ATP-linked Oxygen Consumption Rate Research Articles

Bioenergetic programming of macrophages by the apolipoprotein A-I mimetic peptide 4F.

The apoA-I (apolipoprotein A-I) mimetic peptide 4F favours the differentiation of human monocytes to an alternatively activated M2 phenotype. The goal of the present study was to test whether the 4F-mediated differentiation of MDMs (monocyte-derived macrophages) requires the induction of an oxidative metabolic programme. 4F treatment induced several genes in MDMs that play an important role in lipid metabolism, including PPARγ (peroxisome-proliferator-activated receptor γ) and CD36. Addition of 4F was associated with a significant increase in FA (fatty acid) uptake and oxidation compared with vehicle treatment. Mitochondrial respiration was assessed by measurement of the OCR (oxygen-consumption rate). 4F increased basal and ATP-linked OCR as well as maximal uncoupled mitochondrial respiration. These changes were associated with a significant increase in ΔΨm (mitochondrial membrane potential). The increase in metabolic activity in 4F-treated MDMs was attenuated by etomoxir, an inhibitor of mitochondrial FA uptake. Finally, addition of the PPARγ antagonist T0070907 to 4F-treated MDMs reduced the expression of CD163 and CD36, cell-surface markers for M2 macrophages, and reduced basal and ATP-linked OCR. These results support our hypothesis that the 4F-mediated differentiation of MDMs to an anti-inflammatory phenotype is due, in part, to an increase in FA uptake and mitochondrial oxidative metabolism.

Bioenergetic differences between MCF-7 and T47D breast cancer cells and their regulation by oestradiol and tamoxifen

Oestrogen receptor α (ERα+) breast tumours rely on mitochondria (mt) to generate ATP. The goal of the present study was to determine how oestradiol (E2) and 4-hydroxytamoxifen (4-OHT) affect cellular bioenergetic function in MCF-7 and T47D ERα+ breast cancer cells in serum-replete compared with dextran-coated charcoal (DCC)-stripped foetal bovine serum (FBS)-containing medium ('serum-starved'). Serum-starvation reduced oxygen consumption rate (OCR), extracellular acidification rate (ECAR), ATP-linked OCR and maximum mt capacity, reflecting lower ATP demand and mt respiration. Cellular respiratory stateapparent was unchanged by serum deprivation. 4-OHT reduced OCR independent of serum status. Despite having a higher mt DNA/nuclear DNA ratio than MCF-7 cells, T47D cells have a lower OCR and ATP levels and higher proton leak. T47D express higher nuclear respiratory factor-1 (NRF-1) and NRF-1-regulated, nuclear-encoded mitochondrial transcription factor TFAM and cytochrome c, but lower levels of cytochrome c oxidase, subunit IV, isoform 1 (COX4, COX4I1). Mitochondrial reserve capacity, reflecting tolerance to cellular stress, was higher in serum-starved T47D cells and was increased by 4-OHT, but was decreased by 4-OHT in MCF-7 cells. These data demonstrate critical differences in cellular energetics and responses to 4-OHT in these two ERα+ cell lines, likely reflecting cancer cell avoidance of apoptosis.

Abstract 1120: Bioenergetics of T cells in the context of adoptive immunotherapy

Abstract Tumor-specific T cell therapy has been used as an experimental approach for an anti-tumor therapy (Sadelain M 2003, Gattinoni L 2006). Adoptive T cell transfer therapies rely on ex vivo T cell isolation, transduction, activation and expansion of autologous tumor-reactive T cell populations prior to patient administration (Yee C 2002, Dudley ME 2003, Bollard CM 2004). The metabolic changes occurring in modified T-cells are the object of growing interest. For example, it has been shown that quiescent T cells display low energetic and biosynthetic demands, generating ATP through the TCA cycle and OXPHOS (oxidative phosphorylation). Activated T cells undergo conversion from a resting to an active state and ATP production switches from OXPHOS to high glycolysis. Our objective was to assess T cell metabolism and evaluate changes in glycolytic and mitochnodrial function that occur during transduction and expansion, prior to administration of T-cells in adoptive immunotherapy protocols. Methods. Human T-cells were isolated from buffy coat using ficoll separation. 48 hours after phytohemagglutinin (PHA) stimulation, cells were transduced with the retroviral vector bearing chimeric antigen receptor (CAR) targeting PSMA (Maher, Brentjens et al. 2002), and transduction efficacy was assessed by FACS. T-cells further were stimulated by exposure to antigen-presenting cells, APC. The metabolic profiles were determined at different steps of T cell stimulation and transduction. A Seahorse XF96 Analyzer was used to measure glycolysis (extracellular acidification rate, ECAR) and the oxygen consumption rate (OCR), a measure of oxidative phosphorylation (OXPHOS). Results: Glycolysis and oxygen consumption was low in resting non-stimulated cells. Both CAR-transduced and non-transduced T cells increased glycolysis (5-fold), oxygen consumption (3-fold) and ATP-linked OCR following PHA stimulation. No significant metabolic differences were observed between CAR-transduced and non-transduced T cells. High glycolytic activity was maintained by T cells over the 25 day course of T cell transduction and expansion. Mitochondrial function (oxygen consumption) declined during this period. Restimulation by exposure to antigen-presenting cells resulted in a small mitochondrial response in transduced T cells, whereas glycolysis remained high and showed no additional response. Conclusion: Glycolysis (ECAR) remains high, whereas oxygen consumption (OCR and OXPHOS) declines during T cell preparation for adoptive T cell therapy. Whether the specific changes in glucose metabolism and mitochondria respiration can influence cytotoxic function remains to be elucidated. Citation Format: Ekaterina Moroz, Maxim Moroz, Inna Serganova, Juan Zurita, Jason Lee, Nisargbhai Shah, Vladimir Ponomarev, Ronald Blasberg. Bioenergetics of T cells in the context of adoptive immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1120. doi:10.1158/1538-7445.AM2014-1120

AKIP1 Expression Modulates Mitochondrial Function in Rat Neonatal Cardiomyocytes

A kinase interacting protein 1 (AKIP1) is a molecular regulator of protein kinase A and nuclear factor kappa B signalling. Recent evidence suggests AKIP1 is increased in response to cardiac stress, modulates acute ischemic stress response, and is localized to mitochondria in cardiomyocytes. The mitochondrial function of AKIP1 is, however, still elusive. Here, we investigated the mitochondrial function of AKIP1 in a neonatal cardiomyocyte model of phenylephrine (PE)-induced hypertrophy. Using a seahorse flux analyzer we show that PE stimulated the mitochondrial oxygen consumption rate (OCR) in cardiomyocytes. This was partially dependent on PE mediated AKIP1 induction, since silencing of AKIP1 attenuated the increase in OCR. Interestingly, AKIP1 overexpression alone was sufficient to stimulate mitochondrial OCR and in particular ATP-linked OCR. This was also true when pyruvate was used as a substrate, indicating that it was independent of glycolytic flux. The increase in OCR was independent of mitochondrial biogenesis, changes in ETC density or altered mitochondrial membrane potential. In fact, the respiratory flux was elevated per amount of ETC, possibly through enhanced ETC coupling. Furthermore, overexpression of AKIP1 reduced and silencing of AKIP1 increased mitochondrial superoxide production, suggesting that AKIP1 modulates the efficiency of electron flux through the ETC. Together, this suggests that AKIP1 overexpression improves mitochondrial function to enhance respiration without excess superoxide generation, thereby implicating a role for AKIP1 in mitochondrial stress adaptation. Upregulation of AKIP1 during different forms of cardiac stress may therefore be an adaptive mechanism to protect the heart.

Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death

BackgroundRecent research has revealed that targeting mitochondrial bioenergetic metabolism is a promising chemotherapeutic strategy. Key to successful implementation of this chemotherapeutic strategy is the use of new and improved mitochondria-targeted cationic agents that selectively inhibit energy metabolism in breast cancer cells, while exerting little or no long-term cytotoxic effect in normal cells.MethodsIn this study, we investigated the cytotoxicity and alterations in bioenergetic metabolism induced by mitochondria-targeted vitamin E analog (Mito-chromanol, Mito-ChM) and its acetylated ester analog (Mito-ChMAc). Assays of cell death, colony formation, mitochondrial bioenergetic function, intracellular ATP levels, intracellular and tissue concentrations of tested compounds, and in vivo tumor growth were performed.ResultsBoth Mito-ChM and Mito-ChMAc selectively depleted intracellular ATP and caused prolonged inhibition of ATP-linked oxygen consumption rate in breast cancer cells, but not in non-cancerous cells. These effects were significantly augmented by inhibition of glycolysis. Mito-ChM and Mito-ChMAc exhibited anti-proliferative effects and cytotoxicity in several breast cancer cells with different genetic background. Furthermore, Mito-ChM selectively accumulated in tumor tissue and inhibited tumor growth in a xenograft model of human breast cancer.ConclusionsWe conclude that mitochondria-targeted small molecular weight chromanols exhibit selective anti-proliferative effects and cytotoxicity in multiple breast cancer cells, and that esterification of the hydroxyl group in mito-chromanols is not a critical requirement for its anti-proliferative and cytotoxic effect.

Toxic effects of T-2 toxin and deoxynivalenol on the mitochondrial electron transport system of cardiomyocytes in rats

The in vitro effects of 2 representative mycotoxins, T-2 toxin and deoxynivalenol (DON), of trichothecene group on the electron transport system (ETS) of mitochondria in rat cardiomyocytes were investigated by measuring oxygen consumption rates (OCR). The ATP-linked OCR and the reserve capacity (RC) of the mitochondria ETS were quantified by a "mitochondria stress test" which was estimated by the OCR responses to oligomycin and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, with an extracellular flux analyzer. The basal OCR was significantly inhibited by the application of T-2 toxin at concentrations of 6 × 10⁻¹ to 6 × 10⁻⁵ μM and DON at concentrations of 0.78 to 100 μM for 24 hr. The threshold of cardiomyocyte toxicity was estimated to be between 6.0 × 10⁻⁶ and 6.0 × 10⁻⁵ μM for T-2 toxicity on both ATP-linked OCR and RC and between 0.39 and 0.78 μM on ATP-linked OCR or between 1.56 and 3.13 μM on RC for DON. The decrease in OCR of cardiomyocytes exposed to T-2 toxin with a concentration of 6.0 × 10⁻³ and 6.0 × 10⁻⁴ μM was significantly inhibited by antioxidants, catalase and vitamin C. In conclusion, the present study demonstrated, through the direct and real-time measurement of respiratory function in mitochondria, that a marked inhibition of mitochondrial ETS function in cardiomyocytes was induced by T-2 toxin and DON and that the mitochondrial dysfunction by T-2 toxin was largely associated with oxidative stress.

Distinct effects of rotenone, 1-methyl-4-phenylpyridinium and 6-hydroxydopamine on cellular bioenergetics and cell death.

Parkinson’s disease is characterized by dopaminergic neurodegeneration and is associated with mitochondrial dysfunction. The bioenergetic susceptibility of dopaminergic neurons to toxins which induce Parkinson’s like syndromes in animal models is then of particular interest. For example, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP+), and 6-hydroxydopamine (6-OHDA), have been shown to induce dopaminergic cell death in vivo and in vitro. Exposure of animals to these compounds induce a range of responses characteristics of Parkinson’s disease, including dopaminergic cell death, and Reactive Oxygen Species (ROS) production. Here we test the hypothesis that cellular bioenergetic dysfunction caused by these compounds correlates with induction of cell death in differentiated dopaminergic neuroblastoma SH-SY5Y cells. At increasing doses, rotenone induced significant cell death accompanied with caspase 3 activation. At these concentrations, rotenone had an immediate inhibition of mitochondrial basal oxygen consumption rate (OCR) concomitant with a decrease of ATP-linked OCR and reserve capacity, as well as a stimulation of glycolysis. MPP+ exhibited a different behavior with less pronounced cell death at doses that nearly eliminated basal and ATP-linked OCR. Interestingly, MPP+, unlike rotenone, stimulated bioenergetic reserve capacity. The effects of 6-OHDA on bioenergetic function was markedly less than the effects of rotenone or MPP+ at cytotoxic doses, suggesting a mechanism largely independent of bioenergetic dysfunction. These studies suggest that these dopaminergic neurotoxins induce cell death through distinct mechanisms and differential effects on cellular bioenergetics.