Huetter 2004 Biochem J

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Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380:919-28.

» PMID: 15018610 Open Access; pdf

Huetter E, Renner K, Pfister G, Stoeckl P, Jansen-Duerr P, Gnaiger E (2004) Biochem J

Abstract: Limitation of lifespan in replicative senescence is related to oxidative stress, which is probably both the cause and consequence of impaired mitochondrial respiratory function. The respiration of senescent human diploid fibroblasts was analysed by high-resolution respirometry. To rule out cell-cycle effects, proliferating and growth-arrested young fibroblasts were used as controls. Noncoupled respiration, as normalized to citrate synthase activity, remained unchanged, reflecting a constant mitochondrial Electron transfer-pathway (ET-pathway) capacity. Oligomycin-inhibited LEAK respiration, however, was significantly increased in mitochondria of senescent cells, indicating a lower coupling of electron transfer to phosphorylation of ADP to ATP. In contrast, growth-arrested young fibroblasts exhibited a higher coupling control compared with proliferating controls. In intact cells, partial uncoupling (dyscoupling) may lead to either decreased oxidative ATP production or a compensatory increase in ROUTINE respiration. To distinguish between these alternatives, we subtracted oligomycin-inhibited respiration from ROUTINE respiration, which allowed us to determine the part of respiratory activity coupled with ATP production. Despite substantial differences in the respiratory acceptor control ratio, ranging from 4 to 11 in the different experimental groups, a fixed proportion of ET-capacity was maintained for coupled oxidative phosphorylation in all the experimental groups. This finding indicates that the senescent cells fully compensate for increased proton leakage by enhanced electron-transfer activity in the ROUTINE state. These results provide a new insight into age-associated defects in mitochondrial function and compensatory mechanisms in intact cells.

Keywords: Aging, Coupling state, Mitochondria, Oxidative stress, Primary human fibroblast, Respiration, Senescence.

O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck Jansen-Duerr P, DE Regensburg Renner-Sattler K


Labels: MiParea: Respiration, mt-Biogenesis;mt-density  Pathology: Aging;senescence  Stress:Oxidative stress;RONS  Organism: Human  Tissue;cell: Fibroblast  Preparation: Intact cells  Enzyme: Marker enzyme  Regulation: Coupling efficiency;uncoupling, mt-Membrane potential, Uncoupler  Coupling state: LEAK, ROUTINE, ET 

HRR: Oxygraph-2k, O2k-Protocol 

SUIT-003 O2 ce D009, SUIT-003 

Coupling control protocol

Coupling control protocol and nomenclature

An update on nomenclature
ceCCP intact cells (fibroblasts)
LEAK vs ETS


High-resolution respirometry with the ceCCP

Cell coupling control protocol: respiration in human primary fibroblasts. Left: High-resolution respirometry in senescent cells (0.2×106 cells/ml). Arrows show steps in the titration regime of the coupling control protocol, inducing the following respiratory states: ROUTINE (R; routine state in cell-culture medium); LEAK (L; inhibition of ATP synthase by 1 μg/ml oligomycin); ETS (E; maximal stimulation by uncoupling of oxidative phosphorylation in four subsequent titrations of the uncoupler, U, FCCP (2.5–4 μM final concentration); ROX (rotenone, Rot, and antimycin A; residual oxygen consumption). Right: ROX-corrected oxygen flow per million cells, showing different coupling control in senescent versus young cells by plotting LEAK respiration as a function of ETS efficiency. Every data point is a single run in an O2k chamber. Modified after Huetter_2004_Biochem J.



Comment

  • In the context of another study (MiP2005 Abstract), we compared respiration of intact (ce) and permeabilized (pce) fibroblasts with excellent results. Therefore, permeabilized cells provide a validated model to extend mitochondrial studies of senescence. In fact, the fibroblasts in the present study were permeabilized for the JC1 experiments, to eliminate the confounding effect of the plasma membrane potential.