Glycine N-methyltransferase modulates mitochondria-derived formate utilization and central carbon metabolism during methotrexate therapy

Mutations or reduced expression in glycine N-methyltransferase ( GNMT ) has been linked to liver diseases. Methotrexate (MTX) is commonly prescribed for the treatment of human rheumatic diseases. We previously demonstrated that MTX selectively inhibits the partitioning of mitochondria derived formate pathways, including glycine cleavage system (GCS). We also demonstrated that GNMT expression is essential for methyl group supply, intracellular folate homeostasis and MTX toxicity. We tested the hypothesis that GNMT regulates the metabolic response to low-dose methotrexate by altering mitochondrial one-carbon metabolism, thereby influencing glycine-derived one-carbon partitioning, DNA methylation, and central carbon metabolism. Metabolic alterations of mitochondrial one carbon metabolism and TCA cycle were investigated in liver derived cell-lines with/without human GNMT expression, as well as in wild-type (WT, Gnmt +/+ ) and GNMT knockout (KO, Gnmt -/- ) mouse models treated with MTX using stable isotopic tracers and GC/MS. MTX inhibited deoxythymidylate (dTMP) synthesis from mitochondria derived formate in both WT and Gnmt -/- mice bone marrow. Deletion of Gnmt in mice decreased enrichment in 5-methyl-2′-deoxycytidine (5-mdC) but not dTMP synthesis from GCS using [2- 13 C] glycine tracer. Interestingly, MTX inhibited 5-mdC enrichments in Gnmt +/+ but promoted that in Gnmt -/- in the bone marrow, indicating gene-drug interactions in GCS activity. MTX decreased liver pyruvate concentrations in both Gnmt +/+ and Gnmt -/- , whereas MTX increased plasma citrate concentration in Gnmt +/+ but not in Gnmt -/- . MTX suppressed liver alpha-ketoglutarate but increased malate concentrations in Gnmt +/+ but not Gnmt -/- . The present study provides evidence on the interactions between MTX and GNMT function and provides new insights on how this essential gene may affect mitochondria derived formate dependent pathways and central carbon metabolism during MTX therapy. In patients taking long-term MTX therapy, genetic factors such as hepatic GNMT function should be taken into consideration.