In hepatocytes isolated from fasted rats, 2,5-anhydromannitol inhibits gluconeogenesis from lactate plus pyruvate and from substrates that enter the gluconeogenic pathway as triose phosphate. This fructose analog has no effect, however, on gluconeogenesis from xylitol, a substrate that enters the pathway primarily as fructose 6-phosphate. The sensitivity of gluconeogenesis to 2,5-anhydromannitol depends on the substrate metabolized; concentrations of 2,5-anhydromannitol required for 50% inhibition increase in the order lactate plus pyruvate < dihydroxyacetone < glycerol < sorbitol < fructose. The inhibition by 2,5-anhydromannitol of gluconeogenesis from dihydroxyacetone is accompanied by an increase in lactate formation and by two distinct crossovers in gluconeogenic-glycolytic metabolite patterns i.e., increases in pyruvate concentrations with decreases in phosphoenolpyruvate and increases in fructose-1,6-bisphosphate concentrations with little change in fructose 6-phosphate. In addition, 2,5-anhydromannitol blocks the ability of glucagon to stimulate gluconeogenesis and inhibit lactate production from dihydroxyacetone. 2,5-Anhydromannitol decreases cellular fructose 2,6-bisphosphate content in hepatocytes; therefore the effects of the fructose analog are not mediated by fructose 2,6-bisphosphate, a naturally occurring allosteric regulator. 2,5-Anhydromannitol also inhibits gluconeogenesis in hepatocytes isolated from fasted diabetic rats, but higher concentrations of the analog are required.2,5-Anhydro-D-mannitol (2,5-AM-ol), an analog of P-D-fructose locked in the furan ring structure, is phosphorylated by fructokinase to form 2,5-AM-ol-l-P (1, 2).Because 2,5-AM-ol is symmetrical, the monophosphate product can be considered an analog of both fructose-i-P and fructose-6-P (3). Because of the stability of its ring structure, 2,5-AMI-ol monophosphate cannot be cleaved bv aldolase in a manner similar to that of fructose-l-P, nor can it act as a substrate for phosphoglucoisomerase and be converted to glucose-6-P in a manner similar to that of fructose-6-P. 2,5-AM-ol monophosphate is a substrate for phosphofructokinase 1 (4, 5). The resulting product, 2,5-AM-ol bisphosphate, is an analog of 8-fructose-1,6-P2 rather than a-fructose-1,6-P2 and, as such, is not hydrolyzed readily by fructose-1,6-bisphosphatase, which prefers the a anomer (6). The bisphosphate compound, thus, should accumulate within the cell. In vitro experiments have shown that 2,5-AM-ol bisphosphate is a competitive inhibitor of fructose-1,6-bisphosphatase (7,8). In view of the described findings, the potential of 2,5-AM-ol to act as a regulator of gluconeogenesis and glycolysis was examined in isolated rat hepatocytes.METHODS AND MATERIALS Synthesis of 2,5-AM-ol. 2,5-AM-ol was prepared as in ref.9 except the crystallization step was omitted. The crude 2,5-AM1-ol, in 5 mM ammonium borate (pH 9), was purified on Dowex-I-X-8 (borate) (10). The unretained material, after deionization and repeated concentration by evaporation of methanol, showe...
