Fructose 2,6-bisphosphate (Fru-2,6-P2) plays an important role in the regulation of major carbohydrate fluxes as both allosteric activator and inhibitor of target enzymes. To examine the role of Fru-2,6-P2 in the regulation of hepatic carbohydrate metabolism in vivo, Fru-2,6-P2 levels were elevated in ADM mice with adenovirus-mediated overexpression of a double mutant bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (n = 6), in comparison to normal control mice (control, n = 6). The rates of hepatic glycogen synthesis in the ADM and control mouse liver in vivo were measured using new advances in 13C NMR including 3D localization in conjunction with [1-13C]glucose infusion. In addition to glycogen C1, the C6 and C2-C5 signals were measured simultaneously for the first time in vivo, which provide the basis for the estimation of direct and indirect synthesis of glycogen in the liver. The rate of label incorporation into glycogen C1 was not different between the control and ADM group, whereas the rate of label incorporation into glycogen C6 signals was in the ADM group 5.6 +/- 0.5 micro mol.g-1.h-1, which was higher than that of the control group of 3.7 +/- 0.5 micro mol.g-1.h-1 (P < 0.02). The rates of net glycogen synthesis, determined by the glycogen C2-C5 signal changes, were twofold higher in the ADM group (P = 0.04). The results provide direct in vivo evidence that the effects of elevated Fru-2,6-P2 levels in the liver include increased glycogen storage through indirect synthesis of glycogen. These observations provide a key to understanding the mechanisms by which elevated hepatic Fru-2,6-P2 levels promote reduced hepatic glucose production and lower blood glucose in diabetes mellitus.