Elucidation of the role of fructose 2,6-bisphosphate in the regulation of glucose fluxes in mice using in vivo13C NMR measurements of hepatic carbohydrate metabolism

Abstract

Fructose 2,6-bisphosphate (Fru-2,6-P 2 ) 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-P 2 in the regulation of hepatic carbohydrate metabolism in vivo, Fru-2,6-P 2 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 ¼ 6), in comparison to normal control mice (control, n ¼ 6). The rates of hepatic glycogen synthesis in the ADM and control mouse liver in vivo were measured using new advances in 13 C NMR including 3D localization in conjunction with [1-13 C]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 lmolAEg, which was higher than that of the control group of 3.7 ± 0.5 lmolAEg )1 AEh )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 ¼ 0.04). The results provide direct in vivo evidence that the effects of elevated Fru-2,6-P 2 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-P 2 levels promote reduced hepatic glucose production and lower blood glucose in diabetes mellitus.

The Role of Insulin in the Regulation of PEPCK and Gluconeogenesis In Vivo

“…During physiological hyperinsulinemia associated with refeeding in the rat, the F2,6P 2 concentration was rapidly increased, yet GNG flux to G6P was not altered. 55 Conversely, insulinmediated increases in F2,6P 2 stimulated glycolysis in mice 60 and rats. 61,62 Thus, in agreement with findings, it appears that glycolysis is far more sensitive to insulin (and F2,6P 2 ) in vivo than is GNG flux to G6P.…”

Reduction of Hepatic Glucose Production as a Therapeutic Target in the Treatment of Diabetes

“…This is a strong evidence to support the idea that glycolysis plays an important role in the regulation of HGP. In addition, the flux through PFK-1 may contribute to glycogenesis through the indirect pathway, which is evident by the synthesis of C6-labeled glycogen in the liver after C1-labeled glucose infusion [19]. C1-labeled glucose is converted to three carbon carbohydrates, which are then can be converted back to C6-glucose-6-phosphate through gluconeogenic pathway or, strictly, glucose-6-phosphategenic pathway, and thereby synthesize C6-glycogen.…”

“…However, the in vivo use of these compounds may not be efficacious. Recent studies have brought the viability of FBPase as a target for inhibition of gluconeogenesis into question [19]. This is because of the observation that FBPase activity, reflected by gluconeogenic flux, is not inhibited even under the combined hyperglycemic, hyperinsulinemic and high F-2,6-P 2 states.…”

Design of fructose-2,6-bisphosphatase inhibitors: A novel virtual screening approach

“…The F-2,6-P 2 has been found to be involved in maintaining blood glucose level by two mechanisms, i.e. (i) by inhibiting the activity of enzyme FBPase-1 and thus discontinuing the process of gluconeogenesis and (ii) by activating the enzyme PFK-1 involved in glycolysis [9]. Recent studies revealed that increased F-2,6-P 2 overcomes insulin resistance and also reduces obesity [10].…”