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Figure S1. STS-induced death is enhanced in UCP2hi MIA PaCa-2 and UCP2hi U-87 MG cells.

Cancer cells tilt their energy production away from oxidative phosphorylation (OXPHOS) toward glycolysis during malignant progression, even when aerobic metabolism is available. Reversing this phenomenon, known as the Warburg effect, may offer a generalized anticancer strategy. In this study, we show that overexpression of the mitochondrial membrane transport protein UCP2 in cancer cells is sufficient to restore a balance toward oxidative phosphorylation and to repress malignant phenotypes. Altered expression of glycolytic and oxidative enzymes mediated the effects of this metabolic shift. Notably, UCP2 overexpression increased signaling from the master energy-regulating kinase, adenosine monophosphate-activated protein kinase, while downregulating expression of hypoxia-induced factor. In support of recent new evidence about UCP2 function, we found that UCP2 did not function in this setting as a membrane potential uncoupling protein, but instead acted to control routing of mitochondria substrates. Taken together, our results define a strategy to reorient mitochondrial function in cancer cells toward OXPHOS that restricts their malignant phenotype. Cancer Res; 74(14); 3971–82. ©2014 AACR.

HIV-1 eradication is hindered by viral persistence in cell reservoirs, established not only in circulatory CD4+T-cells but also in tissue-resident macrophages. The nature of macrophage reservoirs and mechanisms of persistence despite combined anti-retroviral therapy (cART) remain unclear. Using genital mucosa from cART-suppressed HIV-1-infected individuals, we evaluated the implication of macrophage immunometabolic pathways in HIV-1 persistence. We demonstrate that ex vivo, macrophage tissue reservoirs contain transcriptionally active HIV-1 and viral particles accumulated in virus-containing compartments, and harbor an inflammatory IL-1R+S100A8+MMP7+M4-phenotype prone to glycolysis. Reactivation of infectious virus production and release from these reservoirs in vitro are induced by the alarmin S100A8, an endogenous factor produced by M4-macrophages and implicated in “sterile” inflammation. This process metabolically depends on glycolysis. Altogether, inflammatory M4-macrophages form a major tissue reservoir of replication-competent HIV-1, which reactivate viral production upon autocrine/paracrine S100A8-mediated glycolytic stimulation. This HIV-1 persistence pathway needs to be targeted in future HIV eradication strategies.

Addition of hydrogen peroxide (H2O2) is a method commonly used to trigger cellular oxidative stress. However, the doses used (often hundreds of micromolar) are disproportionally high with regard to physiological oxygen concentration (low micromolar). In this study using polarographic measurement of oxygen concentration in cellular suspensions we show that H2O2 addition results in O2 release as expected from catalase reaction. This reaction is fast enough to, within seconds, decrease drastically H2O2 concentration and to annihilate it within a few minutes. Firstly, this is likely to explain why recording of oxidative damage requires the high concentrations found in the literature. Secondly, it illustrates the potency of intracellular antioxidant (H2O2) defense. Thirdly, it complicates the interpretation of experiments as subsequent observations might result from high/transient H2O2 exposure and/or from the diverse possible consequences of the O2 release.

Embryonic stem cells (ESC) have the unique ability to differentiate into all three germ cell layers. ESC transition through different states of pluripotency in response to growth factor signals and environmental cues before becoming terminally differentiated. Here, we demonstrated, by a multi-omic strategy, that the deubiquitinase USP9X regulates the developmental potential of ESC, and their transition from a naive to a more developmentally advance, or primed, state of pluripotency. We show that USP9X facilitates developmental gene expression and induces modifications of the mitochondrial bioenergetics, including decreased routing of pyruvate towards its oxidation and reduced respiration. In addition, USP9X binds to the pluripotency factor ESRRB, regulates its abundance and the transcriptional levels of a subset of its target genes. Finally, under permissive culture conditions, depletion of Usp9X accelerates cell differentiation in all cell lineages. We thus identified a new regulator of naive pluripotency and show that USP9X couples ESRRB pluripotency transcriptional network and cellular metabolism, both of which are important for ESC fate and pluripotency.

Sulfide (H 2 S in the gas form) is the third gaseous transmitter found in mammals. However, in contrast to nitric oxide (NO) or carbon monoxide (CO), sulfide is oxidized by a sulfide quinone reductase and generates electrons that enter the mitochondrial respiratory chain arriving ultimately at cytochrome oxidase, where they combine with oxygen to generate water. In addition, sulfide is also a strong inhibitor of cytochrome oxidase, similar to NO, CO and cyanide. The balance between the electron donor and the inhibitory role of sulfide is likely controlled by sulfide and oxygen availability. The present study aimed to evaluate if and how sulfide release and oxidation impacts on the cellular affinity for oxygen. Results : i ) when sulfide delivery approaches the maximal sulfide oxidation rate cells become exquisitely dependent on oxygen; ii ) a positive feedback makes the balance between sulfide-releasing and -oxidizing rates the relevant parameter rather than the absolute values of these rates, and; iii ) this altered dependence on oxygen is detected with sulfide concentrations that remain in the low micromolar range. Conclusions : i ) within the context of continuous release of sulfide stemming from cellular metabolism, alterations in the activity of the sulfide oxidation pathway fine-tunes the cell's affinity for oxygen, and; ii ) a decrease in the expression of the sulfide oxidation pathway greatly enhances the cell's dependence on oxygen concentration.

Until recently, hydrogen sulfide (H2S) was exclusively viewed a toxic gas and an environmental hazard, with its toxicity primarily attributed to the inhibition of mitochondrial Complex IV, resulting in a shutdown of mitochondrial electron transport and cellular ATP generation. Work over the last decade established multiple biological regulatory roles of H2S, as an endogenous gaseous transmitter. H2S is produced by cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). In striking contrast to its inhibitory effect on Complex IV, recent studies showed that at lower concentrations, H2S serves as a stimulator of electron transport in mammalian cells, by acting as a mitochondrial electron donor. Endogenous H2S, produced by mitochondrially localized 3-MST, supports basal, physiological cellular bioenergetic functions; the activity of this metabolic support declines with physiological aging. In specialized conditions (calcium overload in vascular smooth muscle, colon cancer cells), CSE and CBS can also associate with the mitochondria; H2S produced by these enzymes, serves as an endogenous stimulator of cellular bioenergetics. The current article overviews the biochemical mechanisms underlying the stimulatory and inhibitory effects of H2S on mitochondrial function and cellular bioenergetics and discusses the implication of these processes for normal cellular physiology. The relevance of H2S biology is also discussed in the context of colonic epithelial cell physiology: colonocytes are exposed to high levels of sulfide produced by enteric bacteria, and serve as a metabolic barrier to limit their entry into the mammalian host, while, at the same time, utilizing it as a metabolic ‘fuel’. Linked Articles This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8