The metabolite alpha-ketoglutarate extends lifespan by inhibiting the ATP synthase and TOR

Metabolism and ageing are intimately linked. Compared to ad libitum feeding, dietary restriction (DR) or calorie restriction (CR) consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms 1,2 . Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits 3,4 . Recently, several metabolites have been identified that modulate ageing 5,6 with largely undefined molecular mechanisms. Here we show that the tricarboxylic acid (TCA) cycle intermediate α ketoglutarate ( α -KG) extends the lifespan of adult C. elegans . ATP synthase subunit beta is identified as a novel binding protein of α -KG using a small-molecule target identification strategy called DARTS (drug affinity responsive target stability) 7 . The ATP synthase, also known as Complex V of the mitochondrial electron transport chain (ETC), is the main cellular energy-generating machinery and is highly conserved throughout evolution 8,9 . Although complete loss of mitochondrial function is detrimental, partial suppression of the ETC has been shown to extend C. elegans lifespan 10–13 . We show that α -KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α -KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α -KG requires ATP synthase subunit beta and is dependent on the target of rapamycin (TOR) downstream. Endogenous α -KG levels are increased upon starvation and α -KG does not extend the lifespan of DR animals, indicating that α -KG is a key metabolite that mediates longevity by DR. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator, and DR in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases. HT115 atp-2 dsRNA. day 2 and day 3 at least was to every sample and animals were lysed by alternate boil/freeze cycles. Lysed animals were centrifuged at 14,000 rpm for 10 min at 4 °C to pellet debris, and supernatant was collected for oxyblot analysis. Protein concentration of samples was determined by the 660 nm Protein Assay and normalized for all samples. Carbonylation of proteins in each sample was detected the OxyBlot Protein Oxidation Detection

[1]  A. Vazquez,et al.  Disruption of wild-type IDH1 suppresses D-2-hydroxyglutarate production in IDH1-mutated gliomas. , 2013, Cancer research.

[2]  M. Jung,et al.  Synthesis of the 1-monoester of 2-ketoalkanedioic acids, for example, octyl α-ketoglutarate. , 2012, The Journal of organic chemistry.

[3]  David B. Allison,et al.  Impact of caloric restriction on health and survival in rhesus monkeys from the NIA study , 2012, Nature.

[4]  X. Correig,et al.  Metabolomics Approach for Analyzing the Effects of Exercise in Subjects with Type 1 Diabetes Mellitus , 2012, PloS one.

[5]  Kevin W Eliceiri,et al.  NIH Image to ImageJ: 25 years of image analysis , 2012, Nature Methods.

[6]  K. Bornfeldt,et al.  Coordinate Regulation of Lipid Metabolism by Novel Nuclear Receptor Partnerships , 2012, PLoS genetics.

[7]  Evan G. Williams,et al.  NCoR1 Is a Conserved Physiological Modulator of Muscle Mass and Oxidative Function , 2011, Cell.

[8]  M. Brand,et al.  High Throughput Microplate Respiratory Measurements Using Minimal Quantities Of Isolated Mitochondria , 2011, PloS one.

[9]  M. Brand,et al.  Assessing mitochondrial dysfunction in cells , 2011, The Biochemical journal.

[10]  B. Viollet,et al.  AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1 , 2011, Nature Cell Biology.

[11]  Bin Wang,et al.  Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. , 2011, Cancer cell.

[12]  R. Olsen,et al.  Identification of Direct Protein Targets of Small Molecules , 2010, ACS chemical biology.

[13]  M. Hansen,et al.  A Role for Autophagy in the Extension of Lifespan by Dietary Restriction in -1 , 2011 .

[14]  A. Alberti,et al.  The autophagosomal protein LGG-2 acts synergistically with LGG-1 in dauer formation and longevity in C. elegans , 2010, Autophagy.

[15]  C. Kenyon.  The genetics of ageing , 2010, Nature.

[16]  J. Pelletier,et al.  Target identification using drug affinity responsive target stability (DARTS) , 2009, Proceedings of the National Academy of Sciences.

[17]  Tanja Diemer,et al.  Oxaloacetate supplementation increases lifespan in Caenorhabditis elegans through an AMPK/FOXO‐dependent pathway , 2009, Aging cell.

[18]  B. Kennedy,et al.  The TOR pathway comes of age. , 2009, Biochimica et biophysica acta.

[19]  J. Ahnn,et al.  C. elegans behavior of preference choice on bacterial food , 2009, Molecules and cells.

[20]  Z. Zhai,et al.  The HIF-1 Hypoxia-Inducible Factor Modulates Lifespan in C. elegans , 2009, PloS one.

[21]  Sterling C. Johnson,et al.  Caloric Restriction Delays Disease Onset and Mortality in Rhesus Monkeys , 2009, Science.

[22]  Marco Pahor,et al.  Rapamycin fed late in life extends lifespan in genetically heterogeneous mice , 2009, Nature.

[23]  M. Kaeberlein,et al.  Measuring Caenorhabditis elegans Life Span on Solid Media , 2009, Journal of visualized experiments : JoVE.

[24]  D. Louis,et al.  Faculty Opinions recommendation of Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha. , 2009 .

[25]  F. Sobott,et al.  Application of a proteolysis/mass spectrometry method for investigating the effects of inhibitors on hydroxylase structure. , 2009, Journal of medicinal chemistry.

[26]  E. Greer,et al.  Different dietary restriction regimens extend lifespan by both independent and overlapping genetic pathways in C. elegans , 2009, Aging cell.

[27]  S. Mango,et al.  The Target of Rapamycin Pathway Antagonizes pha-4/FoxA to Control Development and Aging , 2008, Current Biology.

[28]  Christopher J Schofield,et al.  Expanding chemical biology of 2-oxoglutarate oxygenases. , 2008, Nature chemical biology.

[29]  W. Kaelin.  Faculty Opinions recommendation of Cell-permeating alpha-ketoglutarate derivatives alleviate pseudohypoxia in succinate dehydrogenase-deficient cells. , 2008 .

[30]  M. Driscoll,et al.  A Role for Autophagy in the Extension of Lifespan by Dietary Restriction in C. elegans , 2008, PLoS genetics.

[31]  L. Avery,et al.  Dual roles of autophagy in the survival of Caenorhabditis elegans during starvation. , 2007, Genes & development.

[32]  H. Aguilaniu,et al.  PHA-4/Foxa mediates diet-restriction-induced longevity of C. elegans , 2007, Nature.

[33]  G. Ruvkun,et al.  Lifespan Regulation by Evolutionarily Conserved Genes Essential for Viability , 2007, PLoS genetics.

[34]  Seung-Jae V. Lee,et al.  Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans , 2007, Aging cell.

[35]  Min Wu,et al.  Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. , 2007, American journal of physiology. Cell physiology.

[36]  Matthew J. Brauer,et al.  Conservation of the metabolomic response to starvation across two divergent microbes , 2006, Proceedings of the National Academy of Sciences.

[37]  M. Hall,et al.  TOR Signaling in Growth and Metabolism , 2006, Cell.

[38]  D. Guertin,et al.  Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR Complex , 2005, Science.

[39]  D. Hall,et al.  Autophagy Genes Are Essential for Dauer Development and Life-Span Extension in C. elegans , 2003, Science.

[40]  D. Hardie,et al.  Management of cellular energy by the AMP‐activated protein kinase system , 2003, FEBS letters.

[41]  Gary Ruvkun,et al.  A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity , 2003, Nature Genetics.

[42]  Andrew G Fraser,et al.  Rates of Behavior and Aging Specified by Mitochondrial Function During Development , 2002, Science.

[43]  J. Avruch,et al.  TOR Deficiency in C. elegans Causes Developmental Arrest and Intestinal Atrophy by Inhibition of mRNA Translation , 2002, Current Biology.

[44]  S. Mango,et al.  Regulation of Organogenesis by the Caenorhabditis elegans FoxA Protein PHA-4 , 2002, Science.

[45]  S K Burley,et al.  Hierarchical phosphorylation of the translation inhibitor 4E-BP1. , 2001, Genes & development.

[46]  Michael I. Wilson,et al.  C. elegans EGL-9 and Mammalian Homologs Define a Family of Dioxygenases that Regulate HIF by Prolyl Hydroxylation , 2001, Cell.

[47]  D. Pilgrim,et al.  Mitochondrial Respiratory Chain Deficiency inCaenorhabditis elegans Results in Developmental Arrest and Increased Life Span* , 2001, The Journal of Biological Chemistry.

[48]  Takeshi Noda,et al.  LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing , 2000, The EMBO journal.

[49]  D L Riddle,et al.  Genetic, behavioral and environmental determinants of male longevity in Caenorhabditis elegans. , 2000, Genetics.

[50]  B. Lakowski,et al.  The genetics of caloric restriction in Caenorhabditis elegans. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[51]  A. Fire,et al.  Specific interference by ingested dsRNA , 1998, Nature.

[52]  S. Snyder,et al.  RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[53]  G. Thomas,et al.  The modular phosphorylation and activation of p70s6k , 1997, FEBS letters.

[54]  P. Boyer.  The ATP synthase--a splendid molecular machine. , 1997, Annual review of biochemistry.

[55]  Jan Pieter Abrahams,et al.  Structure at 2.8 Â resolution of F1-ATPase from bovine heart mitochondria , 1994, Nature.

[56]  N. Munakata.  [Genetics of Caenorhabditis elegans]. , 1989, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[57]  J. Davies,et al.  Molecular Biology of the Cell , 1983, Bristol Medico-Chirurgical Journal.

[58]  E. Kosenko,et al.  Metabolites of citric acid cycle, carbohydrate and phosphorus metabolism, and related reactions, redox and phosphorylating states of hepatic tissue, liver mitochondria and cytosol of the pigeon, under normal feeding and natural nocturnal fasting conditions. , 1982, Comparative biochemistry and physiology. B, Comparative biochemistry.