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 13C metabolic flux analysis (13C-MFA) to an established cell line with diminished FH activity (FHdim) and parental HEK293 cells. FHdim 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 FHdim cells. Consistently, FHdim cells exhibited higher glucose dependence for ATP production and higher resistance to an ATP synthase inhibitor. In summary, using FHdim 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 13C-MFA can provide direct and quantitative information on metabolic alterations induced by FH defect.

• Authors: Shingo Noguchi, Hirokazu Ishikawa, Kenichi Wakita, Fumio Matsuda, Hiroshi Shimizu
• Journal: Scientific reports (Sci Rep) Vol. 10 Issue 1 Pg. 13065 (08 03 2020) ISSN: 2045-2322 [Electronic] England
• PMID: 32747645 (Publication Type: Journal Article)

Chemical References

• Carbon Isotopes
• Glutamine
• Adenosine Triphosphate
• Fumarate Hydratase
• Carbon-13
• Glucose

Topics

• Adenosine Triphosphate (metabolism)
• Base Sequence
• Carbon Isotopes
• Cell Survival (drug effects)
• Cytosol (drug effects, metabolism)
• Extracellular Space (metabolism)
• Fumarate Hydratase (genetics, metabolism)
• Glucose (pharmacology)
• Glutamine (pharmacology)
• HEK293 Cells
• Humans
• Isotope Labeling
• Metabolic Flux Analysis
• Mitochondria (drug effects, metabolism)
• Time Factors