Calorie restriction (CR) induces a metabolic shift towards mitochondrial respiration; however, molecular mechanisms underlying CR remain unclear. Recent studies suggest that CR-induced mitochondrial activity is associated with nitric oxide (NO) production. To understand the role of mitochondria in CR, we identify and study Saccharomyces cerevisiae mutants with increased NO levels as potential CR mimics. Analysis of the top 17 mutants demonstrates a correlation between increased NO, mitochondrial respiration, and longevity. Interestingly, treating yeast with NO donors such as GSNO (S-nitrosoglutathione) is sufficient to partially mimic CR to extend lifespan. CR-increased NO is largely dependent on mitochondrial electron transport and cytochrome c oxidase (COX). Although COX normally produces NO under hypoxic conditions, CR-treated yeast cells are able to produce NO under normoxic conditions. Our results suggest that CR may derepress some hypoxic genes for mitochondrial proteins that function to promote the production of NO and the extension of lifespan. 1. Introduction Calorie restriction (CR) extends lifespan in a variety of organisms and has also been shown to ameliorate many age-associated diseases such as diabetes and cancers [1–3]. However, the molecular mechanisms underlying CR-induced beneficial effects are still not fully understood. Owing to a short lifespan and well-established molecular genetic techniques, the budding yeast Saccharomyces cerevisiae represents a powerful system to identify new components in CR signaling pathways and to study these factors at the molecular/genetic level. Yeast lifespan can be studied in two distinct ways: replicative lifespan (RLS) and chronological lifespan (CLS). RLS measures the number of cell divisions an individual yeast cell undergoes before senescence (division potential), whereas CLS measures the length of time cells remain viable at a nondividing state (postmitotic survival). RLS may serve as a model to understand the mechanisms of replicative senescence such as in stem cells, while CLS may be more relevant to postmitotic cell senescence in adult animals [4, 5]. Moderate CR can be imposed in yeast by reducing the glucose concentration from 2% to 0.5% in rich media [6–9], which extends both RLS and CLS. In yeast, CR is suggested to function through reducing the activities of conserved nutrient-sensing pathways. Decreasing the activities of the Ras-cAMP/PKA (cyclic AMP-activated protein kinase A) pathway, Sch9 (homolog of mammalian S6K kinases) and Tor1 kinases have been shown to mimic CR and extend
References
[1]
W. Weindruch and R. L. Walford, The Retardation of Aging and Diseases by Dietary Restriction, Charles C. Thomas, Springfield, Ill, USA, 1998.
[2]
G. S. Roth, D. K. Ingram, and M. A. Lane, “Caloric restriction in primates and relevance to humans,” Annals of the New York Academy of Sciences, vol. 928, pp. 305–315, 2001.
[3]
L. Guarente, “Mitochondria-A Nexus for Aging, Calorie Restriction, and Sirtuins?” Cell, vol. 132, no. 2, pp. 171–176, 2008.
[4]
P. Fabrizio and V. D. Longo, “The chronological life span of Saccharomyces cerevisiae,” Aging Cell, vol. 2, no. 2, pp. 73–81, 2003.
[5]
C. Skinner and S.-J. Lin, “Effects of calorie restriction on life span of microorganisms,” Applied Microbiology and Biotechnology, vol. 88, pp. 817–828, 2010.
[6]
S. J. Lin, P. A. Defossez, and L. Guarente, “Requirement of NAD and SIR2 for life-span extension by calorie restriction in saccharomyces cerevisiae,” Science, vol. 289, no. 5487, pp. 2126–2128, 2000.
[7]
E. Easlon, F. Tsang, I. Dilova et al., “The dihydrolipoamide acetyltransferase is a novel metabolic longevity factor and is required for calorie restriction-mediated life span extension,” Journal of Biological Chemistry, vol. 282, no. 9, pp. 6161–6171, 2007.
[8]
D. L. Smith Jr., J. M. McClure, M. Matecic, and J. S. Smith, “Calorie restriction extends the chronological lifespan of Saccharomyces cerevisiae independently of the Sirtuins,” Aging Cell, vol. 6, no. 5, pp. 649–662, 2007.
[9]
M. Wei, P. Fabrizio, J. Hu et al., “Life span extension by calorie restriction depends on Rim15 and transcription factors downstream of Ras/PKA, Tor, and Sch9.,” PLoS Genetics, vol. 4, no. 1, article e13, 2008.
[10]
P. Fabrizio, F. Pozza, S. D. Pletcher, C. M. Gendron, and V. D. Longo, “Regulation of longevity and stress resistance by Sch9 in yeast,” Science, vol. 292, no. 5515, pp. 288–290, 2001.
[11]
M. Kaeberlein, R. W. Powers, K. K. Steffen et al., “Cell biology: regulation of yeast replicative life span by TOR and Sch9 response to nutrients,” Science, vol. 310, no. 5751, pp. 1193–1196, 2005.
[12]
S. J. Lin, M. Kaeberlein, A. A. Andalis, et al., “Calorie restriction extends life span by shifting carbon toward respiration,” Nature, vol. 418, pp. 344–348, 2002.
[13]
D. Chen, A. D. Steele, S. Lindquist, and L. Guarente, “Medicine: increase in activity during calorie restriction requires Sirt1,” Science, vol. 310, no. 5754, p. 1641, 2005.
[14]
N. A. Bishop and L. Guarente, “Two neurons mediate diet-restriction-induced longevity in C. elegans,” Nature, vol. 447, no. 7144, pp. 545–549, 2007.
[15]
S. H. Panowski, S. Wolff, H. Aguilaniu, J. Durieux, and A. Dillin, “PHA-4/Foxa mediates diet-restriction-induced longevity of C. elegans,” Nature, vol. 447, no. 7144, pp. 550–555, 2007.
[16]
E. Easlon, F. Tsang, C. Skinner, C. Wang, and SU. J. Lin, “The malate-aspartate NADH shuttle components are novel metabolic longevity regulators required for calorie restriction-mediated life span extension in yeast,” Genes and Development, vol. 22, no. 7, pp. 931–944, 2008.
[17]
K. K. Steffen, V. L. MacKay, E. O. Kerr et al., “Yeast life span extension by depletion of 60s ribosomal subunits is mediated by Gcn4,” Cell, vol. 133, no. 2, pp. 292–302, 2008.
[18]
I. Dilova, E. Easlon, and S. J. Lin, “Calorie restriction and the nutrient sensing signaling pathways,” Cellular and Molecular Life Sciences, vol. 64, no. 6, pp. 752–767, 2007.
[19]
S. I. Imai and L. Guarente, “Ten years of NAD-dependent SIR2 family deacetylases: implications for metabolic diseases,” Trends in Pharmacological Sciences, vol. 31, no. 5, pp. 212–220, 2010.
[20]
E. Nisoli, C. Tonello, A. Cardile et al., “Cell biology: calorie restriction promotes mitochondrial biogenesis by inducing the expression of eNOS,” Science, vol. 310, no. 5746, pp. 314–317, 2005.
[21]
T. J. Schulz, K. Zarse, A. Voigt, N. Urban, M. Birringer, and M. Ristow, “Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress,” Cell Metabolism, vol. 6, no. 4, pp. 280–293, 2007.
[22]
K. Zarse, T. J. Schulz, M. Birringer, and M. Ristow, “Impaired respiration is positively correlated with decreased life span in Caenorhabditis elegans models of Friedreich Ataxia,” FASEB Journal, vol. 21, no. 4, pp. 1271–1275, 2007.
[23]
J. M. Zahn, S. Poosala, A. B. Owen et al., “AGEMAP: a gene expression database for aging in mice,” PLoS Genetics, vol. 3, no. 11, article e201, 2007.
[24]
J. P. de Magalh？es, J. Curado, and G. M. Church, “Meta-analysis of age-related gene expression profiles identifies common signatures of aging,” Bioinformatics, vol. 25, no. 7, pp. 875–881, 2009.
[25]
S. I. Imai, “SIRT1 and caloric restriction: an insight into possible trade-offs between robustness and frailty,” Current Opinion in Clinical Nutrition and Metabolic Care, vol. 12, no. 4, pp. 350–356, 2009.
[26]
R. M. Anderson and R. Weindruch, “Metabolic reprogramming, caloric restriction and aging,” Trends in Endocrinology and Metabolism, vol. 21, no. 3, pp. 134–141, 2010.
[27]
R. H. Houtkooper, R. W. Williams, and J. Auwerx, “Metabolic networks of longevity,” Cell, vol. 142, no. 1, pp. 9–14, 2010.
[28]
P. Fabrizio, C. Gattazzo, L. Battistella et al., “Sir2 blocks extreme life-span extension,” Cell, vol. 123, no. 4, pp. 655–667, 2005.
[29]
C. R. Burtner, C. J. Murakami, B. K. Kennedy, and M. Kaeberlein, “A molecular mechanism of chronological aging in yeast,” Cell Cycle, vol. 8, no. 8, pp. 1256–1270, 2009.
[30]
L. Fontana, L. Partridge, and V. D. Longo, “Extending healthy life span-from yeast to humans,” Science, vol. 328, no. 5976, pp. 321–326, 2010.
[31]
M. H. Barros, B. Bandy, E. B. Tahara, and A. J. Kowaltowski, “Higher respiratory activity decreases mitochondrial reactive oxygen release and increases life span in Saccharomyces cerevisiae,” Journal of Biological Chemistry, vol. 279, no. 48, pp. 49883–49888, 2004.
[32]
R. O. Poyton, K. A. Ball, and P. R. Castello, “Mitochondrial generation of free radicals and hypoxic signaling,” Trends in Endocrinology and Metabolism, vol. 20, no. 7, pp. 332–340, 2009.
[33]
C. Wang, C. Skinner, E. Easlon, and SU. J. Lin, “Deleting the 14-3-3 protein Bmh1 extends life span in Saccharomyces cerevisiae by increasing stress response,” Genetics, vol. 183, no. 4, pp. 1373–1384, 2009.
[34]
P. R. Castello, P. S. David, T. McClure, Z. Crook, and R. O. Poyton, “Mitochondrial cytochrome oxidase produces nitric oxide under hypoxic conditions: implications for oxygen sensing and hypoxic signaling in eukaryotes,” Cell Metabolism, vol. 3, no. 4, pp. 277–287, 2006.
[35]
B. Derakhshan, P. C. Wille, and S. S. Gross, “Unbiased identification of cysteine S-nitrosylation sites on proteins,” Nature protocols, vol. 2, no. 7, pp. 1685–1691, 2007.
[36]
A. Balcerczyk, M. Soszynski, and G. Bartosz, “On the specificity of 4-amino-5-methylamino-2′,7′- difluorofluorescein as a probe for nitric oxide,” Free Radical Biology and Medicine, vol. 39, no. 3, pp. 327–335, 2005.
[37]
A. Lewinska and G. Bartosz, “Yeast flavohemoglobin protects against nitrosative stress and controls ferric reductase activity,” Redox Report, vol. 11, no. 5, pp. 231–239, 2006.
[38]
P. R. Castello, K. W. Dong, K. Ball, J. Wojcik, L. Liu, and R. O. Poyton, “Oxygen-regulated isoforms of cytochrome c oxidase have differential effects on its nitric oxide production and on hypoxic signaling,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 24, pp. 8203–8208, 2008.
[39]
E. E. McKee and R. O. Poyton, “Mitochondrial gene expression in Saccharomyces cerevisiae. I. Optimal conditions for protein synthesis in isolated mitochondria,” Journal of Biological Chemistry, vol. 259, no. 14, pp. 9320–9331, 1984.
[40]
L. Liu, A. Hausladen, M. Zeng, L. Que, J. Heitman, and J. S. Stamler, “A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans,” Nature, vol. 410, no. 6827, pp. 490–494, 2001.
[41]
L. Liu, M. Zeng, A. Hausladen, J. Heitman, and J. S. Stamler, “Protection from nitrosative stress by yeast flavohemoglobin,” Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 9, pp. 4672–4676, 2000.
[42]
M. Benhar, M. T. Forrester, D. T. Hess, and J. S. Stamler, “Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins,” Science, vol. 320, no. 5879, pp. 1050–1054, 2008.
[43]
B. Almeida, S. Buttner, S. Ohlmeier et al., “NO-mediated apoptosis in yeast,” Journal of Cell Science, vol. 120, no. 18, pp. 3279–3288, 2007.
[44]
J. C. Pruessner, C. Kirschbaum, G. Meinlschmid, and D. H. Hellhammer, “Two formulas for computation of the area under the curve represent measures of total hormone concentration versus time-dependent change,” Psychoneuroendocrinology, vol. 28, no. 7, pp. 916–931, 2003.
[45]
D. B. Fekedulegn, M. E. Andrew, C. M. Burchfiel et al., “Area under the curve and other summary indicators of repeated waking cortisol measurements,” Psychosomatic Medicine, vol. 69, no. 7, pp. 651–659, 2007.
[46]
N. Cassanova, K. M. O'Brien, B. T. Stahl, T. McClure, and R. O. Poyton, “Yeast flavohemoglobin, a nitric oxide oxidoreductase, is located in both the cytosol and the mitochondrial matrix: effects of respiration, anoxia, and the mitochondrial genome on its intracellular level and distribution,” Journal of Biological Chemistry, vol. 280, no. 9, pp. 7645–7653, 2005.
[47]
R. C. Dickson, “Thematic review series: sphingolipids. New insights into sphingolipid metabolism and function in budding yeast,” The Journal of Lipid Research, vol. 49, pp. 909–921, 2008.
[48]
E. D. Smith, M. Tsuchiya, L. A. Fox et al., “Quantitative evidence for conserved longevity pathways between divergent eukaryotic species,” Genome Research, vol. 18, no. 4, pp. 564–570, 2008.
[49]
R. Bosson, M. Jaquenoud, and A. Conzelmann, “GUP1 of Saccharomyces cerevisiae encodes an O-acyltransferase involved in remodeling of the GPI anchor,” Molecular Biology of the Cell, vol. 17, no. 6, pp. 2636–2645, 2006.
[50]
J. J. Wyrick, F. C. P. Holstege, E. G. Jennings et al., “Chromosomal landscape of nucleosome-dependent gene expression and silencing in yeast,” Nature, vol. 402, no. 6760, pp. 418–421, 1999.
[51]
P. Tongaonkar, S. L. French, M. L. Oakes et al., “Histones are required for transcription of yeast rRNA genes by RNA polymerase I,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 29, pp. 10129–10134, 2005.
[52]
D. C. Hess, C. Myers, C. Huttenhower et al., “Computationally driven, quantitative experiments discover genes required for mitochondrial biogenesis,” PLoS Genetics, vol. 5, no. 3, Article ID e1000407, 2009.
[53]
N. D. Bonawitz, M. Chatenay-Lapointe, Y. Pan, and G. S. Shadel, “Reduced TOR signaling extends chronological life span via increased respiration and upregulation of mitochondrial gene expression,” Cell Metabolism, vol. 5, no. 4, pp. 265–277, 2007.
[54]
D. R. Lorenz, C. R. Cantor, and J. J. Collins, “A network biology approach to aging in yeast,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 4, pp. 1145–1150, 2009.
[55]
K. E. Kwast, P. V. Burke, K. Brown, and R. O. Poyton, “REO1 and ROX1 are alleles of the same gene which encodes a transcriptional repressor of hypoxic genes in Saccharomyces cerevisiae,” Current Genetics, vol. 32, no. 6, pp. 377–383, 1997.
[56]
K. E. Kwast, P. V. Burke, B. T. Staahl, and R. O. Poyton, “Oxygen sensing in yeast: evidence for the involvement of the respiratory chain in regulating the transcription of a subset of hypoxic genes,” Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 10, pp. 5446–5451, 1999.
[57]
R. Fukuda, H. Zhang, J. W. Kim, L. Shimoda, C. V. Dang, and G. Semenza, “HIF-1 Regulates Cytochrome Oxidase Subunits to Optimize Efficiency of Respiration in Hypoxic Cells,” Cell, vol. 129, no. 1, pp. 111–122, 2007.
[58]
A. Mesquita, M. Weinberger, A. Silva et al., “Caloric restriction or catalase inactivation extends yeast chronological lifespan by inducing H2O2 and superoxide dismutase activity,” Proceedings of the National Academy of Sciences of the United States of America, vol. 107, no. 34, pp. 15123–15128, 2010.
[59]
Y. Zhang and N. Hogg, “S-Nitrosothiols: cellular formation and transport,” Free Radical Biology and Medicine, vol. 38, no. 7, pp. 831–838, 2005.
[60]
A. Zuin, M. Carmona, I. Morales-Ivorra et al., “Lifespan extension by calorie restriction relies on the Sty1 MAP kinase stress pathway,” EMBO Journal, vol. 29, no. 5, pp. 981–991, 2010.
[61]
T. I. Lee, N. J. Rinaldi, F. Robert et al., “Transcriptional regulatory networks in Saccharomyces cerevisiae,” Science, vol. 298, no. 5594, pp. 799–804, 2002.
[62]
D. B. Berry and A. P. Gasch, “Stress-activated genomic expression changes serve a preparative role for impending stress in yeast,” Molecular Biology of the Cell, vol. 19, no. 11, pp. 4580–4587, 2008.
[63]
G. Chua, Q. D. Morris, R. Sopko et al., “Identifying transcription factor functions and targets by phenotypic activation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 32, pp. 12045–12050, 2006.
[64]
M. Rep, M. Proft, F. Remize et al., “The Saccharomyces cerevisiae Sko1p transcription factor mediates HOG pathway-dependent osmotic regulation of a set of genes encoding enzymes implicated in protection from oxidative damage,” Molecular Microbiology, vol. 40, no. 5, pp. 1067–1083, 2001.
[65]
R. O. Poyton, P. R. Castello, K. A. Ball, D. K. Woo, and N. Pan, “Mitochondria and hypoxic signaling: a new view,” Annals of the New York Academy of Sciences, vol. 1177, pp. 48–56, 2009.
[66]
M. Ristow and K. Zarse, “How increased oxidative stress promotes longevity and metabolic health: the concept of mitochondrial hormesis (mitohormesis),” Experimental Gerontology, vol. 45, no. 6, pp. 410–418, 2010.
[67]
S. Gao, J. Chen, S. V. Brodsky et al., “Docking of endothelial nitric oxide synthase (eNOS) to the mitochondrial outer membrane: a pentabasic amino acid sequence in the autoinhibitory domain of eNOS targets a proteinase K-cleavable peptide on the cytoplasmic face of mitochondria,” Journal of Biological Chemistry, vol. 279, no. 16, pp. 15968–15974, 2004.
