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

Wen-Nan Huang,Yi-Cheng Wang,Yi-Hsuan Fan,Yi-Ying Lin,Der-Yuan Chen,Yi-Ming Chen,Barry Shane,Nga-Lai Sou,Feng-Yao Tang,Hsin-An Ko,En-Pei Isabel Chiang,Hui-Ming Chih,Yu-Hsuan Huang

doi:10.3390/ijms22031350

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.

Highlights

Formate plays a key role in mammals’ cellular and whole-body metabolism
MTX inhibited the incorporation and utilization of endogenous formate generated from L-[3-13 C]serine, and increased the utilization of 1C moiety from exogenous [13 C]formate for nucleotide synthesis (Figure 1a,b)
The present study demonstrated that clinical use of low-dose MTX impedes 1C metabolism via depletion of mitochondrial formate in vitro and in vivo

Summary

Introduction

Formate plays a key role in mammals’ cellular and whole-body metabolism. The mitochondrial production of formate is a major process for the endogenous generation of folate-related one-carbon (1C) moieties [1]. Formate protects the cytosolic pool of folate cofactors [4] and is used to re-synthesize serine via cytosolic 1C metabolism [5]. In cells with defective mitochondrial 1C metabolism, the cytosolic pathway can compensate for the loss of mitochondrial formate production. When both the cytosolic and mitochondrial pathways are compromised, cells need exogenous formate or endogenous formaldehyde [6] as alternative 1C sources

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Discussion

Conclusion