Direct and quantitative analysis of altered metabolic flux distributions and cellular ATP production pathway in fumarate hydratase-diminished cells

Abstract

Fumarate hydratase (FH) is an enzyme in the tricarboxylic acid (TCA) cycle, biallelic loss-of-function mutations of which are associated with hereditary leiomyomatosis and renal cell cancer. However, how FH defect modulates intracellular metabolic fluxes in human cells has remained unclear. This study aimed to reveal metabolic flux alterations induced by reduced FH activity. We applied 13 c metabolic flux analysis (13 C-MFA) to an established cell line with diminished FH activity (FH dim) and parental HEK293 cells. FH dim cells showed reduced pyruvate import flux into mitochondria and subsequent TCA cycle fluxes. Interestingly, the diminished FH activity decreased FH flux only by about 20%, suggesting a very low need for FH to maintain the oxidative TCA cycle. Cellular ATP production from the TCA cycle was dominantly suppressed compared with that from glycolysis in FH dim cells. Consistently, FH dim cells exhibited higher glucose dependence for ATP production and higher resistance to an ATP synthase inhibitor. In summary, using FH dim cells we demonstrated that FH defect led to suppressed pyruvate import into mitochondria, followed by downregulated TCA cycle activity and altered ATP production pathway balance from the TCA cycle to glycolysis. We confirmed that 13 c-MFA can provide direct and quantitative information on metabolic alterations induced by FH defect. Fumarate hydratase (FH) is one of the enzymes in the tricarboxylic acid (TCA) cycle and catalyses the hydration of fumarate to malate. FH gene mutations causing loss of function are known to be associated with predispositions to hereditary leiomyomatosis and renal cell cancer (HLRCC) 1,2. This syndrome is characterised by cutaneous and uterine leiomyoma and an aggressive form of type 2 papillary renal carcinoma, which is often fatal 3. Since the TCA cycle plays an important role in cellular energy metabolism, FH defect leads to significant metabolic reprogramming. Effects of FH defect on cellular metabolism have been well studied using Fh1 (murine FH)-knockout mouse cells and FH-deficient UOK262 cells derived from HLRCC-associated kidney tumour 4. Yang et al., Frezza et al. and O'Flaherty et al. indirectly demonstrated the downregulated mitochondrial metabolism and the upregulated glycolysis in FH-inactivated cells by measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) 5-7. Via metabolome analysis, Adam et al. and Zheng et al. revealed that FH inactivation altered urea cycle metabolism and caused arginine auxotrophy 8,9. In addition, Gonçalves et al. demonstrated the increased phosphorylation of pyruvate dehydrogenase and restriction of carbon entry from glucose to the TCA cycle in FH-deficient cells by phosphoproteome analysis and 13 C tracer analysis 10. As described above, how FH defect modulates cellular energy metabolism has been addressed by various approaches. However, to the best of our knowledge, a 13 C metabolic flux analysis (13 C-MFA)-based approach has not been applied to address the impact of FH defect on ...

Isotope-Assisted Metabolic Flux Analysis: A Powerful Technique to Gain New Insights into the Human Metabolome in Health and Disease

“…This analysis revealed that NADPH consumption and NADH production were reduced in FH-diminished cells, suggesting that the altered redox balance may have caused decreased proline synthesis from glutamine. This analysis also demonstrated that FH-deficiency shifts ATP production towards glycolysis, allowing for increased resistance to mitochondrial inhibitors [126]. Together, these studies exemplify how iMFA can parse key drivers of cancer reprogramming from the multitude of metabolic alterations that occur.…”

Serum Anti-Fumarate Hydratase Autoantibody as a Biomarker for Predicting Prognosis of Acute-on-Chronic Liver Failure

“…It is reported that decreases of FH can lead to adenosine triphosphate depletion by crippling tricarboxylic acid cycle and oxidative phosphorylation. 25 Therefore, we speculate that because of the involvement of higher frequency and titer of FH, ACLF patients with good prognosis have an advantage in hepatocyte regeneration compared with ACLF patients with poor prognosis.…”

Succinylation of a KEAP1 sensor lysine promotes NRF2 activation

“…For example, depletion of fumarate hydratase (FH) was identified as a top hit in the screen, activating the ARE-GFP reporter, consistent with studies showing fumarate hydratase deficiency increases cellular levels of the KEAP1-reactive metabolite fumarate. 10,11,16 Further, depletion of transketolase (TKT) or transaldolase (TALDO1), two enzymes in the nonoxidative branch of the PPP, also resulted in ARE-GFP reporter activation. This result likely reflects the ability for these enzymes to reversibly catalyze reactions with the glycolytic metabolite Ga3P, which we have previously shown to activate NRF2 through direct modification of KEAP1.…”