Glucokinase Overexpression Restores Glucose Utilization and Storage in Cultured Hepatocytes from Male Zucker Diabetic Fatty Rats

Abstract

Zucker diabetic fatty rats develop type 2 diabetes concomitantly with peripheral insulin resistance. Hepatocytes from these rats and their control lean counterparts have been cultured, and a number of key parameters of glucose metabolism have been determined. Glucokinase activity was 4.5-fold lower in hepatocytes from diabetic rats than in hepatocytes from healthy ones. In contrast, hexokinase activity was about 2-fold higher in hepatocytes from diabetic animals than in healthy ones. Glucose-6-phosphatase activity was not significantly different. Despite the altered ratios of glucokinase to hexokinase activity, intracellular glucose 6-phosphate concentrations were similar in the two types of cells when they where incubated with 1-25 mM glucose. However, glycogen levels and glycogen synthase activity ratio were lower in hepatocytes from diabetic animals. Total pyruvate kinase activity and its activity ratio as well as fructose 2,6-bisphosphate concentration and lactate production were also lower in cells from diabetic animals. All of these data indicate that glucose metabolism is clearly impaired in hepatocytes from Zucker diabetic fatty rats.Glucokinase overexpression using adenovirus restored glucose metabolism in diabetic hepatocytes. In glucokinase-overexpressing cells, glucose 6-phosphate levels increased. Moreover, glycogen deposition was greatly enhanced due to the activation of glycogen synthase. Pyruvate kinase was also activated, and fructose-2,6-bisphosphate concentration and lactate production were increased in glucokinase-overexpressing diabetic hepatocytes. Overexpression of hexokinase I did not increase glycogen deposition. In conclusion, hepatocytes from Zucker diabetic fatty rats showed depressed glycogen and glycolytic metabolism, but glucokinase overexpression improved their glucose utilization and storage.

Defect in glucokinase translocation in Zucker diabetic fatty rats

“…Glycolytic flux is normally activated by the rise in glucose via increasing intracellular G-6-P concentration (35) and/or fructose 2,6-phosphate (F-2,6-P) by activating the 6-phosphofructo-2-kinase domain of the bifunctional enzyme (48). However, Seoane et al (54) reported that, in the physiological range of glucose concentration, the glucose-induced increase in F-2,6-P concentration and lactate production were much lower in hepatocytes isolated from 12-wk-old ZDF rats than from lean rats. Interestingly, they also showed that GK overexpression using adenovirus overcame this defect seen in the diabetic hepatocytes (54).…”

“…However, Seoane et al (54) reported that, in the physiological range of glucose concentration, the glucose-induced increase in F-2,6-P concentration and lactate production were much lower in hepatocytes isolated from 12-wk-old ZDF rats than from lean rats. Interestingly, they also showed that GK overexpression using adenovirus overcame this defect seen in the diabetic hepatocytes (54). Rossetti et al (53) demonstrated that hyperglycemia caused a marked inhibition of HGP, mainly through the suppression of glycogenolysis and the increase in GK flux in conscious rats.…”

“…They also showed that the total GK activity, which caused a half-maximal increase in glycogen synthesis from glucose, is within the physiological range of hepatic GK activity (4). Seoane et al (54) showed that impaired glycogen synthesis from glucose seen in cultured hepatocytes isolated from ZDF rats was overcome by overexpressing GK using adenovirus. Therefore a lack of increasing glycogen synthesis from plasma glucose is also suggestive of a defect in increase in GK flux.…”

Type 2 Diabetes Impairs Splanchnic Uptake of Glucose but Does Not Alter Intestinal Glucose Absorption During Enteral Glucose Feeding

“…However, the recent observation that glucokinase overexpression is sufficient to fully restore hepatocyte glucose uptake and glycogen synthesis in Zucker diabetic rats argues against this possibility (58). Increased rates of glucose-6-phosphatase activity could cause a decrease in net hepatic glucose uptake.…”

Phenotypic Correction of Diabetic Mice by Adenovirus-Mediated Glucokinase Expression

“…This observation led the authors to conclude that the GK gene delivery to a fraction of the whole body was sufficient to improve glucose disposal and provided a rationale for the development of new therapeutic strategies for treatment of diabetes (18). Furthermore, in vitro studies demonstrated that adenovirus-mediated overexpression of GK in hepatocytes from Zucker diabetic fatty rats resulted in an improvement in glucose utilization and storage (19).…”