Differential Metabolic Effects of Adenovirus-mediated Glucokinase and Hexokinase I Overexpression in Rat Primary Hepatocytes

Abstract

The first step of glucose metabolism is the phosphorylation of glucose, catalyzed by the hexokinase family of enzymes. To address the metabolic impact of increasing glucose phosphorylation capacity in liver, rat primary hepatocytes were treated with recombinant adenoviruses containing the cDNAs encoding either rat liver glucokinase (AdCMV-GKL) or rat hexokinase I (Ad-CMV-HKI). Maximal glucose phosphorylation in Ad-CMV-GKL-and AdCMV-HKI-treated hepatocytes was increased 7.1 ؎ 1.2-and 6.3 ؎ 0.8-fold, respectively, over hepatocytes treated with an adenovirus expressing ␤-galactosidase. Glucose usage (measured with 3 and 20 mM 2-[ 3 H]glucose and 5-[ 3 H]glucose) was significantly increased in AdCMV-GKL-treated cells preincubated in 1 or 25 mM glucose. Treatment of hepatocytes with Ad-CMV-HKI also caused enhanced glucose utilization, but the increases were smaller and were less apparent in cells preincubated in high (25 mM) glucose. AdCMV-GKL-treated hepatocytes incubated for 48 h in the presence of variable glucose concentrations had glycogen levels that were maximally 15.0 ؎ 0.6-fold greater than levels in corresponding control cells. AdCMV-HKItreated hepatocytes incubated under similar conditions had unchanged glycogen levels relative to controls. In AdCMV-GKL-treated cells, lactate output was increased to a maximum of 3.0 ؎ 0.4-fold (at 25 mM glucose), glucose oxidation was increased 3.5 ؎ 0.3-fold, and triglyceride production was unchanged relative to untreated cells. Among these three parameters, only lactate production was increased in AdCMV-HKI-treated cells, and then only at low glucose concentrations. We conclude that overexpression of glucokinase has potent effects on glucose storage and utilization in hepatocytes and that these effects are not matched by overexpression of hexokinase I.The hexokinases (HKs), 1 a family of four isozymes, catalyze the conversion of glucose to glucose 6-phosphate (Glc-6-P), the first committed step in glucose metabolism (1). Glucokinase (GK or HK IV) and HK I are both expressed in the pancreas and the liver but differ in their kinetic characteristics, regulation, and metabolic function (1-3). GK has an S 0.5 for glucose in the physiological range (8 -10 mM), and is thought to play a central role in the regulation of glucose-stimulated insulin secretion in the pancreas (2, 4) and the maintenance of glucose homeostasis by the liver (3). HK I has a low K m for glucose (0.1 mM) and has been ascribed roles in the regulation of basal insulin secretion in the ␤-cell (5) and glycolysis and oxidative metabolism in other tissues (1).Using recombinant adenoviruses, Becker et al. (5, 6) demonstrated differential metabolic effects of GK and HK I overexpression in isolated rat islets of Langerhans. GK overexpression had no effect on 2-or 5-[ 3 H]glucose usage, lactate accumulation, glycogen content, or glucose oxidation and minimal effects on glucose-stimulated insulin secretion. Conversely, HK I overexpression significantly increased all of these metabolic parameters except glycogen cont...

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

“…In previous reports we have shown that Glc-6-P produced by GK in cultured hepatocytes (13,28) and in FTO2B cells (14) is more effective in promoting the covalent activation of hepatic GS and the deposition of glycogen than Glc-6-P derived from the catalytic action of HK I. This result suggested that Glc-6-P is compartmentalized in at least two pools in these cells.…”

“…This second pool is also accessible to other enzymes and provides substrate for several metabolic routes. Thus, overexpression of GK enhances not only glycogen deposition, but also glycolysis, both in cultured hepatocytes (28) and in FTO2B cells (29). Glc-6-P from this pool can also be directed to hydrolysis by G6Pase, because overexpression of the catalytic subunit of this system reduces glycogen deposition and lactate production, while it increases hydrolysis of Glc-6-P (21).…”

Long-term overexpression of glucokinase in the liver of transgenic mice leads to insulin resistance

“…A previous report has shown that a short-term adenovirus-mediated six-fold-increase in GK activity lowers blood glucose concentrations at the cost of an increase in circulating triglyceride and FFA levels [3]. In contrast, a slight increase in GK activity (three-fold) had no effect on glycaemia or serum triglyceride levels [3].…”

“…A previous report has shown that a short-term adenovirus-mediated six-fold-increase in GK activity lowers blood glucose concentrations at the cost of an increase in circulating triglyceride and FFA levels [3]. In contrast, a slight increase in GK activity (three-fold) had no effect on glycaemia or serum triglyceride levels [3]. Here we showed that long-term GK overexpression leads to hyperglycaemia, hyperinsulinaemia, hepatic steatosis and increased serum triglyceride levels, resulting in glucose intolerance and lower insulin sensitivity.…”

“…Glucose transport and phosphorylation are the first steps in glucose utilization in the liver, where GK contributes to glucose disposal. Several studies in vitro have shown that GK activation is needed for glycolysis and glycogen synthesis [1,2,3,4]. Similarly, in transgenic liquid N 2 .…”