The Mammalian Target of Rapamycin (mTOR) Pathway Regulates Mitochondrial Oxygen Consumption and Oxidative Capacity

Metabolic rate and the subsequent production of reactive oxygen species are thought to contribute to the rate of aging in a wide range of species. The target of rapamycin (TOR) is a well conserved serine/threonine kinase that regulates cell growth in response to nutrient status. Here we demonstrate that in mammalian cells the mammalian TOR (mTOR) pathway plays a significant role in determining both resting oxygen consumption and oxidative capacity. In particular, we demonstrate that the level of complex formation between mTOR and one of its known protein partners, raptor, correlated with overall mitochondrial activity. Disruption of this complex following treatment with the mTOR pharmacological inhibitor rapamycin lowered mitochondrial membrane potential, oxygen consumption, and ATP synthetic capacity. Subcellular fractionation revealed that mTOR as well as mTOR-raptor complexes can be purified in the mitochondrial fraction. Using two-dimensional difference gel electrophoresis, we further demonstrated that inhibiting mTOR with rapamycin resulted in a dramatic alteration in the mitochondrial phosphoproteome. RNA interference-mediated knockdown of TSC2, p70 S6 kinase (S6K1), raptor, or rictor demonstrates that mTOR regulates mitochondrial activity independently of its previously identified cellular targets. Finally we demonstrate that mTOR activity may play an important role in determining the relative balance between mitochondrial and non-mitochondrial sources of ATP generation. These results may provide insight into recent observations linking the TOR pathway to life span regulation of lower organisms. Metabolic rate and the subsequent production of reactive oxygen species are thought to contribute to the rate of aging in a wide range of species. The target of rapamycin (TOR) is a well conserved serine/threonine kinase that regulates cell growth in response to nutrient status. Here we demonstrate that in mammalian cells the mammalian TOR (mTOR) pathway plays a significant role in determining both resting oxygen consumption and oxidative capacity. In particular, we demonstrate that the level of complex formation between mTOR and one of its known protein partners, raptor, correlated with overall mitochondrial activity. Disruption of this complex following treatment with the mTOR pharmacological inhibitor rapamycin lowered mitochondrial membrane potential, oxygen consumption, and ATP synthetic capacity. Subcellular fractionation revealed that mTOR as well as mTOR-raptor complexes can be purified in the mitochondrial fraction. Using two-dimensional difference gel electrophoresis, we further demonstrated that inhibiting mTOR with rapamycin resulted in a dramatic alteration in the mitochondrial phosphoproteome. RNA interference-mediated knockdown of TSC2, p70 S6 kinase (S6K1), raptor, or rictor demonstrates that mTOR regulates mitochondrial activity independently of its previously identified cellular targets. Finally we demonstrate that mTOR activity may play an important role in determining the relative balance between mitochondrial and non-mitochondrial sources of ATP generation. These results may provide insight into recent observations linking the TOR pathway to life span regulation of lower organisms. For nearly a century it has been appreciated that an organism's intrinsic metabolic rate is an important determinant of life span. This theory, initially known as the “rate of living” hypothesis, has merged with another proposed mechanism for aging first enunciated by Denham Harman (1Harman D. J. Gerontol. 1956; 11: 298-300Crossref PubMed Scopus (6606) Google Scholar) and often called the “free radical theory of aging.” The basis for combining these two hypotheses came from observations demonstrating that mitochondria determine both cellular and organismal metabolic rate and that these organelles also produce a continuous stream of reactive oxygen species. Although both the rate of living and the “free radical” theories of aging remain viable and attractive explanations for determining the rate of aging in a wide range of species, neither hypothesis has been conclusively proven (2Finkel T. Holbrook N.J. Nature. 2000; 408: 239-247Crossref PubMed Scopus (7425) Google Scholar, 3Wallace D.C. Annu. Rev. Genet. 2005; 39: 359-407Crossref PubMed Scopus (2562) Google Scholar, 4Balaban R.S. Nemoto S. Finkel T. Cell. 2005; 120: 483-495Abstract Full Text Full Text PDF PubMed Scopus (3350) Google Scholar). Surprisingly relatively little is known regarding what regulates the intrinsic metabolic rate of an organism. Similarly on a cellular level the molecular regulation of mitochondrial activity is incompletely understood. Resting oxygen consumption presumably is set at a point to meet overall energetic demands. Nonetheless for most cells this basal respiration is considerably below the maximum oxidative capacity of the mitochondria. The maximal oxidative or ATP synthetic capacity can be easily assessed by treating the cell with various chemical uncouplers and thereby producing the maximal degree of respiration. It is important to note that although mitochondria are the most efficient generators of ATP, cells can also produce ATP through the cytosolic metabolism of glucose. This cytosolic process is particularly important during hypoxic conditions. Nonetheless even under normal aerobic laboratory conditions, most cultured cells rely on both aerobic mitochondrial metabolism and aerobic glycolysis to generate their basal ATP needs. Indeed for many cells in culture these two sources of ATP generation each appear to contribute to roughly half of the overall ATP supply (5Sariban-Sohraby S. Magrath I.T. Balaban R.S. Cancer Res. 1983; 43: 4662-4664PubMed Google Scholar). It is presently unclear what determines the relative balance between cytosolic glycolysis and mitochondrial metabolism and why cells with significant unused mitochondrial capacity rely so heavily on less efficient non-mitochondrial energy sources. The use of lower organisms including yeast, flies, and worms has provided significant insight into the molecular mechanisms underlying aging. Both genetic and environmental interventions have been studied as potential modifiers of life span. Interestingly in simple organisms, structural mutations in mitochondrial subunits or knockdown of mitochondrial components has been shown to significantly alter life span albeit in both positive and negative directions (4Balaban R.S. Nemoto S. Finkel T. Cell. 2005; 120: 483-495Abstract Full Text Full Text PDF PubMed Scopus (3350) Google Scholar, 6Kenyon C. Cell. 2005; 120: 449-460Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 7Lee S.S. Lee R.Y. Fraser A.G. Kamath R.S. Ahringer J. Ruvkun G. Nat. Genet. 2003; 33: 40-48Crossref PubMed Scopus (772) Google Scholar, 8Hamilton B. Dong Y. Shindo M. Liu W. Odell I. Ruvkun G. Lee S.S. Genes Dev. 2005; 19: 1544-1555Crossref PubMed Scopus (409) Google Scholar, 9Dillin A. Hsu A.L. Arantes-Oliveira N. Lehrer-Graiwer J. Hsin H. Fraser A.G. Kamath R.S. Ahringer J. Kenyon C. Science. 2002; 298: 2398-2401Crossref PubMed Scopus (801) Google Scholar). Caloric restriction is another non-genetic intervention that appears to be effective in extending life span in a wide range of organisms (10Guarente L. Picard F. Cell. 2005; 120: 473-482Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). The molecular basis for the antiaging effects of caloric restriction remains incompletely understood. In the budding yeast Saccharomyces cerevisiae, reducing the glucose concentration in the culture medium can increase the replicative life span of the organism by 20-40% (11Lin S.J. Defossez P.A. Guarente L. Science. 2000; 289: 2126-2128Crossref PubMed Scopus (1488) Google Scholar, 12Lin S.J. Kaeberlein M. Andalis A.A. Sturtz L.A. Defossez P.A. Culotta V.C. Fink G.R. Guarente L. Nature. 2002; 418: 344-348Crossref PubMed Scopus (864) Google Scholar). Using a collection of yeast strains harboring single gene deletions, it was shown recently that the life span extension occurring with very low extracellular glucose requires the target of rapamycin (TOR) 2The abbreviations used are: TOR, target of rapamycin; mTOR, mammalian target of rapamycin; raptor, regulatory associated protein of mTOR; rictor, rapamycin-insensitive companion of mTOR; TMRM, tetramethylrhodamine methyl ester; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhdrazone; TSC2, tuberous sclerosis complex 2; p70 S6K1 or S6K1, ribosomal protein S6 kinase isoform 1; 4E-BP1, eukaryotic translation initiation factor eIF4E-binding protein 1; HEK-293T, human embryonic kidney-293T; Cy2, -3, or -5, cyanine dye 2, 3, or 5; RNAi, RNA interference; CHAPS, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry. pathway (13Kaeberlein M. Powers III, R.W. Steffen K.K. Westman E.A. Hu D. Dang N. Kerr E.O. Kirkland K.T. Fields S. Kennedy B.K. Science. 2005; 310: 1193-1196Crossref PubMed Scopus (1034) Google Scholar). A subsequent study suggested that this pathway is also involved in the chronological life span of yeast (14Powers III, R.W. Kaeberlein M. Caldwell S.D. Kennedy B.K. Fields S. Genes Dev. 2006; 20: 174-184Crossref PubMed Scopus (775) Google Scholar). These results are also supported by earlier observations in Caenorhabditis elegans and Drosophila where it was demonstrated that RNAi-mediated knockdown of the TOR pathway can lead to life span extension in these organisms (15Vellai T. Takacs-Vellai K. Zhang Y. Kovacs A.L. Orosz L. Muller F. Nature. 2003; 426: 620Crossref PubMed Scopus (847) Google Scholar, 16Kapahi P. Zid B.M. Harper T. Koslover D. Sapin V. Benzer S. Curr. Biol. 2004; 14: 885-890Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar). The TOR pathway is a well conserved pathway from yeast to mammalian cells (17Sarbassov D.D. Ali S.M. Sabatini D.M. Curr. Opin. Cell Biol. 2005; 17: 596-603Crossref PubMed Scopus (1330) Google Scholar, 18Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4745) Google Scholar). Evidence suggests that this pathway is sensitive to both the energetic supply and demand of the cell. TOR itself is a large serine/threonine protein kinase of ∼280 kDa and forms a multisubunit complex with numerous protein partners. In mammalian cells two distinct complexes have been identified: mTORC1 in which mTOR is bound to the protein partner raptor and mTORC2 in which mTOR is bound to another protein partner called rictor (17Sarbassov D.D. Ali S.M. Sabatini D.M. Curr. Opin. Cell Biol. 2005; 17: 596-603Crossref PubMed Scopus (1330) Google Scholar, 18Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4745) Google Scholar). These protein complexes appear to have distinct biological functions. Given the role of metabolism in aging and the role of the TOR pathway in life span determination, in this study we sought to understand the role of mTOR in regulating mitochondrial function and activity. Cell Culture and Reagents—Jurkat T cell leukemia clone E6-1 (ATCC, Manassas, VA) was maintained in RPMI 1640 medium containing 10% fetal calf serum; HEK-293T (ATCC) was maintained in Dulbecco's modified Eagle's medium containing 10% calf serum. In general, cell sorting was performed in Hanks' balanced salt solution. Nucleofection of small interfering RNA (non-targeting control 2, TSC2, S6K, raptor, and rictor small interfering RNA from Dharmacon, Lafayette, CO) was performed according to the manufacturer's protocol (Nucleofector kit V, Amaxa, Gaithersburg, MD), and cells were routinely analyzed 72 h after transfection. Using a control expression plasmid encoding for green fluorescent protein we could demonstrate that ∼80% of Jurkat and HEK-293T cells could be this cells were with carbonyl cyanide or rapamycin from and of for were as mTOR, raptor, S6K1 p70 S6K1, ribosomal protein 4E-BP1, and from Cell rictor and or was performed by of mTOR complexes was performed as previously D.D. Ali S.M. H. P. Sabatini D.M. Curr. Biol. 2004; 14: Full Text Full Text PDF PubMed Scopus Google Scholar) the mTOR from and cytosolic were with the mitochondria kit for cultured cells of the was assessed by for and and Cell potential was assessed the dye tetramethylrhodamine methyl at a concentration of for this significant is thought to be 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). was performed with the was from an laser to For sorting of cells with mitochondrial membrane potential, Jurkat cells were into to the cells with the and For of mitochondrial cells were with for Cell sorting was performed on a from were and were performed Cell was performed by and ATP consumption was the as previously L. S. M. J. J. S. J. 2004; PubMed Scopus Google Scholar). were in culture medium and to a where the of cells were in each of oxygen consumption were under and in the of or was a at for h at an of and of For the maximum of was used and into The respiration shown are of a single that are of at of oxygen consumption were a oxygen as we have previously S. Finkel T. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). was the of oxygen consumption and production for the of the of ATP from mitochondrial respiration and aerobic glycolysis as previously (5Sariban-Sohraby S. Magrath I.T. Balaban R.S. Cancer Res. 1983; 43: 4662-4664PubMed Google Scholar). ATP was the ATP kit according to the manufacturer's from cells were of in at for A was performed to and D. PubMed Scopus Google Scholar). The was and in of a containing and each of the control and were on with and an of a of of the control and of were with as previously J. 2004; PubMed Scopus Google Scholar). The first was the for a of The were an and the were on a gel at for h under a was performed function with was used for gel protein with and on matrix-assisted laser desorption as previously J. 2004; PubMed Scopus Google Scholar). were analyzed the Full mass were first by was the for was performed as recently S. S. Balaban R.S. 2006; PubMed Scopus Google Scholar). were in the role of mTOR in mitochondrial metabolism and sought a to the in metabolic rate for cells in to this we used cell sorting to of cells on the of mitochondrial membrane potential the mitochondrial dye the of cells that can be by is we a recently that for the of oxygen consumption of cells in L. S. M. J. J. S. J. 2004; PubMed Scopus Google Scholar). This with an and a for of metabolic the of cells for oxygen consumption by nearly of to study a Jurkat T cell this cell is routinely in and to and by Jurkat cells were initially with the mitochondrial dye and on or used a to oxygen or cells were to and the oxygen consumption of cells was under normal growth or in the with or maximal oxidative in was a dramatic difference in the basal metabolic rate as well as the oxidative capacity of these This for and oxygen consumption is in of relative Nonetheless we the relative metabolic rate of Jurkat cells the with an oxygen we a between these and The basal level of ATP was also in the cells increase to 3, on these results we was that of mTOR activity be correlated to these in oxygen consumption and oxidative capacity. in were in the level of mTOR or the protein raptor in Jurkat cell In we that the degree of mTOR-raptor complex formation correlated with the metabolic Similarly the level of of known mTOR as p70 S6 kinase and to also with the metabolic further mTOR-raptor was an important of mitochondrial metabolism we use of a pharmacological inhibitor of mTOR-raptor complex formation The of mTOR-raptor complexes with rapamycin is with observations D.D. Ali S.M. H. P. Sabatini D.M. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Y. K. N. S. C. J. K. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). with a role for the mTOR-raptor complex in regulating mitochondrial treatment of Jurkat cells with rapamycin resulted in a significant in mitochondrial membrane potential Similarly of oxygen consumption and oxidative capacity of Jurkat cells with and rapamycin treatment revealed that inhibiting mTOR-raptor complex formation metabolic rate and overall oxidative capacity capacity is thought to an intrinsic of the mitochondria that mTOR could with the mitochondria. Interestingly the of mTOR is well although a has demonstrated that a of mTOR with the mitochondria S. A. 2002; PubMed Scopus Google Scholar). with that in fractionation revealed that mTOR and raptor could both be identified in the mitochondrial we could also mTOR-raptor complexes from both the cytosolic and mitochondrial Given that the mTOR-raptor complex appears to with the mitochondria and the of the mTOR-raptor complex with mitochondrial we sought to we could to the mitochondria following of this performed two-dimensional difference gel of mitochondria from and mitochondrial could be by this of control and rapamycin revealed large difference in the level of associated with oxidative or This in between control and suggested that the in ATP synthetic capacity following rapamycin treatment was most to In of this a of to in their point with a in and of the in their point following rapamycin treatment could be identified by subsequent mass The identified to be for involved in metabolism or treatment to with the of these metabolic a also with the known role of rapamycin as an mTOR kinase Nonetheless it is unclear the identified or of mTOR or in these in or the that these of numerous mitochondrial was further supported by of two-dimensional from mitochondrial from and The of the has been shown previously to with the degree of protein S. S. Balaban R.S. 2006; PubMed Scopus Google Scholar, K. L.A. 2003; PubMed Scopus Google Scholar, Y. A. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). to a of and as treatment with rapamycin to the level of Although rapamycin is as a inhibitor of the mTOR-raptor we sought and to alter the level of complex formation to the regulatory role of mTOR activity on mitochondrial first use of observations demonstrating that mTOR activity is by the gene (17Sarbassov D.D. Ali S.M. Sabatini D.M. Curr. Opin. Cell Biol. 2005; 17: 596-603Crossref PubMed Scopus (1330) Google Scholar, 18Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4745) Google Scholar, K. T. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). expression of for resulted in a significant in protein the of mTOR or raptor with knockdown also activity (17Sarbassov D.D. Ali S.M. Sabatini D.M. Curr. Opin. Cell Biol. 2005; 17: 596-603Crossref PubMed Scopus (1330) Google Scholar, 18Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4745) Google Scholar, Y. A. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). In cell knockdown appear to Interestingly knockdown also resulted in an increase in mTOR-raptor complex formation with a role for mTOR in regulating mitochondrial knockdown basal oxygen consumption as well as oxidative capacity in oxygen consumption were also following knockdown in cell and of these results is that mTOR an increase in energetic demand by protein translation an increase in S6K1 activity. In an to this we S6K1 by demonstrated in this intervention on mitochondrial In knockdown of raptor resulted in a in oxygen consumption and knockdown of rictor oxygen consumption and oxidative capacity and Interestingly of mTOR-raptor complex were in rictor knockdown cells as previously in cell raptor and rictor for to mTOR D.D. Ali S.M. H. P. Sabatini D.M. Curr. Biol. 2004; 14: Full Text Full Text PDF PubMed Scopus Google Scholar). The alteration in mitochondrial metabolism with TSC2, S6K1, raptor, or rictor knockdown was associated with significant in mitochondrial mass as assessed by and or by with a membrane fluorescent of mitochondrial mass Similarly although inhibiting mTOR can we alteration in the cell in various the in mitochondrial respiration significantly the relative balance of ATP generation. are two for ATP generation in mammalian the first is aerobic respiration in the the the metabolism of glucose in the In control Jurkat and of respiration and production revealed that these cells a little less half of their ATP from mitochondrial metabolism a for cells maintained in culture (5Sariban-Sohraby S. Magrath I.T. Balaban R.S. Cancer Res. 1983; 43: 4662-4664PubMed Google Scholar). of and raptor appears to alter this In particular, knockdown cells of their basal ATP from raptor knockdown cells of their ATP from this of ATP from mitochondrial respiration and aerobic of ATP production in a In we have provided that mTOR activity and mTOR-raptor complex formation is correlated with mitochondrial In particular, cells for in resting mitochondrial respiration in their mTOR-raptor complexes and mTOR activity. Similarly mTOR-raptor complexes by pharmacological with rapamycin or with resulted in a in are at two explanations for this The first is that mTOR activity a significant energetic on the cell presumably through an increase in protein ribosomal that results in a reactive increase in mitochondrial activity. The is that mTOR can mitochondrial metabolism in what be as a these two be Although we conclusively which mechanism we that observations provide for a This regulation is supported by mTOR-raptor associated with the and of mTOR-raptor resulted in a significant alteration in the mitochondrial phosphoproteome. mTOR-raptor the of basal oxygen consumption also the oxidative capacity of the a that is to an intrinsic mitochondrial Finally the alteration in mitochondrial metabolism were to in energetic demand we the relative balance of aerobic to ATP generation to be we that cellular demand or mitochondrial metabolism and cytosolic glycolysis or so their Although oxygen consumption have with to that knockdown of raptor and appears to alter the relative of cytosolic glycolysis to mitochondrial These in further a regulatory role of mTOR on mitochondrial activity. performed respiration the of have used mitochondria. of is that in cells the level of aerobic ATP production oxygen and the mitochondrial membrane potential This is most that increase the of ATP in mitochondria that through the in a of membrane A mitochondrial membrane potential in the of respiration the that the overall capacity of the mitochondria to produce ATP has been This is also supported by that oxidative capacity as a function of mitochondrial membrane these observations the potential of cells with intrinsic in mitochondrial activity and capacity and mitochondria from Although both S6K1 and are important of the mTOR demonstrate that the of mTOR to oxygen consumption and oxidative capacity appears to be of these known For although knockdown oxygen consumption it appear in Jurkat cells to significantly Similarly knockdown of S6K1 on overall oxygen Interestingly a recent has demonstrated that have a increase in their rate of This large increase in as well as an increase in to an increase in oxygen consumption in these F. M. Picard F. M. M. S. J. G. Nature. 2004; PubMed Scopus Google Scholar). it is important to that in metabolic rate both intrinsic cellular as including mitochondrial and as well as a of and overall results have suggested that mitochondrial function can mTOR activity by a known as (17Sarbassov D.D. Ali S.M. Sabatini D.M. Curr. Opin. Cell Biol. 2005; 17: 596-603Crossref PubMed Scopus (1330) Google Scholar, 18Wullschleger S. Loewith R. Hall M.N. Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4745) Google Scholar, S. S. Balaban R.S. 2006; PubMed Scopus Google Scholar, D.D. Ali S.M. H. P. Sabatini D.M. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). that this pathway is also in Jurkat cells as that lower mitochondrial membrane potential mTOR activity rapamycin and results that the regulation is also these results that mitochondria can to mTOR and that mTOR may in mitochondrial activity It is that a may be for energy supply and demand during and important as cell Finally is that mitochondrial metabolism may play an important role in aging (2Finkel T. Holbrook N.J. Nature. 2000; 408: 239-247Crossref PubMed Scopus (7425) Google Scholar, 3Wallace D.C. Annu. Rev. Genet. 2005; 39: 359-407Crossref PubMed Scopus (2562) Google Scholar, 4Balaban R.S. Nemoto S. Finkel T. Cell. 2005; 120: 483-495Abstract Full Text Full Text PDF PubMed Scopus (3350) Google Scholar). have that in yeast, C. and of the TOR pathway results in life extension (13Kaeberlein M. Powers III, R.W. Steffen K.K. Westman E.A. Hu D. Dang N. Kerr E.O. Kirkland K.T. Fields S. Kennedy B.K. Science. 2005; 310: 1193-1196Crossref PubMed Scopus (1034) Google Scholar, III, R.W. Kaeberlein M. Caldwell S.D. Kennedy B.K. Fields S. Genes Dev. 2006; 20: 174-184Crossref PubMed Scopus (775) Google Scholar, T. Takacs-Vellai K. Zhang Y. Kovacs A.L. Orosz L. Muller F. Nature. 2003; 426: 620Crossref PubMed Scopus (847) Google Scholar, 16Kapahi P. Zid B.M. Harper T. Koslover D. Sapin V. Benzer S. Curr. Biol. 2004; 14: 885-890Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar). of a large of mutations in C. elegans has to a hypothesis that these genetic may function by energetic from the mitochondria and S. Dev. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). that mTOR activity with oxygen consumption and to the balance between glycolysis and aerobic metabolism appears with that are to and for the to and for on oxygen consumption to and A. for to for and to for with the