Tracing Metabolic Fate of Mitochondrial Glycine Cleavage System Derived Formate In Vitro and In Vivo

Abstract

Folate-mediated one-carbon (1C) metabolism is a major target of many therapies in human diseases. Studies have focused on the metabolism of serine 3-carbon as it serves as a major source for 1C units. The serine 3-carbon enters the mitochondria transferred by folate cofactors and eventually converted to formate and serves as a major building block for cytosolic 1C metabolism. Abnormal glycine metabolism has been reported in many human pathological conditions. The mitochondrial glycine cleavage system (GCS) catalyzes glycine degradation to CO2 and ammonium, while tetrahydrofolate (THF) is converted into 5,10-methylene-THF. GCS accounts for a substantial proportion of whole-body glycine flux in humans, yet the particular metabolic route of glycine 2-carbon recycled from GCS during mitochondria glycine decarboxylation in hepatic or bone marrow 1C metabolism is not fully investigated, due to the limited accessibility of human tissues. Labeled glycine at 2-carbon was given to humans and primary cells in previous studies for investigating its incorporations into purines, its interconversion with serine, or the CO2 production in the mitochondria. Less is known on the metabolic fate of the glycine 2-carbon recycled from the GCS; hence, a model system tracing its metabolic fate would help in this regard. We took the direct approach of isotopic labeling to further explore the in vitro and in vivo metabolic fate of the 2-carbon from [2-13C]glycine and [2-13C]serine. As the 2-carbon of glycine and serine is decarboxylated and catabolized via the GCS, the original 13C-labeled 2-carbon is transferred to THF and yield methyleneTHF in the mitochondria. In human hepatoma cell-lines, 2-carbon from glycine was found to be incorporated into deoxythymidine (dTMP, dT + 1), M + 3 species of purines (deoxyadenine, dA and deoxyguanine, dG), and methionine (Met + 1). In healthy mice, incorporation of GCS-derived formate from glycine 2-carbon was found in serine (Ser + 2 via cytosolic serine hydroxy methyl transferase), methionine, dTMP, and methylcytosine (mC + 1) in bone marrow DNA. In these experiments, labeled glycine 2-carbon directly incorporates into Ser + 1, A + 2, and G + 2 (at C2 and C8 of purine) in the cytosol. It is noteworthy that since the serine 3-carbon is unlabeled in these experiments, the isotopic enrichments in dT + 1, Ser + 2, dA + 3, dG + 3, and Met + 1 solely come from the 2-carbon of glycine/serine recycled from GCS, re-enters the cytosolic 1C metabolism as formate, and then being used for cytosolic syntheses of serine, dTMP, purine (M + 3) and methionine. Taken together, we established model systems and successfully traced the metabolic fate of mitochondrial GCS-derived formate from glycine 2-carbon in vitro and in vivo. Nutritional supply significantly alters formate generation from GCS. More GCS-derived formate was used in hepatic serine and methionine syntheses, whereas more GCS-derived formate was used in dTMP synthesis in the bone marrow, indicating that the utilization and partitioning of GCS-derived 1C unit are tissue-specific. These approaches enable better understanding concerning the utilization of 1C moiety generated from mitochondrial GCS that can help to further elucidate the role of GCS in human disease development and progression in future applications. More studies on GCS using these approaches are underway.

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

“…We recently established a feasible approach to trace the utilization of 1C moiety from mitochondrial GCS using [2- 13 C]glycine [ 21 ]. Labeled glycine at carbon two has been given to humans to investigate its direct incorporation into purines and excretion as uric acid [ 22 ] rather than its metabolic route via the mitochondria GCS.…”

“…Labeled glycine at 2-carbon was given to humans to investigate its direct incorporations into purines and excreted as uric acid [ 22 ], rather than investigating its metabolic route via the mitochondria GCS. In our [2- 13 C]glycine experiment, the original 13 C-labeled glycine 2-carbon is transferred to THF to yield 13 C-labeled methyleneTHF, that ultimately produces 13 C-labeled formate; this 1C unit can be utilized in cytosolic biosyntheses for nucleotides, serine, methionine, adoMet, and methyl-cytosine [ 21 ].…”

Metformin Is a Pyridoxal-5′-phosphate (PLP)-Competitive Inhibitor of SHMT2

“…Serine labeled with 13 C at carbon 3 transfer 13 C to 5,10-CH 2 -THF only via the SHMT activity, whereas glycine cleavage would donate carbon 2 ( 12 C) of serine, thereby excluding any putative carbon contribution from a functional glycine cleavage system. We therefore decided to trace how metformin treatment impacted endogenous formate utilization by monitoring the metabolic fate of serine 3-carbon using labeling experiments with [3-13 C]serine as the tracer ( [32,[50][51][52]; Figure 5B). We additionally tested whether metformin could regulate the mitochondrial SHMT2-channeled serine-to-formate flux beyond its reported capacity to uncouple mCI activity from MTHFD2 functioning [32] by overexpressing the metformin-resistant Saccharomyces cerevisiae NADH dehydrogenase NDI1, which can correct functional defects in mCI [16].…”

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

“…Tetrahydrofolate (THF) is a metabolic cofactor that carries and activates single carbons for the synthesis of purine and thymidine nucleotides [39] and homocysteine remethylation to methionine [40]. Folate-mediated one-carbon metabolism is compartmentalized in the mitochondria, nucleus, and cytoplasm of eukaryotic cells [41].…”