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Inhibition of gluconeogenesis by hypoglycin in the rat. Evidence for inhibition of glucose-6-phosphatase in vivo.

ABSTRACT: Treatment of rats with hypoglycaemic doses of hypoglycin has been shown to abolish the relative detritiation of [2-3H,U-14C]glucose [Osmundsen, Billington, Taylor & Sherratt (1978) Biochem. J. 170, 337-342], indicating that both the Cori and the glucose/glucose 6-phosphate cycles were inhibited in vivo. This inhibition was confirmed and, in addition, it was shown that the conversion in vivo of both [14C]lactate and [14C]fructose into glucose was decreased after hypoglycin treatment. These results suggest that hypoglycin poisoning results in the inhibition in vivo of glucose-6-phosphatase activity, which participates in the overall inhibition of gluconeogenesis and hypoglycaemia. Clofibrate feeding apparently protected the rats against the inhibition of the fructose-to-glucose conversion by hypoglycin. However, in isolated hepatocytes prepared from hypoglycin-treated rats, the conversion of [14C]fructose into glucose and the recycling of [2-3H,U-14C]glucose were not different from that in control hepatocytes. This suggests that the inhibition was lost during preparation of the hepatocytes. The direct measurement of glucose-6-phosphatase activity showed that it was inhibited when measured in concentrated, but not dilute, homogenates prepared from hypoglycin-treated rats.

SUBMITTER: Hue L

PROVIDER: S-EPMC1147484 | biostudies-other | 1986 Dec

REPOSITORIES: biostudies-other

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Project description:Pyrophosphate (PPi) is produced by anabolic reactions and serves as an energy donor in the cytosol of plant cells; however, its accumulation to toxic levels disrupts several common biosynthetic pathways and is lethal. Before acquiring photosynthetic capacity, young seedlings must endure a short but critical heterotrophic period, during which they are nourished solely by sugar produced from seed reserves by the anabolic process of gluconeogenesis. Previously, we reported that excess PPi in H+-PPase-knockout fugu5 mutants of Arabidopsis thaliana severely compromised gluconeogenesis. However, the precise metabolic target of PPi inhibition in vivo remained elusive. Here, CE-TOF MS analyses of major metabolites characteristic of gluconeogenesis from seed lipids showed that the Glc6P;Fru6P level significantly increased and that Glc1P level was consistently somewhat higher in fugu5 compared to wild type. In contrast, the UDP-Glc level decreased significantly in the mutants. Importantly, specific removal of PPi in fugu5, and thus in AVP1pro:IPP1 transgenic lines, restored the Glc1P and the Glc6P;Fru6P levels, increased the UDP-Glc level ~2.0-fold, and subsequently increased sucrose synthesis. Given the reversible nature of the Glc1P/UDP-Glc reaction, our results indicate that UGP-Glc pyrophosphorylase is the major target when excess PPi exerts inhibitory effects in vivo. To validate our findings, we analyzed metabolite responses using a mathematical theory called structural sensitivity analysis (SSA), in which the responses of concentrations in reaction systems to perturbations in enzyme activity are determined from the structure of the network alone. A comparison of our experimental data with the results of pure structural theory predicted the existence of unknown reactions as the necessary condition for the above metabolic profiles, and confirmed the above results. Our data support the notion that H+-PPase plays a pivotal role in cytosolic PPi homeostasis in plant cells. We propose that the combination of metabolomics and SSA is powerful when seeking to identify and predict metabolic targets in living cells.

Project description:BackgruoundSodium-dependent glucose cotransporter 2 (SGLT2) mediates glucose reabsorption in the renal proximal tubules, and SGLT2 inhibitors are used as therapeutic agents for treating type 2 diabetes mellitus. This study aimed to elucidate the effects and mechanisms of SGLT2 inhibition on hepatic glucose metabolism in both serum deprivation and serum supplementation states.MethodsHuh7 cells were treated with the SGLT2 inhibitors empagliflozin and dapagliflozin to examine the effect of SGLT2 on hepatic glucose uptake. To examine the modulation of glucose metabolism by SGLT2 inhibition under serum deprivation and serum supplementation conditions, HepG2 cells were transfected with SGLT2 small interfering RNA (siRNA), cultured in serum-free Dulbecco's modified Eagle's medium for 16 hours, and then cultured in media supplemented with or without 10% fetal bovine serum for 8 hours.ResultsSGLT2 inhibitors dose-dependently decreased hepatic glucose uptake. Serum deprivation increased the expression levels of the gluconeogenesis genes peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), glucose 6-phosphatase (G6pase), and phosphoenolpyruvate carboxykinase (PEPCK), and their expression levels during serum deprivation were further increased in cells transfected with SGLT2 siRNA. SGLT2 inhibition by siRNA during serum deprivation induces nuclear localization of the transcription factor forkhead box class O 1 (FOXO1), decreases nuclear phosphorylated-AKT (p-AKT), and p-FOXO1 protein expression, and increases phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK) protein expression. However, treatment with the AMPK inhibitor, compound C, reversed the reduction in the protein expression levels of nuclear p- AKT and p-FOXO1 and decreased the protein expression levels of p-AMPK and PEPCK in cells transfected with SGLT2 siRNA during serum deprivation.ConclusionThese data show that SGLT2 mediates glucose uptake in hepatocytes and that SGLT2 inhibition during serum deprivation increases gluconeogenesis via the AMPK/AKT/FOXO1 signaling pathway.

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Inhibition of gluconeogenesis by hypoglycin in the rat. Evidence for inhibition of glucose-6-phosphatase in vivo.

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