We investigated the impact of GLUT2 gene inactivation on the regulation of hepatic glucose metabolism during the fed to fast transition. In control and GLUT2-null mice, fasting was accompanied by a ϳ10-fold increase in plasma glucagon to insulin ratio, a similar activation of liver glycogen phosphorylase and inhibition of glycogen synthase and the same elevation in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. In GLUT2-null mice, mobilization of glycogen stores was, however, strongly impaired. This was correlated with glucose-6-phosphate (G6P) levels, which remained at the fed values, indicating an important allosteric stimulation of glycogen synthase by G6P. These G6P levels were also accompanied by a paradoxical elevation of the mRNAs for L-pyruvate kinase. Reexpression of GLUT2 in liver corrected the abnormal regulation of glycogen and L-pyruvate kinase gene expression. Interestingly, GLUT2-null livers were hyperplasic, as revealed by a 40% increase in liver mass and 30% increase in liver DNA content. Together, these data indicate that in the absence of GLUT2, the G6P levels cannot decrease during a fasting period. This may be due to neosynthesized glucose entering the cytosol, being unable to diffuse into the extracellular space, and being phosphorylated back to G6P. Because hepatic glucose production is nevertheless quantitatively normal, glucose produced in the endoplasmic reticulum may also be exported out of the cell through an alternative, membrane traffic-based pathway, as previously reported (Guillam, M.-T., Burcelin, R., and Thorens, B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12317-12321). Therefore, in fasting, GLUT2 is not required for quantitative normal glucose output but is necessary to equilibrate cytosolic glucose with the extracellular space. In the absence of this equilibration, the control of hepatic glucose metabolism by G6P is dominant over that by plasma hormone concentrations.Hepatic glucose production is an essential physiological function required to prevent hypoglycemia in the postprandial or fasted states. The last two steps in hepatic glucose release consist of the hydrolysis of glucose-6-phosphate (G6P) 1 into glucose and release of glucose from the cells. Hydrolysis of G6P is catalyzed by glucose-6-phosphatase, an enzyme located in the lumen of the endoplasmic reticulum (1). Classically, the pathway for glucose release has been viewed as involving glucose diffusion back into the cytosol through an as yet uncharacterized glucose carrier and release in the extracellular space by transport through the plasma membrane glucose transporter GLUT2 (2). Recently, however, we described the existence of an alternative pathway for glucose release, which coexists in normal hepatocytes with the GLUT2-dependent pathway (3). We showed that in the absence of GLUT2, although facilitated diffusion of glucose across the hepatocyte plasma membrane was decreased by greater than 95%, the rate of hepatic glucose production was normal and at least 10-fold higher than the remaining...