Folinate Supplementation Ameliorates Methotrexate Induced Mitochondrial Formate Depletion In Vitro and In Vivo

Abstract

(1) Background: Antifolate methotrexate (MTX) is the most common disease-modifying antirheumatic drug (DMARD) for treating human rheumatoid arthritis (RA). The mitochondrial-produced formate is essential for folate-mediated one carbon (1C) metabolism. The impacts of MTX on formate homeostasis in unknown, and rigorously controlled kinetic studies can greatly help in this regard. (2) Methods: Combining animal model (8-week old female C57BL/6JNarl mice, n = 18), cell models, stable isotopic tracer studies with gas chromatography/mass spectrometry (GC/MS) platforms, we systematically investigated how MTX interferes with the partitioning of mitochondrial and cytosolic formate metabolism. (3) Results: MTX significantly reduced de novo deoxythymidylate (dTMP) and methionine biosyntheses from mitochondrial-derived formate in cells, mouse liver, and bone marrow, supporting our postulation that MTX depletes mitochondrial 1C supply. Furthermore, MTX inhibited formate generation from mitochondria glycine cleavage system (GCS) both in vitro and in vivo. Folinate selectively rescued 1C metabolic pathways in a tissue-, cellular compartment-, and pathway-specific manner: folinate effectively reversed the inhibition of mitochondrial formate-dependent 1C metabolism in mouse bone marrow (dTMP, methionine, and GCS) and cells (dTMP and GCS) but not methionine synthesis in liver/liver-derived cells. Folinate failed to fully recover hepatic mitochondrial-formate utilization for methionine synthesis, suggesting that the efficacy of clinical folinate rescue in MTX therapy on hepatic methionine metabolism is poor. (4) Conclusion: Conducting studies in mouse and cell models, we demonstrate novel findings that MTX specifically depletes mitochondrial 1C supply that can be ameliorated by folinate supplementation except for hepatic transmethylation. These results imply that clinical use of low-dose MTX may particularly impede 1C metabolism via depletion of mitochondrial formate. The MTX induced systematic and tissue-specific formate depletion needs to be addressed more carefully, and the efficacy of folinate with respect to protecting against such depletion deserves to be evaluated in medical practice.

MTHFR Knockdown Assists Cell Defense against Folate Depletion Induced Chromosome Segregation and Uracil Misincorporation in DNA

“…The impacts of folate depletion/low folate seemed milder in shMTHFR compared to WT and Neg. Folinate has been shown to be effective in rescuing certain impaired 1C metabolic pathways induced by MTX [45] or folate depletion in vivo [47,48]. In the present study, shMTHFR seemed to have better rescuing effects from folinate after initial folate depletion in our cell model.…”

“…Were Amplified after Folinate Supplementation Folate depletion and folinate supplementation change the partitioning of 5,10-CH 2 THF dependent 1C metabolic fluxes between mitochondrial and cytosolic derived formate in vivo [45]. It is plausible that MTHFR can regulate the competition between folatedependent deoxyribonucleotide and SAM biosynthesis by controlling the balance between folate cofactor methyleneTHF (5,10-CH 2 THF) and methylTHF (5-CH 3 THF) [26].…”

“…Folate-depleted cells (-FA) were cultured in RPMI-1640 without folate medium for 11 days. Folinate has been shown to be effective in rescuing certain impaired 1C metabolic pathways induced by methotrexate [45] or folate depletion [47]. The folate-replete and -deplete cells (-FA + folinate group) were cultured in no-folate medium for 8 days and then supplemented with 10 nM folinate for 3 days.…”

“…To investigate the impacts of shMTHFR on purine and thymidylate synthesis under different folates conditions, kinetic experiments were conducted based on our previous studies [5,26,45] with modifications for the present study. Stable isotopic tracer [3-13 C]serine (Cambridge Isotope Laboratories, Woburn, MA, USA) was used to trace the 1-carbon flow in the subsequent experiments as the β-carbon of serine provides the major 1-carbon source in folate metabolic pathways.…”

MAT2A Localization and Its Independently Prognostic Relevance in Breast Cancer Patients

“…Folate-mediated one-carbon metabolism is essential for methylation status [9] and nucleotide biosynthesis [10][11][12]; both are critical in cancer development and therapeutics [13,14]. Methionine is converted to the cellular methyl donor, S-adenosylmethionine (SAM) through the transfer of adenosine from ATP to the methionine sulfur that is catalyzed by methionine adenosyl transferases (MATs).…”