[PMC free article] [PubMed] [Google Scholar]Gameiro PA, Yang JJ, Metelo AM, Perez-Carro R, Baker R, Wang ZW, Arreola A, Rathmell WK, Olumi A, Lopez-Larrubia P, et al
[PMC free article] [PubMed] [Google Scholar]Gameiro PA, Yang JJ, Metelo AM, Perez-Carro R, Baker R, Wang ZW, Arreola A, Rathmell WK, Olumi A, Lopez-Larrubia P, et al. catabolism contributes to toxic NADH accumulation. serine synthesis, which produces cytoplasmic NADH, nearly ceased (Supplementary Fig. 4I) (Diehl et al., 2019). Open in a separate window Physique 4: High NADH/NAD shuts off other NADH sources but not serine catabolism A: Decrease in [U-13C]glucose access into TCA cycle with increasing cellular NADH/NAD. B: Decrease in [U-13C]glutamine oxidation (glutamine is usually instead metabolized reductively, observe Supplementary Fig. 4F). C: Initial increase, followed by decrease, in [U-13C]palmitate contribution to TCA cycle. D: Maintenance of mitochondrial serine catabolism indicated by M+1 dTTP from [2,3,3-2H] serine (observe Fig. 1A). E: Quantification of intracellular NAD(H) concentration with metformin treatment (The increase in NADH/NAD ratio with metformin treatment is largely driven by NADH accumulation). F: Michaelis-Menten plot of pyruvate dehydrogenase (PDH), -ketoglutarate dehydrogenase (KGDH), malate dehydrogenase (MDH) and MTHFD2 enzyme activity with increasing NADH as the competitive inhibitor (mean SD, N=3). Observe also Physique S4 To examine whether mitochondrial serine catabolism is also shut off, we measured thymidine labeling from [2,3,3-2H]serine. Mitochondrial serine catabolism generates M+1 thymidine, whereas the alternative cytosolic pathway generates M+2 thymidine. There was no decrease in the thymidine M+1 labeling portion up to a 5-fold increase in NADH/NAD, a level of respiration impairment sufficient Secretin (rat) to block TCA turning driven by glucose, glutamine and excess fat and serine synthesis (Physique 4D). Yet higher NADH/NAD ratios did result in a partial shift towards cytosolic serine metabolism. Nevertheless, catabolism of serine is usually uniquely strong to rising NADH/NAD. To examine the underlying biochemical mechanism, we examined the sensitivity of pyruvate dehydrogenase (PDH), -ketoglutarate dehydrogenase (KGDH) and malate dehydrogenase (MDH) versus MTHFD2 to increasing NADH. These assays were conducted in the presence of adequate NAD as substrate, which is appropriate given that physiologically we observe large changes in the NADH/NAD ratio driven by increasing NADH, with NAD largely maintained (Physique 4E). While all of the enzymes were subject to product inhibition by NADH, MTHFD2 was at least 10-fold less sensitive, maintaining full activity up to 200 M NADH, a typical level observed in respiration-impaired cells (Physique 4F). Thus, the key redox enzyme of serine catabolism is usually NADH-resistant, and therefore serine catabolism persists even after other NADH production pathways shut off. Loss of mitochondrial serine catabolism paradoxically facilitates cell growth in respiration-impaired cells Serine catabolism is the main cellular source of 1C units, which are required for nucleotide synthesis and therefore growth. Inhibition of 1C metabolism with anti-folates is commonly clinically employed to treat proliferative conditions, including malignancy and autoimmunity (Chabner and Roberts, 2005; Kremer, 1994; Weinblatt et al., 1985). Thus, serine catabolism is usually classically pro-growth. Maintenance of redox homeostasis is usually, however, also Secretin (rat) crucial to cell growth. We wondered if prolonged serine catabolism during respiration impairment might induce a pathologically high NADH/NAD ratio and thereby paradoxically Itga2 impair cell growth. To examine this Secretin (rat) possibility, we analyzed metabolite levels and growth of MTHFD2 knockout cells, in the presence and absence of complex I inhibitors. Strikingly, such cells tended to have a lower NADH/NAD ratio under basal conditions, which was significantly lower than wild-type cells after complex I inhibition with metformin, phenformin, or rotenone (Physique 5A, Supplementary Fig. 5A). Open in a separate window Physique 5: Loss of mitochondrial serine catabolism paradoxically facilitates cell growth in respiration-impaired cells. A: NADH/NAD for wild type and MTHFD2 knockout HCT116 cells metformin (imply SD, N6). B: Schematic of links between NADH/NAD, aspartate, and nucleotide synthesis. C-E: Intracellular aspartate (C), IMP/AMP ratio (D) and cell number (E) for wild type and MTHFD2 deficient HCT116 in the presence and absence of 1 mM metformin (mean SD, panel C&D: N6, panel E: N6). F: Schematic of SHIN1 as inhibitor of SHMT1/2. G: HCT116 cell growth in normal medium or the presence of 1 mM metformin the indicated concentration of the serine catabolism inhibitor SHIN1 (mean SD, N=5). **p 0.01, ***p 0.001 by two-tailed students t test. Observe also Physique S5 In response to.