MilliporeSigma
  • Bcl-xL knockout attenuates mitochondrial respiration and causes oxidative stress that is compensated by pentose phosphate pathway activity.

Bcl-xL knockout attenuates mitochondrial respiration and causes oxidative stress that is compensated by pentose phosphate pathway activity.

Free radical biology & medicine (2017-08-16)
Annika Pfeiffer, Julia Schneider, Diones Bueno, Amalia Dolga, Timo-Daniel Voss, Jan Lewerenz, Verena Wüllner, Axel Methner
ABSTRACT

Bcl-xL is an anti-apoptotic protein that localizes to the outer mitochondrial membrane and influences mitochondrial bioenergetics by controlling Ca2+ influx into mitochondria. Here, we analyzed the effect of mitochondrial Bcl-xL on mitochondrial shape and function in knockout (KO), wild type and rescued mouse embryonic fibroblast cell lines. Mitochondria of KO cells were more fragmented, exhibited a reduced ATP concentration, and reduced oxidative phosphorylation (OXPHOS) suggesting an increased importance of ATP generation by other means. Under steady-state conditions, acidification of the growth medium as a readout for glycolysis was similar, but upon inhibition of ATP synthase with oligomycin, KO cells displayed an instant increase in glycolysis. In addition, forced energy production through OXPHOS by replacing glucose with galactose in the growth medium rendered KO cells more susceptible to mitochondrial toxins. KO cells had increased cellular reactive oxygen species and were more susceptible to oxidative stress, but had higher glutathione levels, which were however more rapidly consumed under conditions of oxidative stress. This coincided with an increased activity and protein abundance of the pentose phosphate pathway protein glucose-6-phosphate dehydrogenase, which generates NADPH necessary to regenerate reduced glutathione. KO cells were also less susceptible to pharmacological inhibition of the pentose phosphate pathway. We conclude that mitochondrial Bcl-xL is involved in maintaining mitochondrial respiratory capacity. Its deficiency causes oxidative stress, which is associated with an increased glycolytic capacity and balanced by an increased activity of the pentose phosphate pathway.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Glutathione Reductase from baker's yeast (S. cerevisiae), ammonium sulfate suspension, 100-300 units/mg protein (biuret)
Sigma-Aldrich
Cytochrome c from equine heart, ≥95% based on Mol. Wt. 12,384 basis
Sigma-Aldrich
Anti-Actin Antibody, clone C4, ascites fluid, clone C4, Chemicon®