ABSTRACT2,5-Anhydromannitol decreases lactate formation and 3H2O formation from [5-3H]glucose in isolated rat hepatocytes metabolizing high concentrations of glucose. The inhibition of glycolysis is accompanied by a slight decrease in the cellular content of fructose-6-P and a more substantial decrease in the cellular content of fructose-1,6-P2, with no change in the content of glucose-6-P. The 3H20 release data and changes in hexosephosphate distribution indicate possible inhibitions at phosphofructokinase-1 and phosphoglucose isomerase. 2,5-Anhydromannitol also inhibits glycolysis in Ehrlich ascites cells, but the tumor cells, unlike hepatocytes, must be treated with 2,5-anhydromannitol prior to exposure to glucose to obtain the inhibition. The decrease in 3H20 formation from [5-3H]glucose and the metabolite pattern that results from the addition of low concentrations (c0.25 mM) of 2,5-anhydromannitol indicate an inhibition at phosphofructokinase-1 that cannot be attributed to a decrease in the cellular content of fructose-2,6-P2. Higher concentrations (-0.5 mM) of 2,5-anhydromannitol cause a substantial decrease in the cellular content of ATP that is accompanied by decreases in the content of glucose-6-P and fructose-6-P and transient increases in fructose-1,6-P2. In Ehrlich ascites cells, 2,5-anhydromannitol is metabolized to 2,5-anhydromannitol mono-and bisphosphate. The inhibition of glycolysis caused by 2,5-anhydromannitol decreases with time, because the phosphorylated metabolites formed during the preliminary incubation in the absence of glucose are rapidly dephosphorylated during the incubation in the presence of glucose.2,5-Anhydro-D-mannitol (2,5-AM-ol) is an analog of the 13-furanose form of D-fructose that lacks the C-2 hydroxyl and is thus locked in the furan ring structure (for structure, see ref. 1). Incubation of isolated rat hepatocytes with 2,5-AMol results in a rapid increase in 2,5-AM-ol-1-P and a slower accumulation of 2,5-AM-ol-1,6-P2 (2). Under these conditions, the intracellular concentration of fructose-2,6-P2 (Fru-2,6-P2), a potent activator of phosphofructokinase-1 (PFK-1), is decreased (1), probably because of the accumulation of 2,5-AM-ol-1-P. 2,5-AM-ol-1,6-P2 is a poor substitute for Fru-2,6-P2 as an activator of PFK-1, and at high concentrations it can act as a product inhibitor (2). Since the formation of either of the phosphorylated metabolites of 2,5-AM-ol could cause an inhibition of flux through PFK-1, the effects of 2,5-AM-ol on glycolysis in isolated rat hepatocytes and in Ehrlich ascites tumor cells were examined. A portion of this work has been presented (3).
MATERIALS AND METHODSSynthesis of 2,5-AM-ol. Unlabeled 2,5-AM-ol was synthesized and purified as described (1) Isolation and Incubation of Hepatocytes. Cells were isolated from livers of normal rats fasted 24 hr and were incubated as described in ref. 5 in the presence of 1.3 mM Ca2 .Isolation and Incubation of Ehrlich Ascites Cells. Ehrlich ascites tumor cells, originally obtained from Efraim Racker, were maintain...