Glucose-6-phosphate transport was investigated in rat or human liver microsomal vesicles using rapid filtration and light-scattering methods. Upon addition of glucose-6-phosphate, rat liver microsomes accumulated the radioactive tracer, reaching a steady-state level of uptake. In this phase, the majority of the accumulated tracer was glucose, but a significant intraluminal glucose-6-phosphate pool could also be observed. The extent of the intravesicular glucose pool was proportional with glucose-6-phosphatase activity. The relative size of the intravesicular glucose-6-phosphate pool (irrespective of the concentration of the extravesicular concentration of added glucose-6-phosphate) expressed as the apparent intravesicular space of the hexose phosphate was inversely dependent on glucose-6-phosphatase activity. The increase of hydrolysis by elevating the extravesicular glucose-6-phosphate concentration or temperature resulted in lower apparent intravesicular glucose-6-phosphate spaces and, thus, in a higher transmembrane gradient of glucose-6-phosphate concentrations. In contrast, inhibition of glucose-6-phosphate hydrolysis by vanadate, inactivation of glucose-6-phosphatase by acidic pH, or genetically determined low or absent glucose-6-phosphatase activity in human hepatic microsomes of patients suffering from glycogen storage disease type 1a led to relatively high intravesicular glucose-6-phosphate levels. Glucose-6-phosphate transport investigated by light-scattering technique resulted in similar traces in control and vanadate-treated rat microsomes as well as in microsomes from human patients with glycogen storage disease type 1a. It is concluded that liver microsomes take up glucose-6-phosphate, constituting a pool directly accessible to intraluminal glucose-6-phosphatase activity. In addition, normal glucose-6-phosphate uptake can take place in the absence of the glucose-6-phosphatase enzyme protein, confirming the existence of separate transport proteins.

The expression of a luciferase reporter gene under the control of the human glucose 6-phosphatase gene promoter was stimulated by both dexamethasone and dibutyryl cAMP in H4IIE hepatoma cells. A cis-active element located between nucleotides -161 and -152 in the glucose 6-phosphatase gene promoter was identified and found to be necessary for both basal reporter-gene expression and induction of expression by both dibutyryl cAMP and dexamethasone. Nucleotides -161 to -152 were functionally replaced by the consensus sequence for a cAMP response element. An antibody against the cAMP response element-binding protein caused a supershift in gel-electrophoretic-mobility-shift assays using an oligonucleotide probe representing the glucose 6-phosphatase gene promoter from nucleotides -161 to -152. These results strongly indicate that in H4IIE cells the glucose 6-phosphatase gene-promoter sequence from -161 to -152 is a cAMP response element which is important for the regulation of transcription of the glucose 6-phosphatase gene by both cAMP and glucocorticoids.

The transport of glucose-6-phosphate (G6P), glucose, and orthophosphate into liver microsomes, isolated from six patients with various subtypes of type 1 glycogen storage disease (GSD), was measured using a light-scattering method. We found that G6P, glucose, and phosphate could all cross the microsomal membrane, in four cases of type 1a GSD. In contrast, liver microsomal transport of G6P and phosphate was deficient in the GSD 1b and 1c patients, respectively. These results support the involvement of multiple proteins (and genes) in GSD type 1. The results obtained with the light-scattering method are in accordance with conventional kinetic analysis of the microsomal glucose-6-phosphatase system. Therefore, this technique could be used to directly diagnose type 1b and 1c GSD.