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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...

Dehydroepiandrosterone (DHEA), an intermediate in the biosynthesis of testosterone and estrogens, exerts several physiological effects not involving the sex hormones. When fed to rats it induces the thermogenic enzymes mitochondrial sn-glycerol-3-phosphate dehydrogenase and cytosolic malic enzyme in their livers. Animals and humans, and their excised tissues, are known to hydroxylate DHEA at several positions and to interconvert 7a-hydroxy-DHEA, 7,8-hydroxy-DHEA, 7-oxo-DHEA, and the corresponding derivatives of androst-5-enediol. We report here that these 7-oxygenated derivatives are active inducers of these thermogenic enzymes in rats and that the 7-oxo derivatives are more active than the parent steroids. We postulate that the 7cr-hydroxy and 7-oxo derivatives are on a metabolic pathway from DHEA to more active steroid hormones. These 7-oxo steroids have potential as therapeutic agents because of their increased activity and because they are not convertible to either testosterone or estrogens.We wish to report the biological activity of some known and some newly discovered steroids structurally related to dehydroepiandrosterone (androst-5-ene-313-ol-17-one; DHEA). DHEA is produced in the adrenals and brain and is the most abundant steroid in the blood of adult humans; it reaches a maximum concentration at 20-25 years of age and declines thereafter. It circulates mainly as the sulfate ester but the ester and free steroid are metabolically interconverted (1, 2). DHEA is an intermediate in the metabolic conversion of cholesterol to testosterone and estrogens and it also exerts several physiological effects independent of the sex hormones. In relatively large doses, it causes weight loss in genetically obese (3) and normal (4) animals without affecting food intake; it depresses blood cholesterol levels in men (5), rats (6), and dogs (7); it decreases blood sugar concentration in diabetic mice (8); it enhances resistance of mice to viral infections (9, 10); it reduces the incidence of spontaneous (11, 12) and carcinogen-induced (13) tumors in mice; and it improves memory in aged mice (14). Body weight responses in humans treated with DHEA are questionable (5,(15)(16)(17)(18)(19).The diversity of the responses to DHEA and the large doses (-0.5% of the diet for animals) required to elicit most of them led to the postulate that DHEA might be the precursor of steroid hormones other than the sex hormones and that these metabolites might be active in smaller doses and might display specificity for one, or only a few, of the effects described above.We therefore synthesized known and possible metabolites of DHEA in the hope of finding compounds with greater biological activity. Another objective was to find metabolites of DHEA that retained the activities of the parent except for the ability to form androgens and estrogens. DHEA should not be used by women for extended periods because it increases their circulating testosterone and dihydrotestosterone manyfoldThe publication costs of this article were defrayed in p...

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L-Serine metabolism in rat liver was investigated, focusing on the relative contributions of the three pathways, one initiated by L-serine dehydratase (SDH), another by serine: pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Because serine hydroxymethyltransferase is responsible for the interconversion between serine and glycine, SDH, SPT/AGT, and GCS were considered to be the metabolic exits of the serine-glycine pool. In vitro, flux through SDH was predominant in both 24-h starved and glucagontreated rats. Flux through SPT/AGT was enhanced by glucagon administration, but even after the induction, its contribution under quasi-physiological conditions (1 mM L-serine and 0.25 mM pyruvate) was about 1 ⁄10 of that through SDH. Flux through GCS accounted for only several percent of the amount of L-serine metabolized. Relative contributions of SDH and SPT/AGT to gluconeogenesis from L-serine were evaluated in vivo based on the principle that 3 H at the 3 position of L-serine is mostly removed in the SDH pathway, whereas it is largely retained in the SPT/AGT pathway. The results showed that SPT/AGT contributed only 10-20% even after the enhancement of its activity by glucagon. These results suggested that SDH is the major metabolic exit of L-serine in rat liver.

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For isolated rat hepatocytes, glucagon, The presence in liver of both phosphofructokinase (EC 2.7.1.11) and fructose diphosphatase (EC 3.1.3.11) provides for the possibility of a substrate cycle and the apparent futile hydrolysis of ATP. Until recently, it was considered that the reactions of glycolysis and gluconeogenesis were mutually exclusive and that minimal flux occurred through phosphofructokinase during periods of gluconeogenesis in liver. In recent studies in this laboratory significant rates of flux through phosphofructokinase were observed concomitant with gluconeogenesis (1) and these rates were directly proportional to the extracellular glucose concentration. Thus, at physiologically normal glucose concentrations (i.e., 5.5 mM), the rate of flux through phosphofructokinase varied from 4 to 36% of the flux rate through fructose diphosphatase, depending upon the nature and concentration of the gluconeogenic substrate.Furthermore, it was found that glucagon and cAMP stimulate glucose formation from galactose, dihydroxyacetone, xylitol, and fructose by influencing the rate of reactions involving fructose 6-phosphate (1). Both agents were found to inhibit flux through phosphofructokinase and simultaneously to enhance flux through fructose diphosphatase.The identification of a glucagon-mediated site of regulation of hepatic gluconeogenesis between triose phosphate and glucose (1) supports earlier speculation from this laboratory (2-4) and others (5-9) that a control site occurs in this region. In addition, the identification of a control site at phosphofructokinase-fructose diphosphatase may explain observations by Haugaard and Haugaard (10), repeated and confirmed recently by Tepperman and Tepperman (11), that glucagon specifically acts to inhibit incorporation of carbon from glucose, but not fructose, into fatty acids in rat liver slices.In the present work further regulatory control at the phosphofructokinase-fructose diphosphatase site is examined.Both epinephrine and cGMP are effective stimulants of gluconeogenesis and appear to act in a manner similar to that previously described for glucagon and cAMIP (1

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