Advertisement

Cellular and Molecular Life Sciences

, Volume 70, Issue 11, pp 2015–2029 | Cite as

A novel AMPK-dependent FoxO3A-SIRT3 intramitochondrial complex sensing glucose levels

  • Alessia Peserico
  • Fulvio Chiacchiera
  • Valentina Grossi
  • Antonio Matrone
  • Dominga Latorre
  • Marta Simonatto
  • Aurora Fusella
  • James G. Ryall
  • Lydia W. S. Finley
  • Marcia C. Haigis
  • Gaetano Villani
  • Pier Lorenzo Puri
  • Vittorio Sartorelli
  • Cristiano SimoneEmail author
Research Article

Abstract

Reduction of nutrient intake without malnutrition positively influences lifespan and healthspan from yeast to mice and exerts some beneficial effects also in humans. The AMPK-FoxO axis is one of the evolutionarily conserved nutrient-sensing pathways, and the FOXO3A locus is associated with human longevity. Interestingly, FoxO3A has been reported to be also a mitochondrial protein in mammalian cells and tissues. Here we report that glucose restriction triggers FoxO3A accumulation into mitochondria of fibroblasts and skeletal myotubes in an AMPK-dependent manner. A low-glucose regimen induces the formation of a protein complex containing FoxO3A, SIRT3, and mitochondrial RNA polymerase (mtRNAPol) at mitochondrial DNA-regulatory regions causing activation of the mitochondrial genome and a subsequent increase in mitochondrial respiration. Consistently, mitochondrial transcription increases in skeletal muscle of fasted mice, with a mitochondrial DNA-bound FoxO3A/SIRT3/mtRNAPol complex detectable also in vivo. Our results unveil a mitochondrial arm of the AMPK-FoxO3A axis acting as a recovery mechanism to sustain energy metabolism upon nutrient restriction.

Keywords

Glucose restriction FoxO3A AMPK SIRT3 OXPHOS 

Abbreviations

GR

Glucose restriction

FoxO

Forkhead-box O

AMPK

AMP-activated kinase

FoxO binding sites

FHRE

mtDNA

Mitochondrial DNA

MEFs

Mouse embryonic fibroblasts

mtRNAPol

Mitochondrial RNA polymerase

CR

Calorie restriction

HG

High glucose medium

LG

Low glucose medium

CC

Compound C

NAM

Nicotinamide

DNP

2,4-Dinitrophenol

FED

Feeding

FAST

Fasting

AcK

Anti-acetyl lysine

Notes

Acknowledgments

We thank Dr. Francesco Paolo Jori for his helpful discussion during the preparation of the manuscript and editorial assistance, Dr. Roberta Ledonne for preparing the illustrations and editing this manuscript, Drs. Michele Petruzzeli and Daniele Di Giandomenico for technical assistance, Dr. Antonio Moschetta for discussion, Dr. Karen Arden for generously providing Foxo3A−/− MEFs and Drs. Lucisano and Pellegrini (Unit of Biostatistics, Consorzio Mario Negri Sud) for statistical analysis. Image acquisition and image data analysis were performed at the Advanced Light and Electron Microscopy Facility of the Consorzio Mario Negri Sud. This work was partially supported by a ‘My First Grant 2007’ and an ‘Investigator Grant 2010’ (IG10177) (to C.S.) from the Italian Association for Cancer Research (AIRC).

Supplementary material

18_2012_1244_MOESM1_ESM.tif (15.6 mb)
Supplementary Fig. 1 FoxO3A accumulates into the mitochondria upon GR (LG 24h), as shown by immunogold labeling of murine C2C12-derived and murine NIH-3T3 fibroblasts (black dots represent gold particles recognizing FoxO3A immunocomplexes). (TIFF 15990 kb)
18_2012_1244_MOESM2_ESM.tif (15.6 mb)
Supplementary Fig. 2. Upon GR (LG 24h), but not glutamine deprivation or oxidative stress, FoxO3A localizes to mitochondria in primary IMR90 human fibroblasts, as shown by immunofluorescence analysis using the indicated antibodies and probes. (TIFF 15991 kb)
18_2012_1244_MOESM3_ESM.tif (19.3 mb)
Supplementary Fig. 3 a-d GR induces the time-dependent upregulation of mitochondrial genes both at the RNA (a, c, d) and the protein level (b) in murine NIH-3T3 (a, b), C2C12-derived terminally differentiated myotubes (c) and primary IMR90 human fibroblasts (d). (a, c, d) black bars: ATPase 6 and 8 genes; white bars: COI, COII, and COIII genes; diagonally hatched bars: ND1, ND2, ND3, ND4, ND4L, ND5, and ND6 genes; checkered bars: cytochrome b gene. The dotted line corresponds to the expression levels detected in cells cultured in standard glucose conditions (HG). Data are presented as mean ± SEM. (TIFF 19782 kb)
18_2012_1244_MOESM4_ESM.tif (14.6 mb)
Supplementary Fig. 4 a GR-induced time-dependent upregulation of mitochondrial genes occurs in the absence of mitochondrial biogenesis in C2C12-derived myotubes (LG 24h), primary IMR90 (LG 48h), and NIH-3T3 cells (LG 48h). Data are presented as mean ± SEM. b The efficacy of FoxO3A genetic ablation by a specific siRNA was confirmed by densitometric analysis of Western-blot signals (FoxO3A protein levels were reduced 62% to 79% in both HG and LG conditions compared to control siRNAs). (TIFF 14984 kb)
18_2012_1244_MOESM5_ESM.tif (15.6 mb)
Supplementary Fig. 5 a The efficacy of AMPK genetic ablation by a specific siRNA was confirmed by Western-blot analysis. b, c AMPK activation in HG does not affect mitochondrial biogenesis in C2C12-derived myotubes (b) and NIH-3T3 cells (c), as revealed by quantification of the total amount of mitochondrial DNA 24h after the addition of AICAR, metformin or resveratrol. d Nicotinamide does not influence mitochondrial content in myotubes upon low-glucose conditions (LG 12h). Data are presented as mean ± SEM. (TIFF 15990 kb)
18_2012_1244_MOESM6_ESM.tif (15.6 mb)
Supplementary Fig. 6. ChIP experiments showing that sirtuin inhibition using nicotinamide (NAM) reduces FoxO3A binding to mtDNA in NIH-fibroblasts (FHRE#1, LG 16h). Unrel: unrelated antibody (anti-IgG). Data are presented as mean ± SEM. (TIFF 15986 kb)
18_2012_1244_MOESM7_ESM.tif (15.6 mb)
Supplementary Fig. 7. a, b Immunogold labeling of NIH-3T3 fibroblasts showing that (a) FoxO3A accumulates into the mitochondria in response to GR and AMPK exogenous activation (AICAR) and (b) AMPK ablation by a specific siRNA prevents FoxO3A accumulation even in the presence of low glucose conditions or addition of AICAR (black dots represent gold particles recognizing FoxO3A immunocomplexes). (TIFF 15954 kb)
18_2012_1244_MOESM8_ESM.tif (15.6 mb)
Supplementary Fig. 7. a, b Immunogold labeling of NIH-3T3 fibroblasts showing that (a) FoxO3A accumulates into the mitochondria in response to GR and AMPK exogenous activation (AICAR) and (b) AMPK ablation by a specific siRNA prevents FoxO3A accumulation even in the presence of low-glucose conditions or addition of AICAR (black dots represent gold particles recognizing FoxO3A immunocomplexes). (TIFF 15954 kb)
18_2012_1244_MOESM9_ESM.tif (19.3 mb)
Supplementary Fig. 8. Overnight fasting activates AMPK in tibialis and EDL skeletal muscles (18h FAST). (TIFF 19780 kb)
18_2012_1244_MOESM10_ESM.docx (31 kb)
Supplementary material 10 (DOCX 30 kb)

References

  1. 1.
    McCay CM, Crowell MF, Maynard LA (1935) The effect of retarded growth upon the length of life span and upon the ultimate body size. J Nutr 10:63–79Google Scholar
  2. 2.
    Kirkwood TB, Shanley DP (2005) Food restriction, evolution and ageing. Mech Ageing Dev 126:1011–1016PubMedCrossRefGoogle Scholar
  3. 3.
    Fontana L, Partridge L, Longo VD (2010) Extending healthy life span—from yeast to humans. Science 328:321–326PubMedCrossRefGoogle Scholar
  4. 4.
    Schulz TJ, Zarse K, Voigt A, Urban N, Birringer M, Ristow M (2007) Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress. Cell Metab 6:280–293PubMedCrossRefGoogle Scholar
  5. 5.
    Li Y, Liu L, Tollefsbol TO (2010) Glucose restriction can extend normal cell lifespan and impair precancerous cell growth through epigenetic control of hTERT and p16 expression. FASEB J 5:1442–1453CrossRefGoogle Scholar
  6. 6.
    Li Y, Tollefsbol TO (2011) p16INK4a suppression by glucose restriction contributes to human cellular lifespan extension through SIRT1-mediated epigenetic and genetic mechanism. PLOS One 6:e17421, 1–13Google Scholar
  7. 7.
    Greer EL, Dowlatshahi D, Banko MR, Villen J, Hoang K, Blanchard D, Gygi SP, Brunet A (2007) An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C. elegans. Curr Biol 17:1646–1656PubMedCrossRefGoogle Scholar
  8. 8.
    Williams DS, Cash A, Hamadani L, Diemer T (2009) Oxaloacetate supplementation increases lifespan in Caenorhabditis elegans through an AMPK/FOXO-dependent pathway. Aging Cell 8:765–768PubMedCrossRefGoogle Scholar
  9. 9.
    Chiacchiera F, Simone C (2010) The AMPK-FoxO3A axis as a target for cancer treatment. Cell Cycle 6:1091–1096CrossRefGoogle Scholar
  10. 10.
    Willcox BJ, Donlon TA, He Q, Chen R, Grove JS, Yano K, Masaki KH, Willcox DC, Rodriguez B, Curb JD (2008) FOXO3A genotype is strongly associated with human longevity. Proc Natl Acad Sci USA 105:13987–13992PubMedCrossRefGoogle Scholar
  11. 11.
    Flachsbart F, Caliebe A, Kleindorp R, Blanché H, von Eller-Eberstein H, Nikolaus S, Schreiber S, Nebel A (2009) Association of FOXO3A variation with human longevity confirmed in German centenarians. Proc Natl Acad Sci USA 106:2700–2705PubMedCrossRefGoogle Scholar
  12. 12.
    Anselmi CV, Malovini A, Roncarati R, Novelli V, Villa F, Condorelli G, Bellazzi R, Puca AA (2009) Association of the FOXO3A locus with extreme longevity in a southern Italian centenarian study. Rejuvenation Res 12:95–104PubMedCrossRefGoogle Scholar
  13. 13.
    Pawlikowska L, Hu D, Huntsman S, Sung A, Chu C, Chen C, Joyner AH, Schork NJ, Hsueh WC, Reiner AP, Psaty BM, Atzmon G, Barzilai N, Cummings SR, Browner WS, Kwok PY, Ziv E (2009) Association of common genetic variation in the insulin/IGF1 signaling pathway with human longevity. Aging Cell 8:460–472PubMedCrossRefGoogle Scholar
  14. 14.
    Li Y, Wang WJ, Cao H, Lu J, Wu C, Hu FY, Guo J, Zhao L, Yang F, Zhang YX, Li W, Zheng GY, Cui H, Chen X, Zhu Z, He H, Dong B, Mo X, Zeng Y, Tian XL (2009) Genetic association of FOXO1A and FOXO3A with longevity trait in Han Chinese populations. Hum Mol Genet 18:4897–4904PubMedCrossRefGoogle Scholar
  15. 15.
    Calnan DR, Brunet A (2008) The FoxO code. Oncogene 27(16):2276–2288PubMedCrossRefGoogle Scholar
  16. 16.
    Van der Horst A, Burgering BM (2007) Stressing the role of FoxO proteins in lifespan and disease. Nat Rev Mol Cell Biol 8:440–450PubMedCrossRefGoogle Scholar
  17. 17.
    Jacobs KM, Pennington JD, Bisht KS, Aykin-Burns N, Kim HS, Mishra M, Sun L, Nguyen P, Ahn BH, Leclerc J, Deng CX, Spitz DR, Gius D (2008) SIRT3 interacts with the daf-16 homolog FOXO3a in the mitochondria, as well as increases FOXO3a-dependent gene expression. Int J Biol Sci 4:291–299PubMedCrossRefGoogle Scholar
  18. 18.
    Lombard DB, Alt FW, Cheng HL, Bunkenborg J, Streeper RS, Mostoslavsky R, Kim J, Yancopoulos G, Valenzuela D, Murphy A, Yang Y, Chen Y, Hirschey MD, Bronson RT, Haigis M, Guarente LP Jr, Fareser RV, Weissman S, Verdin E, Schwer B (2007) Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation. Mol Cell Biol 27:8807–8814PubMedCrossRefGoogle Scholar
  19. 19.
    Clayton DA (1984) Transcription of the mammalian mitochondrial genome. Annu Rev Biochem 53:573–594PubMedCrossRefGoogle Scholar
  20. 20.
    Kim HS, Patel K, Muldoon-Jacobs K, Bisht KS, Aykin-Burns N, Pennington JD, van der Meer R, Nguyen P, Savage J, Owens KM, Vassilopoulos A, Ozden O, Park SH, Singh KK, Abdulkadir SA, Spitz DR, Deng CX, Gius D (2010) SIRT3 is a mitochondria-localized tumor suppressor required for maintenance of mitochondrial integrity and metabolism during stress. Cancer Cell 17:41–52PubMedCrossRefGoogle Scholar
  21. 21.
    Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y, Tran H, Ross SE, Mostoslavsky R, Cohen HY, Hu LS, Cheng HL, Jedrychowski MP, Gygi SP, Sinclair DA, Alt FW, Greenberg ME (2004) Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 303:2011–2015PubMedCrossRefGoogle Scholar
  22. 22.
    Andrikopoulos S, Blair AR, Deluca N, Fam BC, Proietto J (2008) Evaluating the glucose tolerance test in mice. Am J Physiol Endocrinol Metab 295:E1323–E1332PubMedCrossRefGoogle Scholar
  23. 23.
    Meyer C, Dostou JM, Welle SL, Gerich JE (2002) Role of human liver, kidney, and skeletal muscle in postprandial glucose homeostasis. Am J Physiol Endocrinol Metab 282:E419–E427PubMedGoogle Scholar
  24. 24.
    DeFronzo RA (1997) Pathogenesis of type 2 diabetes: metabolic and molecular implications for identifying genes. Diabetes Rev 5:177–269Google Scholar
  25. 25.
    Guarente L (2008) Mitochondria-a nexus for aging, calorie restriction, and sirtuins? Cell 132:171–176PubMedCrossRefGoogle Scholar
  26. 26.
    Greer EL, Oskoui PR, Banko MR, Maniar JM, Gygi MP, Gygi SP, Brunet A (2007) The energy sensor AMP-activated protein kinase directly regulates the mammalian FOXO3 transcription factor. J Biol Chem 282:30107–30119PubMedCrossRefGoogle Scholar
  27. 27.
    Greer EL, Banko MR, Brunet A (2009) AMP-activated protein kinase and FoxO transcription factors in dietary restriction-induced longevity. Ann NY Acad Sci 1170:688–692PubMedCrossRefGoogle Scholar
  28. 28.
    Chiacchiera F, Matrone A, Ferrari E, Ingravallo G, Lo Sasso G, Murzilli S, Petruzzelli M, Salvatore L, Moschetta A, Simone C (2009) p38alpha blockade inhibits colorectal cancer growth in vivo by inducing a switch from HIF1alpha- to FoxO-dependent transcription. Cell Death Differ 16:1203–1214PubMedCrossRefGoogle Scholar
  29. 29.
    Chiacchiera F, Simone C (2009) Inhibition of p38alpha unveils an AMPK-FoxO3A axis linking autophagy to cancer-specific metabolism. Autophagy 5:1030–1033PubMedCrossRefGoogle Scholar
  30. 30.
    Matrone A, Grossi V, Chiacchiera F, Fina E, Cappellari M, Caringella AM, Di Naro E, Loverro G, Simone C (2010) p38alpha is required for ovarian cancer cell metabolism and survival. Int J Gynecol Cancer 20:203–211PubMedCrossRefGoogle Scholar
  31. 31.
    Psarra AM, Sekeris CE (2008) Nuclear receptors and other nuclear transcription factors in mitochondria: regulatory molecules in a new environment. Biochim Biophys Acta 1783:1–11PubMedCrossRefGoogle Scholar
  32. 32.
    Ingram DK, Zhu M, Mamczarz J, Zou S, Lane MA, Roth GS, deCabo R (2006) Calorie restriction mimetics: an emerging research field. Aging Cell 5:97–108PubMedCrossRefGoogle Scholar
  33. 33.
    Narkar VA, Downes M, Yu RT, Embler E, Wang YX, Banayo E, Mihaylova MM, Nelson MC, Zou Y, Juguilon H, Kang H, Shaw RJ, Evans RM (2008) AMPK and PPARdelta agonists are exercise mimetics. Cell 134:405–415PubMedCrossRefGoogle Scholar
  34. 34.
    Civitarese AE, Carling S, Heilbronn LK, Hulver MH, Ukropcova B, Deutsch WA, Smith SR, Ravussin E (2007) Calorie restriction increases muscle mitochondrial biogenesis in healthy humans. PLoS Med 4:e76PubMedCrossRefGoogle Scholar
  35. 35.
    Smith DL Jr, Nagy TR, Allison DB (2010) Calorie restriction: what recent results suggest for the future of ageing research. Eur J Clin Invest 40:440–450PubMedCrossRefGoogle Scholar
  36. 36.
    Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, Prabhu VV, Allard JS, Lopez-Lluch G, Lewis K, Pistell PJ, Poosala S, Becker KG, Boss O, Gwinn D, Wang M, Ramaswamy S, Fishbein KW, Spencer RG, Lakatta EG, Le Couteur D, Shaw RJ, Navas P, Puigserver P, Ingram DK, de Cabo R, Sinclair DA (2006) Resveratrol improves health and survival of mice on a high-calorie diet. Nature 444:337–342PubMedCrossRefGoogle Scholar
  37. 37.
    Park CB, Larsson NG (2011) Mitochondrial DNA mutations in disease and aging. J Cell Biol 193:809–818PubMedCrossRefGoogle Scholar
  38. 38.
    Simone C, Forcales SV, Hill DA, Imbalzano AN, Latella L, Puri PL (2004) p38 pathway targets SWI-SNF chromatin-remodeling complex to muscle-specific loci. Nat Genet 36:738–743PubMedCrossRefGoogle Scholar
  39. 39.
    Serra C, Palacios D, Mozzetta C, Forcales SV, Morantte I, Ripani M, Jones DR, Du K, Jhala US, Simone C, Puri PL (2007) Functional interdependence at the chromatin level between the MKK6/p38 and IGF1/PI3K/AKT pathways during muscle differentiation. Mol Cell 28:200–213PubMedCrossRefGoogle Scholar
  40. 40.
    Lucocq JM, Habermann A, Watt S, Backer JM, Mayhew TM, Griffiths G (2004) A rapid method for assessing the distribution of gold labeling on thin sections. J Histochem Cytochem 52:991–1000PubMedCrossRefGoogle Scholar
  41. 41.
    Villani G, Attardi G (2007) Polarographic assays of respiratory chain complex activity. Methods Cell Biol 80:121–133PubMedCrossRefGoogle Scholar
  42. 42.
    Villani G, Greco M, Papa S, Attardi G (1998) Low reserve of cytochrome c oxidase capacity in vivo in the respiratory chain of a variety of human cell types. J Biol Chem 273:31829–31836PubMedCrossRefGoogle Scholar
  43. 43.
    Hochberg Y (1988) A sharper Bonferroni procedure for multiple tests of significance. Biometrika 75:800–803CrossRefGoogle Scholar

Copyright information

© Springer Basel 2012

Authors and Affiliations

  • Alessia Peserico
    • 1
  • Fulvio Chiacchiera
    • 1
  • Valentina Grossi
    • 2
  • Antonio Matrone
    • 1
  • Dominga Latorre
    • 3
  • Marta Simonatto
    • 4
  • Aurora Fusella
    • 5
  • James G. Ryall
    • 6
  • Lydia W. S. Finley
    • 7
  • Marcia C. Haigis
    • 7
  • Gaetano Villani
    • 3
  • Pier Lorenzo Puri
    • 4
  • Vittorio Sartorelli
    • 6
  • Cristiano Simone
    • 1
    • 8
    Email author
  1. 1.Laboratory of Signal-dependent Transcription, Department of Translational Pharmacology (DTP)Consorzio Mario Negri SudSanta Maria Imbaro (Ch)Italy
  2. 2.Cancer Genetics LaboratoryIRCCS “S. de Bellis”Castellana Grotte (Ba)Italy
  3. 3.Department of Medical Biochemistry, Biology and PhysicsUniversity of BariBariItaly
  4. 4.Dulbecco Telethon Institute (DTI), IRCCS Fondazione Santa LuciaRomeItaly
  5. 5.Electron Microscopy and Tomography Facility, Department of Translational Pharmacology (DTP)Consorzio Mario Negri SudSanta Maria Imbaro (Ch)Italy
  6. 6.Laboratory of Muscle Stem Cells and Gene Regulation, NIAMS, NIHBethesdaUSA
  7. 7.The Paul F. Glenn Labs for the Biological Mechanisms of Aging, Department of PathologyHarvard Medical SchoolBostonUSA
  8. 8.Division of Medical Genetics, DIMOUniversity of BariBariItaly

Personalised recommendations