytic activity of the muscle isoform under physiological conditions, an assay designated “active phosphorylase assay” was used. This assay was based on the phosphorylase a assay, except that 10 mM glucose, 0.2 mM AMP, and 2 mM ATP were substituted for caffeine. Statistical analysis was performed using Student’s paired t test
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