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Vitamin C Deficiency Activates the Purine Nucleotide Cycle in Zebrafish*

Open AccessPublished:December 13, 2011DOI:https://doi.org/10.1074/jbc.M111.316018
      Vitamin C (ascorbic acid, AA) is a cofactor for many important enzymatic reactions and a powerful antioxidant. AA provides protection against oxidative stress by acting as a scavenger of reactive oxygen species, either directly or indirectly by recycling of the lipid-soluble antioxidant, α-tocopherol (vitamin E). Only a few species, including humans, guinea pigs, and zebrafish, cannot synthesize AA. Using an untargeted metabolomics approach, we examined the effects of α-tocopherol and AA deficiency on the metabolic profiles of adult zebrafish. We found that AA deficiency, compared with subsequent AA repletion, led to oxidative stress (using malondialdehyde production as an index) and to major increases in the metabolites of the purine nucleotide cycle (PNC): IMP, adenylosuccinate, and AMP. The PNC acts as a temporary purine nucleotide reservoir to keep AMP levels low during times of high ATP utilization or impaired oxidative phosphorylation. The PNC promotes ATP regeneration by converting excess AMP into IMP, thereby driving forward the myokinase reaction (2ADP → AMP + ATP). On the basis of this finding, we investigated the activity of AMP deaminase, the enzyme that irreversibly deaminates AMP to form IMP. We found a 47% increase in AMP deaminase activity in the AA-deficient zebrafish, complementary to the 44-fold increase in IMP concentration. These results suggest that vitamin C is crucial for the maintenance of cellular energy metabolism.

      Introduction

      Since the discovery of vitamin C (ascorbic acid, AA)
      The abbreviations used are: AA
      ascorbic acid
      HIF
      hypoxia-induced factor
      α-T
      α-tocopherol
      MDA
      malondialdehyde
      PCA
      principal component analysis
      DA
      discriminant analysis
      AMPD
      AMP deaminase
      GlyPhCh
      glycerophosphocholine
      AMPK
      AMP-activated protein kinase.
      and its function as an antiscorbutic micronutrient in humans, it has become increasingly evident that AA plays significant roles in disease prevention beyond scurvy (
      • Traber M.G.
      • Stevens J.F.
      Vitamins C and E. Beneficial effects from a mechanistic perspective.
      ). AA is involved in neurotransmitter biosynthesis and nitric oxide bioavailability (
      • May J.M.
      How does ascorbic acid prevent endothelial dysfunction.
      ), in gene transcription mediated by hypoxia-induced factor (HIF) (
      • Boulahbel H.
      • Durán R.V.
      • Gottlieb E.
      Prolyl hydroxylases as regulators of cell metabolism.
      ,
      • Pagé E.L.
      • Chan D.A.
      • Giaccia A.J.
      • Levine M.
      • Richard D.E.
      Hypoxia-inducible factor-1α stabilization in nonhypoxic conditions. Role of oxidation and intracellular ascorbate depletion.
      ,
      • Schofield C.J.
      • Ratcliffe P.J.
      Oxygen sensing by HIF hydroxylases.
      ), in maintenance of proper redox status by acting as an antioxidant (
      • Buettner G.R.
      • Schafer F.Q.
      Free radicals, oxidants, and antioxidants.
      ,
      • Halliwell B.
      Antioxidant defence mechanisms. From the beginning to the end (of the beginning).
      ,
      • Kalyanaraman B.
      • Darley-Usmar V.M.
      • Wood J.
      • Joseph J.
      • Parthasarathy S.
      Synergistic interaction between the probucol phenoxyl radical and ascorbic acid in inhibiting the oxidation of low density lipoprotein.
      ) and facilitating the recycling of vitamin E (α-tocopherol, α-T) (
      • Bruno R.S.
      • Leonard S.W.
      • Atkinson J.
      • Montine T.J.
      • Ramakrishnan R.
      • Bray T.M.
      • Traber M.G.
      Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation.
      ,
      • Buettner G.R.
      The pecking order of free radicals and antioxidants: lipid peroxidation, α-tocopherol, and ascorbate.
      ). One of the difficulties with investigating the functions of AA in vivo is that animals commonly used for laboratory research, unlike humans, are able to synthesize AA. Humans depend on dietary intake of AA because they lack the functional gene for gulonolactone oxidase, the enzyme responsible for conversion of gulonolactone into AA (
      • Nishikimi M.
      • Fukuyama R.
      • Minoshima S.
      • Shimizu N.
      • Yagi K.
      Cloning and chromosomal mapping of the human nonfunctional gene for L-gulono-γ-lactone oxidase, the enzyme for L-ascorbic acid biosynthesis missing in man.
      ). Gulonolactone oxidase knockout (Gulo−/−) mice have been used to study the effects of AA (
      • Harrison F.E.
      • Meredith M.E.
      • Dawes S.M.
      • Saskowski J.L.
      • May J.M.
      Low ascorbic acid and increased oxidative stress in gulo(−/−) mice during development.
      ,
      • Telang S.
      • Clem A.L.
      • Eaton J.W.
      • Chesney J.
      Depletion of ascorbic acid restricts angiogenesis and retards tumor growth in a mouse model.
      ). The zebrafish (Danio rerio) is a unique and novel model for studies of AA function. We chose zebrafish because this and other teleost species, like humans, lack a functional gulonolactone oxidase gene (
      • Moreau R.
      • Dabrowski K.
      ). Other beneficial attributes include its small size (< 1 g body weight) and short life span (
      • Dodd A.
      • Curtis P.M.
      • Williams L.C.
      • Love D.R.
      Zebrafish. Bridging the gap between development and disease.
      ,
      • Wixon J.
      Featured organism. Danio rerio, the zebrafish.
      ,
      • Udvadia A.J.
      • Linney E.
      Windows into development. Historic, current, and future perspectives on transgenic zebrafish.
      ), as well as enabling assessment of integrative, whole animal effects. We hypothesized that specific metabolic pathways are at risk if AA and/or α-T are not sufficiently available. Our approach was to let our biological system respond to the insult (AA or α-T deficiency) and then pursue the underlying mechanism by exploiting the advantages of the zebrafish model. The objective of this study was to determine how and to what extent vitamin C and vitamin E deficiency affect metabolism in zebrafish. We quantified the impact of vitamin C and E status on the zebrafish metabolome by high-resolution tandem mass spectrometry using an untargeted metabolomics approach. Here we report the changes in metabolic profiles in relation to vitamin status and show that AA deficiency elicits stress responses in zebrafish that resemble oxidative stress responses in laboratory rodents and in humans. Our findings suggest that zebrafish are an appropriate model for studying the effects of vitamin C on metabolism.

      REFERENCES

        • Traber M.G.
        • Stevens J.F.
        Vitamins C and E. Beneficial effects from a mechanistic perspective.
        Free Radic. Biol. Med. 2011; 51: 1000-1013
        • May J.M.
        How does ascorbic acid prevent endothelial dysfunction.
        Free Radic. Biol. Med. 2000; 28: 1421-1429
        • Boulahbel H.
        • Durán R.V.
        • Gottlieb E.
        Prolyl hydroxylases as regulators of cell metabolism.
        Biochem. Soc. Trans. 2009; 37: 291-294
        • Pagé E.L.
        • Chan D.A.
        • Giaccia A.J.
        • Levine M.
        • Richard D.E.
        Hypoxia-inducible factor-1α stabilization in nonhypoxic conditions. Role of oxidation and intracellular ascorbate depletion.
        Mol. Biol. Cell. 2008; 19: 86-94
        • Schofield C.J.
        • Ratcliffe P.J.
        Oxygen sensing by HIF hydroxylases.
        Nat. Rev. Mol. Cell Biol. 2004; 5: 343-354
        • Buettner G.R.
        • Schafer F.Q.
        Free radicals, oxidants, and antioxidants.
        Teratology. 2000; 62: 234
        • Halliwell B.
        Antioxidant defence mechanisms. From the beginning to the end (of the beginning).
        Free Radic. Res. 1999; 31: 261-272
        • Kalyanaraman B.
        • Darley-Usmar V.M.
        • Wood J.
        • Joseph J.
        • Parthasarathy S.
        Synergistic interaction between the probucol phenoxyl radical and ascorbic acid in inhibiting the oxidation of low density lipoprotein.
        J. Biol. Chem. 1992; 267: 6789-6795
        • Bruno R.S.
        • Leonard S.W.
        • Atkinson J.
        • Montine T.J.
        • Ramakrishnan R.
        • Bray T.M.
        • Traber M.G.
        Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation.
        Free Radic. Biol. Med. 2006; 40: 689-697
        • Buettner G.R.
        The pecking order of free radicals and antioxidants: lipid peroxidation, α-tocopherol, and ascorbate.
        Arch. Biochem. Biophys. 1993; 300: 535-543
        • Nishikimi M.
        • Fukuyama R.
        • Minoshima S.
        • Shimizu N.
        • Yagi K.
        Cloning and chromosomal mapping of the human nonfunctional gene for L-gulono-γ-lactone oxidase, the enzyme for L-ascorbic acid biosynthesis missing in man.
        J. Biol. Chem. 1994; 269: 13685-13688
        • Harrison F.E.
        • Meredith M.E.
        • Dawes S.M.
        • Saskowski J.L.
        • May J.M.
        Low ascorbic acid and increased oxidative stress in gulo(−/−) mice during development.
        Brain Res. 2010; 1349: 143-152
        • Telang S.
        • Clem A.L.
        • Eaton J.W.
        • Chesney J.
        Depletion of ascorbic acid restricts angiogenesis and retards tumor growth in a mouse model.
        Neoplasia. 2007; 9: 47-56
        • Moreau R.
        • Dabrowski K.
        Dabrowski K. Ascorbic Acid in Aquatic Organisms: Status and Perspectives. CRC Press, Boca Raton, Florida2001: 13-32
        • Dodd A.
        • Curtis P.M.
        • Williams L.C.
        • Love D.R.
        Zebrafish. Bridging the gap between development and disease.
        Hum. Mol. Genet. 2000; 9: 2443-2449
        • Wixon J.
        Featured organism. Danio rerio, the zebrafish.
        Yeast. 2000; 17: 225-231
        • Udvadia A.J.
        • Linney E.
        Windows into development. Historic, current, and future perspectives on transgenic zebrafish.
        Dev. Biol. 2003; 256: 1-17
        • Miller G.W.
        • Labut E.M.
        • Lebold K.M.
        • Floeter A.
        • Tanguay R.L.
        • Traber M.G.
        J. Nutr. Biochem. 2011; (in press)
        • Lebold K.M.
        • Jump D.B.
        • Miller G.W.
        • Wright C.L.
        • Labut E.M.
        • Barton C.L.
        • Tanguay R.L.
        • Traber M.G.
        Vitamin E deficiency decreases long-chain PUFA in zebrafish (Danio rerio).
        J. Nutr. 2011; 141: 2113-2118
        • Podda M.
        • Weber C.
        • Traber M.G.
        • Packer L.
        Simultaneous determination of tissue tocopherols, tocotrienols, ubiquinols, and ubiquinones.
        J. Lipid Res. 1996; 37: 893-901
        • Frei B.
        • England L.
        • Ames B.N.
        Ascorbate is an outstanding antioxidant in human blood plasma.
        Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 6377-6381
        • Hong Y.L.
        • Yeh S.L.
        • Chang C.Y.
        • Hu M.L.
        Total plasma malondialdehyde levels in 16 Taiwanese college students determined by various thiobarbituric acid tests and an improved high-performance liquid chromatography-based method.
        Clin. Biochem. 2000; 33: 619-625
        • Young I.S.
        • Trimble E.R.
        Measurement of malondialdehyde in plasma by high performance liquid chromatography with fluorimetric detection.
        Ann. Clin. Biochem. 1991; 28: 504-508
        • Lushchak V.I.
        • Husak V.V.
        • Storey J.M.
        • Storey K.B.
        AMP-deaminase from goldfish white muscle. Regulatory properties and redistribution under exposure to high environmental oxygen level.
        Fish Physiol. Biochem. 2009; 35: 443-452
        • Sims B.
        • Powers R.E.
        • Sabina R.L.
        • Theibert A.B.
        Elevated adenosine monophosphate deaminase activity in Alzheimer's disease brain.
        Neurobiol. Aging. 1998; 19: 385-391
        • Ames B.N.
        • Cathcart R.
        • Schwiers E.
        • Hochstein P.
        Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer. A hypothesis.
        Proc. Natl. Acad. Sci. U.S.A. 1981; 78: 6858-6862
        • Lowenstein J.M.
        Physiol. Rev. 1972; 52: 382-414
        • Mommsen T.P.
        • Hochachka P.W.
        The purine nucleotide cycle as two temporally separated metabolic units. A study on trout muscle.
        Metabolism. 1988; 37: 552-556
        • Hardie D.G.
        • Hawley S.A.
        AMP-activated protein kinase. The energy charge hypothesis revisited.
        BioEssays. 2001; 23: 1112-1119
        • Krebs H.
        The croonian lecture, 1963. Gluconeogenesis.
        Proc. R. Soc. Lond. B Biol. Sci. 1964; 159: 545-564
        • Oakhill J.S.
        • Steel R.
        • Chen Z.P.
        • Scott J.W.
        • Ling N.
        • Tam S.
        • Kemp B.E.
        AMPK is a direct adenylate charge-regulated protein kinase.
        Science. 2011; 332: 1433-1435
        • Shapiro I.M.
        • Leboy P.S.
        • Tokuoka T.
        • Forbes E.
        • DeBolt K.
        • Adams S.L.
        • Pacifici M.
        Ascorbic acid regulates multiple metabolic activities of cartilage cells.
        Am. J. Clin. Nutr. 1991; 54: 1209S-1213S
        • Bohensky J.
        • Leshinsky S.
        • Srinivas V.
        • Shapiro I.M.
        Chondrocyte autophagy is stimulated by HIF-1 dependent AMPK activation and mTOR suppression.
        Pediatr. Nephrol. 2010; 25: 633-642
        • Lushchak V.I.
        • Storey K.B.
        Purification and characteristics of AMP-deaminase from trout white muscle.
        Biokhimiya. 1995; 60: 270-277
        • van Waarde A.
        Operation of the purine nucleotide cycle in animal tissues.
        Biol. Rev. Camb. Philos. Soc. 1988; 63: 259-298
        • Rundell K.W.
        • Tullson P.C.
        • Terjung R.L.
        AMP deaminase binding in rat skeletal muscle after high-intensity running.
        J. Appl. Physiol. 1993; 74: 2004-2006
        • Tavazzi B.
        • Amorini A.M.
        • Fazzina G.
        • Di Pierro D.
        • Tuttobene M.
        • Giardina B.
        • Lazzarino G.
        Oxidative stress induces impairment of human erythrocyte energy metabolism through the oxygen radical-mediated direct activation of AMP-deaminase.
        J. Biol. Chem. 2001; 276: 48083-48092
        • Padh H.
        Cellular functions of ascorbic acid.
        Biochem. Cell Biol. 1990; 68: 1166-1173
        • Ji H.
        • Om A.D.
        • Yoshimatsu T.
        • Umino T.
        • Nakagawa H.
        • Sakamoto S.
        Effect of dietary ascorbate on lipogenesis and lipolysis activities in black sea bream, Acanthopagrus schlegelii.
        Fish Physiol. Biochem. 2010; 36: 749-755
        • Sok D.E.
        Ascorbate-induced oxidative inactivation of Zn2+-glycerophosphocholine choline phosphodiesterase.
        J. Neurochem. 1998; 70: 1167-1174
        • Duggan G.E.
        • Joan Miller B.
        • Jirik F.R.
        • Vogel H.J.
        Metabolic profiling of vitamin C deficiency in Gulo−/− mice using proton NMR spectroscopy.
        J. Biomol. NMR. 2011; 49: 165-173
        • Isherwood F.A.
        • Chen Y.T.
        • Mapson L.W.
        Isolation of D-glyceric acid from cress seedlings and its relationship to the synthesis of L-ascorbic acid.
        Biochem. J. 1954; 56: 15-21