Many tumour cells have elevated rates of glucose uptake but reduced rates of oxidative phosphorylation. This persistence of high lactate production by tumours in the presence of oxygen, known as aerobic glycolysis, was first noted by Otto Warburg more than 75 yr ago. How tumour cells establish this altered metabolic phenotype and whether it is essential for tumorigenesis is as yet unknown. Here we show that a single switch in a splice isoform of the glycolytic enzyme pyruvate kinase is necessary for the shift in cellular metabolism to aerobic glycolysis and that this promotes tumorigenesis. Tumour cells have been shown to express exclusively the embryonic M2 isoform of pyruvate kinase. Here we use short hairpin RNA to knockdown pyruvate kinase M2 expression in human cancer cell lines and replace it with pyruvate kinase M1. Switching pyruvate kinase expression to the M1 (adult) isoform leads to reversal of the Warburg effect, as judged by reduced lactate production and increased oxygen consumption, and this correlates with a reduced ability to form tumours in nude mouse xenografts. These results demonstrate that M2 expression is necessary for aerobic glycolysis and that this metabolic phenotype provides a selective growth advantage for tumour cells in vivo.

SUMMARY Cellular senescence permanently arrests cell proliferation, often accompanied by a multi-faceted senescence-associated secretory phenotype (SASP). Loss of mitochondrial function can drive age-related declines in the function of many post-mitotic tissues, but little is known about how mitochondrial dysfunction affects mitotic tissues. We show here that several manipulations that compromise mitochondrial function in proliferating human cells induce a senescence growth arrest with a modified SASP that lacks the IL-1-dependent inflammatory arm. Cells that underwent mitochondrial dysfunction-associated senescence (MiDAS) had lower NAD+/NADH ratios, which caused both the growth arrest and prevented the IL-1-associated SASP through AMPK-mediated p53 activation. Progeroid mice that rapidly accrue mtDNA mutations accumulated senescent cells with a MiDAS SASP in vivo, which suppressed adipogenesis and stimulated keratinocyte differentiation in cell culture. Our data identify a distinct senescence response and provide a mechanism by which mitochondrial dysfunction can drive aging phenotypes.

mTOR is a central regulator of cellular growth and metabolism. Using metabolic profiling and numerous small-molecule probes, we investigated whether mTOR affects immediate control over cellular metabolism by posttranslational mechanisms. Inhibiting the FKBP12/rapamycin-sensitive subset of mTOR functions in leukemic cells enhanced aerobic glycolysis and decreased uncoupled mitochondrial respiration within 25 min. mTOR is in a complex with the mitochondrial outer-membrane protein Bcl-xl and VDAC1. Bcl-xl, but not VDAC1, is a kinase substrate for mTOR in vitro, and mTOR regulates the association of Bcl-xl with mTOR. Inhibition of mTOR not only enhances aerobic glycolysis, but also induces a state of increased dependence on aerobic glycolysis in leukemic cells, as shown by the synergy between the glycolytic inhibitor 2-deoxyglucose and rapamycin in decreasing cell viability.metabolomics ͉ mitochondria ͉ chemical biology m TOR functions as a multichannel processor in a cellular nutrient-sensing network by receiving multiple inputs derived from distinct environmental cues and directing different outputs to appropriate downstream effectors. Upstream regulators of mTOR are primarily mitogens, nutrients, and energy (1). mTOR exists in 2 separate protein complexes: the mitogen-, nutrient-, and rapamycin-sensitive mTOR complex 1 (mTORC1) and the mitogen-sensitive and nutrient-and rapamycin-insensitive mTORC2 (2). mTOR regulates key cellular processes, including mRNA translation, ribosome biogenesis, autophagy, and metabolism. The most extensively studied targets of mTOR are the translation regulators S6K1 and 4E-BP1 (3). Yeast cells treated with rapamycin mount an immediate (within 20 min) and widespread transcriptional response that results in metabolic reprogramming characteristic of the diauxic shift (4). Mammalian cells do not display an immediate and widespread transcriptional response to mTOR inhibition by rapamycin (5). Inhibition of mTORC1 using either rapamycin or RNA-mediated interference of proteins involved in the signaling network decreases mitochondrial respiration and levels of fully uncoupled respiration, independent of S6K1 and 4EBP1 (6). Recently, rapamycin was reported to decrease the transcription of genes involved in mitochondrial oxidative function by disrupting a complex involving TORC1, YY1, and PGC-1␣, thereby preventing the coactivation of YY1 by PGC-1␣ (7). The authors suggested this transcriptional mechanism as the means by which mTOR controls mitochondrial function; however, treatment with rapamycin does not result in decreased mitochondrial content (6) even after 8 h.Here we describe an immediate change in mitochondrial function following the inhibition of mTOR. Using global physiological profiling, we show that inhibition of mTOR has immediate effects on carbon and mitochondrial metabolism in a leukemic cell line. Rapamycin-treated leukemic cells display reduced mitochondrial function, resulting in energy production via enhanced aerobic glycolysis in preference over mitochondrial respiration....

Weinberg and coworkers have used serial transduction of a human, primary fibroblast cell line with the catalytic domain of human telomerase, large T antigen, small T antigen, and an oncogenic allele of H-ras to study stages leading toward a fully transformed cancerous state. We performed a three-dimensional screening experiment using 4 cell lines, 5 small-molecule perturbagens (2-deoxyglucose, oxamate, oligomycin, rapamycin, and wortmannin), and a large number of metabolic measurements. Hierarchical clustering was performed to obtain signatures of the 4 cell lines, 24 cell states, 5 perturbagens, and a number of metabolic parameters. Analysis of these signatures and sensitivities of the cell lines to the perturbagens provided insights into the bioenergetic states of progressively transformed cell lines, the effect of oncogenes on small-molecule sensitivity, and global physiological responses to modulators of aerobic and anaerobic metabolism. We have gained insight into the relationship between two models of carcinogenesis, one (the Warburg hypothesis) based on increased energy production by glycolysis in cancer cells in response to aberrant respiration, and one based on cancer-causing genes. Rather than being opposing models, the approach described here suggests that these two models are interlinked. The cancercausing genes used in this study appear to increase progressively the cell's dependence on glycolytic energy production and to decrease its dependence on mitochondrial energy production. However, mitochondrial biogenesis appears to have a more complex dependence, increasing to its greatest extent at an intermediate degree of transduction rather than at the fully transformed state.cancer ͉ metabolic profiling ͉ metabolism ͉ small molecules ͉ warburg effect C ancer cells override mechanisms for controlling cellular proliferation, differentiation, and death during malignant transformation. Weinberg and coworkers (1) have shown that ectopic expression of the telomerase catalytic subunit (hTERT) in combination with simian virus 40 large T antigen (LT), small T antigen (ST), and an oncogenic allele of H-ras results in the tumorigenic conversion of normal human epithelial and fibroblast cells (1, 2). In this study, we used the four BJ fibroblast cell, and 4-[hTERT ϩ LT ϩ ST ϩ H-ras], which we abbreviate here as CL1, CL2, CL3, and CL4, respectively. To study physiological changes on the path toward tumorigenic conversion, we performed a three-dimensional screening experiment that yielded a matrix of data derived from variations in cell states, cell measurements, and small molecules (Fig. 1). To assess the bioenergetic status of each cell line CL1-CL4, we selected small-molecule inhibitors of metabolic and nutrient-sensing pathways. Each cell line was incubated individually with (i) DMSO; (ii) oxamic acid, an inhibitor of lactate dehydrogenase, which is an enzyme involved in anaerobic glycolysis; (iii) 2-deoxyglucose, an inhibitor of the glycolysis enzyme hexokinase; (iv) oligomycin, an inhibitor of mitochondrial A...

Emerging metabolomic tools have created the opportunity to establish metabolic signatures of myocardial injury. We applied a mass spectrometry-based metabolite profiling platform to 36 patients undergoing alcohol septal ablation treatment for hypertrophic obstructive cardiomyopathy, a human model of planned myocardial infarction (PMI). Serial blood samples were obtained before and at various intervals after PMI, with patients undergoing elective diagnostic coronary angiography and patients with spontaneous myocardial infarction (SMI) serving as negative and positive controls, respectively. We identified changes in circulating levels of metabolites participating in pyrimidine metabolism, the tricarboxylic acid cycle and its upstream contributors, and the pentose phosphate pathway. Alterations in levels of multiple metabolites were detected as early as 10 minutes after PMI in an initial derivation group and were validated in a second, independent group of PMI patients. A PMI-derived metabolic signature consisting of aconitic acid, hypoxanthine, trimethylamine N-oxide, and threonine differentiated patients with SMI from those undergoing diagnostic coronary angiography with high accuracy, and coronary sinus sampling distinguished cardiac-derived from peripheral metabolic changes. Our results identify a role for metabolic profiling in the early detection of myocardial injury and suggest that similar approaches may be used for detection or prediction of other disease states.

Endogenous circadian clocks orchestrate several metabolic and signaling pathways that are known to modulate lifespan, suggesting clocks as potential targets for manipulation of metabolism and lifespan. We report here that the core circadian clock genes, timeless (tim) and period (per), are required for the metabolic and lifespan responses to DR in Drosophila. Consistent with the involvement of a circadian mechanism, DR enhances the amplitude of cycling of most circadian clock genes, including tim, in peripheral tissues. Mass spectrometry-based lipidomic analysis suggests a role of tim in cycling of specific medium chain triglycerides under DR. Furthermore, overexpression of tim in peripheral tissues improves its oscillatory amplitude and extends lifespan under ad libitum conditions. Importantly, effects of tim on lifespan appear to be mediated through enhanced fat turnover. These findings identify a critical role for specific clock genes in modulating the effects of nutrient manipulation on fat metabolism and aging.

Abstract. The volume change of the Chhota Shigri Glacier (India, 32 • 20 N, 77 • 30 E) between 1988 and 2010 has been determined using in situ geodetic measurements. This glacier has experienced only a slight mass loss between 1988 and 2010 (-3.8 ± 2.0 m w.e. (water equivalent) corresponding to -0.17 pm 0.09 m w.e. yr −1 ). Using satellite digital elevation models (DEM) differencing and field measurements, we measure a negative mass balance (MB) between 1999 and 2010 (-4.8 ± 1.8 m w.e. corresponding to -0.44 ± 0.16 m w.e. yr −1 ). Thus, we deduce a slightly positive or near-zero MB between 1988 and 1999 (+1.0 ± 2.7 m w.e. corresponding to +0.09 ± 0.24 m w.e. yr −1 ). Furthermore, satellite DEM differencing reveals that the MB of the Chhota Shigri Glacier (-0.39 pm 0.15 m w.e. yr −1 ) has been only slightly less negative than the MB of a 2110 km 2 glaciarized area in the Lahaul and Spiti region (-0.44 ± 0.09 m w.e. yr −1 ) during 1999-2011. Hence, we conclude that the ice wastage is probably moderate in this region over the last 22 yr, with near equilibrium conditions during the nineties, and an ice mass loss after. The turning point from balanced to negative mass budget is not known but lies probably in the late nineties and at the latest in 1999. This positive or near-zero MB for Chhota Shigri Glacier (and probably for the surrounding glaciers of the Lahaul and Spiti region) during at least part of the 1990s contrasts with a recent compilation of MB data in the Himalayan range that indicated ice wastage since 1975. However, in agreement with this compilation, we confirm more negative balances since the beginning of the 21st century.

Mitochondrial oxidative phosphorylation (OXPHOS) is central to physiology and disease pathogenesis. To systematically investigate its activity and regulation, we performed a wide range of assays of OXPHOS physiology and nuclear and mitochondrial gene expression across 2490 chemical perturbations in muscle cells. Through mining of the resulting compendium, we discovered that: (1) protein synthesis inhibitors can de-couple coordination of nuclear and mitochondrial transcription; (2) a subset of HMG-CoA reductase inhibitors, in combination with nonselective beta-adrenergic receptor antagonists, can cause mitochondrial toxicity, providing clues into statin-associated myopathy; and (3) structurally diverse microtubule inhibitors stimulate OXPHOS transcription while suppressing reactive oxygen species, via a PGC-1α/ERRα-dependent mechanism, and thus may have utility in treating age-associated degenerative disorders. Our screening compendium is freely available and can be used as a discovery tool for understanding mitochondrial biology and toxicity, and identifying novel therapeutics.