MiP2005: Session 5 - Young Investigator Presentation
Mitochondrial Physiology Network 10.9: 55-57 (2005) - download pdf
Limitation of aerobic metabolism by the phosphorylation system and mitochondrial respiratory capacity of fibroblasts in vivo. The coupled reference state and reinterpretation of the uncoupling control ratio.
Ashley Naimi1,2, A Garedew1,3 , J Troppmair1, R Boushel2, E Gnaiger1
1D. Swarovski Research Lab., Dept. Transplant Surgery, Innsbruck Medical Univ., Austria; 2Dept. Exercise Sci., Concordia Univ., Montreal, Canada; 2OROBOROS INSTRUMENTS, Innsbruck, Austria.. – firstname.lastname@example.org
Oxygen consumption increases between 10-15 fold from resting to maximal in mammals, largely reflecting the rest-work transition in muscle tissues . In a variety of cultured cells, in turn, routine respiration can be stimulated 3-fold by uncoupling oxidative phosphorylation, corresponding to an uncoupling control ratio (UCR) of 3 [2,3]. Uncoupled respiration of intact cells has been considered as the gold-standard for evaluation of maximum capacities in vivo, to avoid any artifacts from the loss of essential metabolites, disruption of the cytoskeletal structure, and channeling of respiratory substrates to the organelles . The definition of a physiological reference state has important implications when interpreting values for the excess capacity of specific enzymes in the electron transport chain, as well as phenotypic effects of various genetic and acquired mitochondrial disorders [4,5]. In the present study, we addressed the problems encountered when using uncoupled respiration as a physiological reference [2-4], and resolved fundamental discrepancies of respiratory capacity in intact and permeabilized cells [4-6].
Using high-resolution respirometry (OROBOROS Oxygraph-2k) and optimized titration regimes with various substrates and inhibitors for evaluation of mitochondrial respiratory function, we measured the oxygen flux in NIH3T3 fibroblasts at a density of 0.3∙106 cells∙ ml-1.
Routine respiration (Cr) measured in fibroblasts suspended in culture medium increased after uncoupling (state Cr,u) by a factor (UCR) of 2.9 ± 0.2 SD. This maximum flux in non-permeabilized cells was 2.5-fold higher than the coupled flux, measured in digitonin permeabilized cells with saturating ADP and complex I substrates (glutamate+malate, state GM3 vs Cr,u; Fig. 1). This confirms previously reported differences . Since the uncoupled cell as a model for maximum coupled respiration (state 3) is questionable, we investigated state 3 respiration and uncoupling with various substrate combinations in permeabilized cells. Uncoupling by FCCP, in the presence of glutamate+malate, stimulated state 3 respiration 2-fold. This flux through complex I was still below uncoupled respiration of intact cells. Complete agreement was reached only with uncoupling and parallel electron input through complexes I and II with glutamate+malate+succinate (GSu versus Cr,u; Fig. 1). Coupled respiration (GS3), however, was merely half of the uncoupled respiratory capacity (Fig. 1). Compared to state 3 with complex I substrates only (GM3), parallel electron input increased respiratory capacity by a factor of 1.3. The additive effect was also shown by comparison with respiration in the presence of rotenone plus succinate (Su). Taken together, (1) the uncoupled state in intact and permeabilized cells showed that combined glutamate and succinate (without rotenone) was essential for adequate substrate supply, (2) the pronounced increase in respiration after uncoupling compared to state 3 indicated the quantitative importance of the phosphorylation system in respiratory control , and (3) the excess capacities of complexes III and IV are at least 1.9 when related to the physiological reference state of coupled respiration, much higher than that derived from uncoupling control ratios . Our results thus resolve controversies on the applicability of permeabilized cells as in vivo models in mitochondrial physiology.
A direct “VO2,max” (state 3) is inaccessible in intact fibroblasts because maximum ADP stimulation cannot be achieved without permeabilization of the plasma membrane. However, agreement between uncoupled respiration in intact and permeabilized cells was obtained with parallel electron input through complexes I and II, which corresponds to the operation of the TCA cycle and the mitochondrial substrate supply in vivo. Not the uncoupled, but the coupled state 3 with parallel electron input thus yields a measure of maximum capacity of mitochondrial respiration. This approach provides a physiological reference state, analogous to the VO2,max obtained by ergometry during maximal aerobic exercise. According to previous interpretations, a UCR of 3 would indicate that routine respiration amounts to merely 33 % of total capacity. Based on our results, however, routine respiration operates much closer to physiological capacity, i.e. at 77 % of the physiological reference state GS3. Maximum respiratory capacity is under tight control by the phosphorylation system, which is eliminated by uncoupling. In this light, conventional applications of the UCR must be reconsidered with reference to accepted concepts of adenylate control in mitochondrial respiration. A comparable pattern of the effects of parallel electron input and uncoupling is observed in permeabilized human muscle biopsies (; Session 1). Since routine coupled and uncoupled respiration cannot be measured in muscle biopsies, cultured cells provide a unique model for contrasting respiratory capacities observed in vivo and after permeabilization. NIH3T3 cells, therefore, provide a valid model for evaluation of the physiological reference state of mitochondrial respiratory capacity. Parallel complex I + II electron input in coupled and uncoupled states represents an important addition to the conventional titration protocols applied for the diagnosis of mitochondrial respiratory control in health and disease.
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