Overexpression of the P46 (T1) translocase component of the glucose-6-phosphatase complex in hepatocytes impairs glycogen accumulation via hydrolysisof glucose 1-phosphate

The final step of gluconeogenesis and glycogenolysis is catalyzed by the gl ucose-6-phosphatase (Glc-6-Pase) enzyme complex, located in the endoplasmic reticulum, The complex consists of a 36-kDa catalytic subunit (P36), a 46- kDa glucose 6-phosphate translocase (P46), and putative glucose and inorgan ic phosphate transporters. Mutations in the genes encoding P36 or P46 have been linked:to glycogen storage diseases type Ia and type Tb, respectively. However, the relative roles of these two proteins in control of the rate o f glucose 6-phosphate hydrolysis have not been defined. To gain insight int o this area, we have constructed a recombinant adenovirus containing the cD NA encoding human P46 (AdCMV-P46) and treated rat hepatocytes with this vir us, or a virus encoding P36 (AdCMV-P36), or the combination of both viruses , resulting in large and equivalent increases in expression of the transgen es within 8-24 h of viral treatment. The overexpressed P46 protein was appr opriately targeted to hepatocyte microsomes and caused a 58% increase in gl ucose 6-phosphate hydrolysis in nondetergent-treated (intact) microsomal pr eparations relative to controls, whereas overexpression of P36 caused a 3.6 -fold increase. Overexpression of P46 caused a 50% inhibition of glycogen a ccumulation in hepatocytes from fasted rats incubated at 25 mM glucose rela tive to cells treated with a control virus (AdCMV-beta GAL), Furthermore, i n hepatocytes from fed rats cultured at 25 mM glucose and then exposed to 1 5 nM glucose, AdCMV-P46 treatment activated glycogenolysis, as indicated by a 50% reduction in glycogen content relative to AdCMV-beta GAL-treated con trols. In contrast, overexpression of P46 had only small effects on glycoly sis, whereas overexpression of P36 had large effects on both glycogen metab olism and glycolysis, even in the presence of co-overexpressed glucokinase, Finally, P46 overexpression enhanced glucose 1-phosphate but not fructose 6-phosphhate hydrolysis in intact microsomes, providing a mechanism by whic h P46 overexpression may exert its preferential effects on glycogen metabol ism.