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

Abstract

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.

GNMT Expression Increases Hepatic Folate Contents and Folate-Dependent Methionine Synthase-Mediated Homocysteine Remethylation

“…We recently discovered in vitro that GNMT expression does not exacerbate methyl group deficiency when the methionine supply is limited; instead it can protect cells from further hypomethylation induced by methionine depletion. Restoring GNMT expression in GNMT diminished cell lines is crucial in maintaining methyl group homeostasis via homocysteine transmethylation and transsulfuration kinetics (41). Adding to the current knowledge, results from the present study further demonstrated evidence that in GNMT-abundant tissue, that is, the liver, normal GNMT function is crucial for optimal folate status and hepatic-folatedependent biochemical reactions in vivo.…”

Turmeric enhancing anti-tumor effect of Rhizoma paridis saponins by influencing their metabolic profiling in tumors of H22 hepatocarcinoma mice

“…Glycine, as the precursor of sarcosine, induced cancer cell invasion [32]. Thus, the decreased glycine and sarcosine inhibited the tumor growth.…”

One-Carbon Metabolism in Prostate Cancer: The Role of Androgen Signaling

“…As discussed above, the AR has important roles in regulating GNMT and the metabolism of sarcosine and the enzymes involved in the diversion of methyl groups into the transsulfuration and polyamine synthesis pathways. Increased GNMT expression leads to increased levels of the methyltransferase inhibitor SAH [200]. Therefore, changes in AR activity indirectly affect methyltransferase activity by modulating the SAM:SAH ratio.…”