Abstract
Glycine N-methyltransferase (GNMT) is a major hepatic enzyme that converts S-adenosylmethionine to S-adenosylhomocysteine while generating sarcosine from glycine, hence it can regulate mediating methyl group availability in mammalian cells. GNMT is also a major hepatic folate binding protein that binds to, and, subsequently, may be inhibited by 5-methyltetrafolate. GNMT is commonly diminished in human hepatoma; yet its role in cellular folate metabolism, in tumorigenesis and antifolate therapies, is not understood completely. In the present study, we investigated the impacts of GNMT expression on cell growth, folate status, methylfolate-dependent reactions and antifolate cytotoxicity. GNMT-diminished hepatoma cell lines transfected with GNMT were cultured under folate abundance or restriction. Folate-dependent homocysteine remethylation fluxes were investigated using stable isotopic tracers and gas chromatography/mass spectrometry. Folate status was compared between wild-type (WT), GNMT transgenic (GNMT(tg)) and GNMT knockout (GNMT(ko)) mice. In the cell model, GNMT expression increased folate concentration, induced folate-dependent homocysteine remethylation, and reduced antifolate methotrexate cytotoxicity. In the mouse models, GNMT(tg) had increased hepatic folate significantly, whereas GNMT(ko) had reduced folate. Liver folate levels correlated well with GNMT expressions (r = 0.53, P = 0.002); and methionine synthase expression was reduced significantly in GNMT(ko), demonstrating impaired methylfolate-dependent metabolism by GNMT deletion. In conclusion, we demonstrated novel findings that restoring GNMT assists methylfolate-dependent reactions and ameliorates the consequences of folate depletion. GNMT expression in vivo improves folate retention and bioavailability in the liver. Studies on how GNMT expression impacts the distribution of different folate cofactors and the regulation of specific folate dependent reactions are underway.
Highlights
Different forms of folate serve as carriers of one-carbon units in DNA synthesis and biological methylation in mammals
We demonstrated that transformed human lymphoblasts with reduced methylenetetrahydrofolate reductase (MTHFR) have advantages in de novo purine synthesis when folate is adequate, but they are more susceptible to S-adenosylmethionine depletion when folate is restricted [21]
In the present study, we demonstrated numerous findings. (a) Restoring Glycine N-methyltransferase (GNMT) in cells with diminished GNMT can improve intracellular folate status. (b) GNMT expression can increase 5-methylTHF-dependent metabolic fluxes in GNMT–deficient cells. (c) GNMT ameliorates the growth retardation induced by folate depletion. (d) Expression of GNMT can protect HepG2 cells from low-dose methotrexate induced apoptosis. (e) In vivo GNMT expression improves folate status presumably owing to increased retention and bioavailability in the liver. (f) Destruction of GNMT in vivo reduces hepatic folate and decreases methylfolate-dependent methionine synthase expression in the liver
Summary
Different forms of folate serve as carriers of one-carbon units in DNA synthesis and biological methylation in mammals. Among all forms of folates, the 5-methyl tetrahydrofolate (5-methyl-THF) is the most abundant that transfers the methyl group to the enzyme methionine synthase, generating methionine from homocysteine remethylation [1]. Glycine N-methyltransferase (GNMT, EC2.1.1.20) is an abundant liver protein that converts S-adenosylmethionine to S-adenosylhomocysteine while generating sarcosine from glycine. The GNMT reaction serves as an alternative pathway to regulate the S-adenosylmethionine to S-adenosylhomocysteine balance and availability of methyl group in mammalian cells [2]. GNMT is a major hepatic folate-binding protein [2,3] that binds to, and, subsequently, may be inhibited by 5-methyl-THF [2]. Loss of heterozygosity within the GNMT gene in the liver tissues of hepatocellular carcinoma patients has been reported, and GNMT alteration appears to be an early event in human hepatocellular carcinoma [11]. Individuals with mutant GNMT and GNMT knockout mice showed that inactivation
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