Metabolic Impact of Adenovirus-mediated Overexpression of the Glucose-6-phosphatase Catalytic Subunit in Hepatocytes

Abstract

Glucose-6-phosphatase (G6Pase) catalyzes the hydrolysis of glucose 6-phosphate (Glu-6-P) to free glucose and, as the last step in gluconeogenesis and glycogenolysis in liver, is thought to play an important role in glucose homeostasis. G6Pase activity appears to be conferred by a set of proteins localized to the endoplasmic reticulum, including a glucose-6-phosphate translocase, a G6Pase phosphohydrolase or catalytic subunit, and glucose and inorganic phosphate transporters in the endoplasmic reticulum membrane. In the current study, we used a recombinant adenovirus containing the cDNA encoding the G6Pase catalytic subunit (AdCMV-G6Pase) to evaluate the metabolic impact of overexpression of the enzyme in primary hepatocytes. We found that AdCMVG6Pase-treated liver cells contain significantly less glycogen and Glu-6-P, but unchanged UDP-glucose levels, relative to control cells. Further, the glycogen synthase activity state was closely correlated with Glu-6-P levels over a wide range of glucose concentrations in both G6Pase-overexpressing and control cells. The reduction in glycogen synthesis in AdCMV-G6Pase-treated hepatocytes is therefore not a function of decreased substrate availability but rather occurs because of the regulatory effects of Glu-6-P on glycogen synthase activity. We also found that AdCMV-G6Pase-treated-cells had significantly lower rates of lactate production and [3-3 H]glucose usage, coupled with enhanced rates of gluconeogenesis and Glu-6-P hydrolysis. We conclude that overexpression of the G6Pase catalytic subunit alone is sufficient to activate flux through the G6Pase system in liver cells. Further, hepatocytes treated with AdCMVG6Pase exhibit a metabolic profile resembling that of liver cells from patients or animals with non-insulin-dependent diabetes mellitus, suggesting that dysregulation of the catalytic subunit of G6Pase could contribute to the etiology of the disease.

Diabetes Affects Similarly the Catalytic Subunit and Putative Glucose-6-phosphate Translocase of Glucose-6-phosphatase

“…Consistent with this idea, a potent inhibitor of p46, chlorogenic acid (22), is currently being studied as a pharmaceutical tool to inhibit hepatic glucose production in diabetes (23). Overexpression of p36 in primary hepatocytes with a recombinant adenovirus (24) resulted in increased flux through G6Pase, suggesting that an increase in the catalytic subunit alone is sufficient to activate G6Pase in the intact cell. The mechanism(s) by which overexpression of p46 might affect p36 catalytic activity is however not known and deserves further studies.…”

Glucose 6-Phosphate Produced by Gluconeogenesis and by Glucokinase Is Equally Effective in Activating Hepatic Glycogen Synthase

“…The intracellular concentration of Glc-6-P was measured using a spectrophotometric assay (20). Glucose concentration in the incubation medium was measured as described (21).…”

Protein Kinase A Phosphorylates Hepatocyte Nuclear Factor-6 and Stimulates Glucose-6-phosphatase Catalytic Subunit Gene Transcription

“…Furthermore, a modest overexpression of G6Pase in rats, again using recombinant adenovirus, resulted in approximately a 1.6 -3-fold increase in hepatic G6Pase enzymatic activity that was associated with glucose intolerance, hyperinsulinemia, decreased hepatic glycogen content, and increased peripheral triglyceride stores, changes similar to those found in early stage type 2 diabetic patients (17). These observations (16,17) suggest that G6Pase is a major control point in the glucose-6-phosphatase system and represents a prime therapeutic target for the treatment of diabetes.…”

“…Several lines of evidence suggest that increased expression of key gluconeogenic enzymes including G6Pase contribute to this increase in hepatic glucose production. Thus, hepatic G6Pase expression is markedly elevated in various diabetic animal models (11)(12)(13)(14)(15), and overexpression of G6Pase in hepatocytes using recombinant adenovirus was associated with enhanced rates of gluconeogenesis as well as defects in glycogen metabolism (16). Furthermore, a modest overexpression of G6Pase in rats, again using recombinant adenovirus, resulted in approximately a 1.6 -3-fold increase in hepatic G6Pase enzymatic activity that was associated with glucose intolerance, hyperinsulinemia, decreased hepatic glycogen content, and increased peripheral triglyceride stores, changes similar to those found in early stage type 2 diabetic patients (17).…”