Dataset Information

Oxidative pentose phosphate pathway and glucose anaplerosis support maintenance of mitochondrial NADPH pool under mitochondrial oxidative stress.

ABSTRACT: Mitochondrial NADPH protects cells against mitochondrial oxidative stress by serving as an electron donor to antioxidant defense systems. However, due to technical challenges, it still remains unknown as to the pool size of mitochondrial NADPH, its dynamics, and NADPH/NADP⁺ ratio. Here, we have systemically modulated production rates of H₂O₂ in mitochondria and assessed mitochondrial NADPH metabolism using iNap sensors, ¹³C glucose isotopic tracers, and a mathematical model. Using sensors, we observed decreases in mitochondrial NADPH caused by excessive generation of mitochondrial H₂O₂, whereas the cytosolic NADPH was maintained upon perturbation. We further quantified the extent of mitochondrial NADPH/NADP⁺ based on the mathematical analysis. Utilizing ¹³C glucose isotopic tracers, we found increased activity in the pentose phosphate pathway (PPP) accompanied small decreases in the mitochondrial NADPH pool, whereas larger decreases induced both PPP activity and glucose anaplerosis. Thus, our integrative and quantitative approach provides insight into mitochondrial NADPH metabolism during mitochondrial oxidative stress.

SUBMITTER: Moon SJ

PROVIDER: S-EPMC7510474 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Oxidative pentose phosphate pathway and glucose anaplerosis support maintenance of mitochondrial NADPH pool under mitochondrial oxidative stress.

Moon Sun Jin SJ Dong Wentao W Stephanopoulos Gregory N GN Sikes Hadley D HD

Bioengineering & translational medicine 20200908 3

Mitochondrial NADPH protects cells against mitochondrial oxidative stress by serving as an electron donor to antioxidant defense systems. However, due to technical challenges, it still remains unknown as to the pool size of mitochondrial NADPH, its dynamics, and NADPH/NADP+ ratio. Here, we have systemically modulated production rates of H2O2 in mitochondria and assessed mitochondrial NADPH metabolism using iNap sensors, 13C glucose isotopic tracers, an ...[more]

PMID: 33005744

Similar Datasets

Project description:The maintenance of advanced malignancies relies on continued activity of driver oncogenes, although their rate-limiting role is highly context-dependent with respect to tumor types and associated genetic alterations. Oncogenic Kras mutation is the signature event in human pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible KrasG12D-driven p53 mutant PDAC mouse model establishes that advanced PDAC remains strictly dependent on continued KrasG12D expression and that KrasG12D serves a vital role in the control of tumor metabolism, through stimulation of glucose uptake and channeling of glucose intermediates through the hexosamine biosynthesis pathway (HBP) and the pentose phosphate pathway (PPP). Notably, these studies reveal that oncogenic Kras regulates ribose biogenesis. Unlike canonical models of PPP-mediated ribose biogenesis, we demonstrate that oncogenic Kras drives intermediates from enhanced glycolytic flux into the non-oxidative arm of the PPP, thereby decoupling ribose biogenesis from NADPNADPH-mediated redox control. Together, this work provides in vivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in Kras-driven PDAC. Primary pancreatic tumor lines were established from p48Cre tetO_LKrasG12D ROSA_rtTAL+ p53L+ mice. Five independent tumor lines (iKras1-5) were used for pancreatic injection into nude mice to generate orthotopic tumors. The mice were kept on doxycycline for 2 weeks until obvious tumor formation. Half of the animals were pulled off doxycycline for 24 hours. Tumors with over 75% cellularity were collected for total RNA prepartion. For in vitro expression profiles, the same five tumor lines were cultured in the presence or absence of doxycycline for 24 hours and total cellular RNA was prepared. For control samples, two independent tumor lines from LSL-KrasG12D p53L+ tumors were cultured in the presence or absence of doxycycline for 24 hours and total cellular RNA was prepared.

Dataset Information

Oxidative pentose phosphate pathway and glucose anaplerosis support maintenance of mitochondrial NADPH pool under mitochondrial oxidative stress.

Publications

Oxidative pentose phosphate pathway and glucose anaplerosis support maintenance of mitochondrial NADPH pool under mitochondrial oxidative stress.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets