Glycine-N Methyltransferase Expression in HepG2 Cells Is Involved in Methyl Group Homeostasis by Regulating Transmethylation Kinetics and DNA Methylation1,2

Mar 18, 2011The Journal of nutrition

Glycine-N methyltransferase helps control methyl group balance by affecting methyl transfer rates and DNA methylation in liver cells

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Abstract

GNMT expression decreased S-adenosylmethionine (SAM) levels and increased S-adenosylhomocysteine (SAH) levels in high or adequate methionine conditions.

GNMT expression is associated with lower homocysteine and greater cysteine concentrations in cells.
In high or adequate l-methionine, GNMT promotes methionine clearance by enhancing homocysteine transsulfuration and remethylation processes.
In low methionine conditions, GNMT does not promote sarcosine synthesis, indicating a mechanism to conserve methyl groups.
Methylated cytidine levels are significantly lower in GNMT inactive cells compared to those expressing GNMT.
GNMT function is linked to alterations in transmethylation kinetics and the regulation of global DNA methylation.

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Glycine-N methyltransferase (GNMT) is a potential tumor suppressor that is commonly inactivated in human hepatoma. We systematically investigated how GNMT regulates methyl group kinetics and global DNA methylation. HepG2 cells (GNMT inactive, GNMT-) and cells transfected with GNMT expressed vector (GNMT+) were cultured in low (10 μmol/L), adequate (100 μmol/L), or high (500 μmol/L) l-methionine, each with 2.27 μmol/L folate. Transmethylation kinetics were studied using stable isotopic tracers and GC-MS. Methylation status was determined by S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) levels, SAM:SAH ratio, DNA methyltransferase (DNMT) activity, and methylated cytidine levels in DNA. Compared with GNMT- cells, GNMT+ cells had lower homocysteine and greater cysteine concentrations. GNMT expression increased methionine clearance by inducing homocysteine transsulfuration and remethylation metabolic fluxes when cells were cultured in high or adequate l-methionine. In contrast, homocysteine remethylation flux was lower in GNMT+ cells than in GNMT- cells and homocysteine transsulfuration fluxes did not differ when cells were cultured in low methionine, suggesting that normal GNMT function helps to conserve methyl groups. Furthermore, GNMT expression decreased SAM and increased SAH levels and reduced DNMT activity in high or adequate, but not low, methionine cultures. In low methionine cultures, restoring GNMT in HepG2 cells did not lead to sarcosine synthesis, which would waste methyl groups. Methylated cytidine levels were significantly lower in GNMT- cells than in GNMT+ cells. In conclusion, we have shown that GNMT affects transmethylation kinetics and SAM synthesis and facilitates the conservation of methyl groups by limiting homocysteine remethylation fluxes.

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Glycine-N Methyltransferase Expression in HepG2 Cells Is Involved in Methyl Group Homeostasis by Regulating Transmethylation Kinetics and DNA Methylation1,2

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