Quantitation of rate control in the rat muscle glycogen synthesis pathway by in vivo NMR spectroscopy

The rate of the muscle glycogen synthesis pathway determines the rate of glucose disposal in humans. The aim of the studies presented in this dissertation is the quantitation of flux control in the glycogen synthesis pathway in rats during insulin stimulation. In vivo nuclear magnetic resonance spectroscopy (NMR) was used to determine the pathway flux and metabolite levels. These values were interpreted within the established framework of metabolic control analysis (MCA) to quantitate flux control by the glucose transport step.; An increase in the plasma glucose concentration ( (glucose) {dollar}sb{lcub}rm plasma{rcub}){dollar} of anesthetized rats did not affect the velocity maximum of any of the steps in the pathway. The (glucose) {dollar}sb{lcub}rm plasma{rcub}{dollar} was increased from 5.45 {dollar}pm{dollar} 0.05mM (1hr) to 13.8 {dollar}pm{dollar} 0.14mM (1hr) while NMR spectra were continuously acquired. This increase in (glucose) {dollar}sb{lcub}rm plasma{rcub}{dollar} did not affect the phosphorylation state (activity) of glycogen synthase (GSase) nor did it affect the energy state of the cell.; The pathway flux control may be calculated from the changes in glycogen synthesis that result from the isolated change in (glucose) {dollar}sb{lcub}rm plasma{rcub}{dollar}. The fractional change in the glycogen synthesis rate is 1.9 {dollar}pm{dollar} 0.2. According to MCA, the control coefficient for glucose transport (C{dollar}sbsp{lcub}rm GT{rcub}{lcub}rm J{rcub}{dollar}) is the ratio of fractional changes in glucose transport to glycogen synthesis, calculated to be 1.2 {dollar}pm{dollar} 0.1. This value indicates that glucose transport is quantitatively flux controlling for glycogen synthesis and those changes in the GSase activity exert little control. These conclusions are corroborated by the relative sensitivities of GSase and another pathway enzyme, hexokinase, to levels of the intermediate metabolite glucose-6-phosphate (G6P): {dollar}rm Csbsp{lcub}GT{rcub}{lcub}J{rcub}/Csbsp{lcub}GSase{rcub}{lcub}J{rcub}{dollar} = 2.; Two main implications of this first quantitation of in vivo flux control are that: (1) it is more probable that the flaw in muscle glycogen synthesis in type II diabetes is in glucose transport, the rate controlling step, rather than GSase; (2) the well-characterized coordination of GSase phosphorylation state with glycogen synthesis flux must not serve to determine the flux, since GSase is not rate controlling. GSase phosphorylation, which is shown in textbooks as a model for pathway regulation by phosphorylation, may instead be serving to regulate the steady-state levels of G6P.