Cardoso 2019 MiP2019
Cultured cells are widely used in research to minimize the use of animal experimentation or human biopsies. The hepatocellular carcinoma cell line Huh7 is an established model to study hepatocyte metabolism. However, application of different culture media, presence and absence of different supplements and different respiratory protocols make it difficult to compare results on a quantitative basis. In this study, we analysed the effects of glucose concentration in culture medium on mitochondrial function of Huh7 cells. High-resolution respirometry (HRR) was applied to study coupling control and electron-transfer (ET) capacity in living cells.
Huh7 cells were cultured in DMEM (Merck, Germany) with low glucose (LG; 5 mM) and high glucose concentration (HG; 25 mM). After 48 h, the cells were trypsinized and added to O2k chambers with final concentrations in the range of 0.25∙106 to 0.68∙106 cells/mL of mitochondrial respiration medium MiR05. Following the measurement of ROUTINE (R) respiration, 5 mM pyruvate was added as an exogenous substrate. Afterwards, 25 mM glucose was added to evaluate the Crabtree effect on ROUTINE respiratory activity. This was followed by evaluation of coupling control  by sequential titration of oligomycin (LEAK respiration) and the uncoupler CCCP (ET-capacity). The addition of 0.5 µM rotenone, inhibiting Complex I and consequently the TCA cycle, led to the measurement of residual oxygen consumption. Cell viability was evaluated by the stimulatory effect of 10 mM succinate and digitonin titration up to maximum O2 flux . Cytochrome c (10 µM) was used to evaluate integrity of mitochondrial outer membrane, and 2.5 µM antimycin A were used to inhibit Complex III and measure residual oxygen consumption after inhibiting ET-pathway-linked respiration.
Huh7 cells in HG medium presented in average 24 ± 8 % decreased ROUTINE respiration in comparison to the LG group. Upon addition of pyruvate, respiration of HG cells increased by 27 ± 5 %, which was significantly higher than in the LG group (15 ± 5 %). Addition of glucose did not affect respiration of HG cells, whereas a Crabtree effect was observed (7 % decrease of respiration) in the LG cells. ET-capacity and ET-coupling efficiency were not significantly different between groups. The cell viability measured by respirometry was not affected by the culture conditions (89 ± 2 %). The final digitonin concentration used to permeabilize the plasma membrane varied from 5 to 20 µg·million cells-1, and no significant damage to the mitochondrial outer membrane (assessed by the cytochrome c test) was observed. No differences in optimum digitonin dose were observed between groups (3.6 µg·million cells-1).
Despite of identical ET-capacities, ROUTINE respiration was lower in cells cultured in HG medium compared to the LG group. This was partially compensated by the addition of pyruvate to the respiration medium. Since a pronounced Crabtree effect was observed only in the LG group, these results collectively suggest that high glucose in culture medium induced a metabolic reprogramming, which can be interpreted as a fixed Crabtree effect independent of an acute glucose dose. These results should be extended by comparing aerobic glycolysis in LG and HG groups.
Labels: MiParea: Respiration
Organism: Human Tissue;cell: Liver Preparation: Intact cells
Coupling state: LEAK, ROUTINE, ET Pathway: ROX HRR: Oxygraph-2k
Affiliations and support
- Cardoso LHD(1), Garcia-Souza LF(1,4), Nindl V(1), Sobotka L(2), Gnaiger E(1,4), Sobotka O(2,3)
- Oroboros Instruments, Innsbruck, AT
- 3rd Dept Internal Medicine, Gerontol Metabolism, Univ Hospital, Fac Medicine Hradec Kralove, CZ
- Dept Physiology, Fac Medicine Hradec Kralove, Charles Univ, CZ
- Daniel Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, AT. - email@example.com
- SO was supported within the framework of Internalisation of research directions of University Hospital in Hradec Kralove, Czech Republic, project number CZ.02.2.69/0.0/0.0/16_027/0008513. This study received funding within the NextGen-O2k project from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770. Contribution to COST Action CA15203 MitoEAGLE funded by the Horizon 2020 Framework Programme of the European Union.
- Gnaiger E, Aasander Frostner E, Abdul Karim N, Abdel-Rahman EA, Abumrad NA, Acuna-Castroviejo D, Adiele RC, et al (2019) Mitochondrial respiratory states and rates. MitoFit Preprint Arch doi:10.26124/mitofit:190001.v6.
- Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70.