The glucose-phosphorylating capacity of liver as measured by three independent assays. Implications for the mechanism of hepatic glycogen synthesis.

M Kuwajima,C B Newgard,D W Foster,J D Mcgarry

doi:10.1016/s0021-9258(19)84459-9

Abstract

In the fasted to fed transition liver glycogen derives mainly from gluconeogenic precursors. Why glucose is not used efficiently as a direct precursor of glycogen has become a controversial issue, in part because of disagreement over the question of how well liver can phosphorylate glucose under conditions prevailing postprandially. To try to resolve the matter the relative merits of two recently described assays, one spectrophotometric (A), the other isotopic (B), for monitoring rates of glucose phosphorylation in the high speed supernatant fraction of liver have been rigorously evaluated. A third method, also isotopic (C), was developed for use with unfractionated as well as fractionated liver homogenates. Using fasted rats and mice from different nutritional backgrounds the glucose-phosphorylating capacity of liver extracts was measured and compared with rates of hepatic glycogen synthesis observed during refeeding. Two of the assays (A and C) provided reliable data at all concentrations of glucose tested (5-100 mM), while method B exhibited shortcomings at lower substrate concentrations. The results clearly establish that in both rats and mice the ability of the liver to phosphorylate glucose at physiological concentrations is sufficient to support only 25-30% of postprandial glycogen synthesis. A limited capacity for glucose phosphorylation probably accounts for the fact that two-thirds of glycogen synthesized with refeeding after a fast is formed by the indirect (gluconeogenic) pathway.

Highlights

From theDepartments of $Internal Medicine and nBiochemistry, University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, Texas 75235 and §Department of Biochemistry and Biophysics, University of Californiu, Sun Francisco, School of Medicine, San Francisco, California 94143
Using fasted rats and mice from different nutritional backgrounds the glucose-phosphorylating capacity of liver extracts wams easured and compared with rates of hepatic glycogen synthesis observed during refeeding
The results clearly estabtlhisaht in both rats and mice the ability of the liver to phosphorylate glucose at physiological concentrationsis sufficient to supporotnly 25-30% of postprandial glycogen synthesis

Summary

DISCUSSION

The firstobjective of this study was to examine the relative merits of two recently described methods for measuring glucose phosphorylation in liver extracts. Under no circumstances did we find the spectrophotometric assay (A) to yield lower values thanthe isotopic procedure (B) The former method proved superior to the latterfor our purpose, which was to assess the total capacity of the tissue to phosphorylate glucose, i.e. the combined activities of hexokinase plus glucokinase, at different substrate concentrations.At low levels of glucose,where the hexokinase component contributes significantly to total phosphorylation, inhibition of hexokinase by accumulating glucose 6-phosphate introduced significanterrorsinto the isotopic assay (B). Hepatic our hands the two procedures yielded similar results at con- with theobserved agreement between the spectrophotometric centrations of glucose sufficient to saturate glucokinase, the method and an independentisotopic assay (C), gives us conprincipal enzyme forglucose phosphorylation inliver. Bothpreparations yielded similar a presumed K,,, value for glucokinase Such an indirect apvalues, indicating that results with highspeed supernatant proach may not bejustifiable when dealing with amixture of fractions provide a valid measurement of total glucose phos- two phosphorylating enzymes (hexokinase and glucokinase) phorylation capacity inwhole liver. Acknowledgments-Wearegrateful to Petra Contreras,Karen Thatcher, and Murphy Daniels for expert technical assistance

Fa7sted

Method