Inhibition by 2,5-anhydromannitol of glycolysis in isolated rat hepatocytes and in Ehrlich ascites cells.

Abstract

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...

Inhibition of glycolysis by 5-amino-4-imidazolecarboxamide riboside in isolated rat hepatocytes

“…Inhibition of glycolysis [19], associated with a decrease in Fru-2,6-P2 [20], has also been reported upon addition of the fructose analogue 2,5-anhydro-D-mannitol to isolated hepatocytes. The decrease in Fru-2,6-P2 is explained by inhibition of PFK 2 by 2,5anhydro-D-mannitol 1-phosphate formed from 2,5-anhydro-Dmannitol [21].…”

“…The decrease in Fru-2,6-P2 is explained by inhibition of PFK 2 by 2,5anhydro-D-mannitol 1-phosphate formed from 2,5-anhydro-Dmannitol [21]. The AICAriboside-induced inhibition of glycolysis differs in several respects from that induced by 2,5-anhydro-Dmannitol: the latter does not influence the activity of glucokinase [19], stimulates that of pyruvate kinase [22], and is less potent, since it maximally decreases the total production of lactate in the presence of 15 mM-glucose by less than 50% [19], as compared with over 90 % inhibition at 100 ,tM-AICAriboside (Fig. 1, upper curve).…”

Impaired glycogen synthesis in hepatocytes from Zucker fatty fa/fa rats: the role of increased phosphorylase activity

“…There is either partitioning of G6P derived from glucose to glycogen synthesis when glycolysis is inhibited or in the presence of increased gluconeogenesis, as would occur with palmitate and gluconeogenic substrates, G6P derived from gluconeogenesis activates glycogen synthase. To eliminate the latter possibility we used 2,5-anhydromannitol which is a potent inhibitor of both glycolysis [30] and gluconeogenesis [31,32]. It also inhibits glycogenolysis [32] but with a lower potency than for glycolysis and has no effect on glycogenolysis at 100 mmol/l (S. Aiston, L. Agius, unpublished results).…”

An energy sensor for control of energy intake

“…However, unlike fructose, the analogue is not further metabolized through glycolysis (see Riquelme et al 1985) and therefore, inorganic phosphate (Pi) is trapped in the phosphorylated forms of 2,5-AM. The depletion of Pi apparently compromises ATP synthesis and enhances the degradation of adenosine nucleotides through disinhibition of deaminase activity (see .…”