Starvation is a strong physiological stimulus of macroautophagy/autophagy. In this study, we addressed the question as to whether it would be possible to measure autophagy in blood cells after nutrient deprivation. Fasting of mice for 48 h (which causes ∼20% weight loss) or starvation of human volunteers for up to 4 d (which causes <2% weight loss) provokes major changes in the plasma metabolome, yet induces only relatively minor alterations in the intracellular metabolome of circulating leukocytes. White blood cells from mice and human volunteers responded to fasting with a marked reduction in protein lysine acetylation, affecting both nuclear and cytoplasmic compartments. In circulating leukocytes from mice that underwent 48-h fasting, an increase in LC3B lipidation (as assessed by immunoblotting and immunofluorescence) only became detectable if the protease inhibitor leupeptin was injected 2 h before drawing blood. Consistently, measurement of an enhanced autophagic flux was only possible if white blood cells from starved human volunteers were cultured in the presence or absence of leupeptin. Whereas all murine leukocyte subpopulations significantly increased the number of LC3B+ puncta per cell in response to nutrient deprivation, only neutrophils from starved volunteers showed signs of activated autophagy (as determined by a combination of multi-color immunofluorescence, cytofluorometry and image analysis). Altogether, these results suggest that white blood cells are suitable for monitoring autophagic flux. In addition, we propose that the evaluation of protein acetylation in circulating leukocytes can be adopted as a biochemical marker of organismal energetic status.

b Université Paris Descartes Sorbonne Paris Cité Paris France

c Université Pierre et Marie Curie Paris France

e Centre d'Investigation Clinique Unité de Recherche Clinique Paris Centre Necker Cochin Assistance Publique Hôpitaux de Paris France

Equipe 11 labellisée Ligue contre le Cancer Centre de Recherche des Cordeliers INSERM U 1138 Paris France

f Sotio a c ; Prague Czech Republic

g Institute of Molecular Biosciences NAWI Graz University of Graz Graz Austria

h BioTechMed Graz Graz Austria

i Pôle de Biologie Hôpital Européen Georges Pompidou AP HP Paris France

j Karolinska Institute Department of Women's and Children's Health Karolinska University Hospital Stockholm Sweden

Metabolomics and Cell Biology Platforms Gustave Roussy Comprehensive Cancer Institute Villejuif France

Zobrazit více v PubMed

Mizushima N, Klionsky DJ. Protein turnover via autophagy: implications for metabolism. Annu Rev Nutr 2007; 27:19-40; PMID:17311494; http://dx.doi.org/10.1146/annurev.nutr.27.061406.093749 PubMed DOI

He C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 2009; 43:67-93; PMID:19653858; http://dx.doi.org/10.1146/annurev-genet-102808-114910 PubMed DOI PMC

Levine B, Mizushima N, Virgin HW. Autophagy in immunity and inflammation. Nature 2011; 469:323-35; PMID:21248839; http://dx.doi.org/10.1038/nature09782 PubMed DOI PMC

Shen HM, Mizushima N. At the end of the autophagic road: an emerging understanding of lysosomal functions in autophagy. Trends Biochem Sci 2014; 39:61-71; PMID:24369758; http://dx.doi.org/10.1016/j.tibs.2013.12.001 PubMed DOI

Galluzzi L, Pietrocola F, Bravo-San Pedro JM, Amaravadi RK, Baehrecke EH, Cecconi F, Codogno P, Debnath J, Gewirtz DA, Karantza V, et al.. Autophagy in malignant transformation and cancer progression. EMBO J 2015; 34:856-80; PMID:25712477; http://dx.doi.org/10.15252/embj.201490784 PubMed DOI PMC

Melendez A, Talloczy Z, Seaman M, Eskelinen EL, Hall DH, Levine B. Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science 2003; 301:1387-91. PubMed

Tavernarakis N, Pasparaki A, Tasdemir E, Maiuri MC, Kroemer G. The effects of p53 on whole organism longevity are mediated by autophagy. Autophagy 2008; 4:870-3; PMID:18728385; http://dx.doi.org/10.4161/auto.6730 PubMed DOI

Eisenberg T, Knauer H, Schauer A, Buttner S, Ruckenstuhl C, Carmona-Gutierrez D, Ring J, Schroeder S, Magnes C, Antonacci L, et al.. Induction of autophagy by spermidine promotes longevity. Nat Cell Biol 2009; 11:1305-14; PMID:19801973; http://dx.doi.org/10.1038/ncb1975 PubMed DOI

Morselli E, Maiuri MC, Markaki M, Megalou E, Pasparaki A, Palikaras K, Criollo A, Galluzzi L, Malik SA, Vitale I. Caloric restriction and resveratrol promote longevity through the Sirtuin-1-dependent induction of autophagy. Cell Death Dis 2010; 1:e10; PMID:21364612; http://dx.doi.org/10.1038/cddis.2009.8 PubMed DOI PMC

Bjedov I, Toivonen JM, Kerr F, Slack C, Jacobson J, Foley A, Partridge L. Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. Cell Metab 2010; 11:35-46; PMID:20074526; http://dx.doi.org/10.1016/j.cmet.2009.11.010 PubMed DOI PMC

Rubinsztein DC, Marino G, Kroemer G. Autophagy and aging. Cell 2011; 146:682-95; PMID:21884931; http://dx.doi.org/10.1016/j.cell.2011.07.030 PubMed DOI

Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell 2013; 153:1194-217; PMID:23746838; http://dx.doi.org/10.1016/j.cell.2013.05.039 PubMed DOI PMC

Madeo F, Zimmermann A, Maiuri MC, Kroemer G. Essential role for autophagy in life span extension. J Clin Invest 2015; 125:85-93; PMID:25654554; http://dx.doi.org/10.1172/JCI73946 PubMed DOI PMC

Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell 2008; 132:27-42; PMID:18191218; http://dx.doi.org/10.1016/j.cell.2007.12.018 PubMed DOI PMC

Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, Agholme L, Agnello M, Agostinis P, Aguirre-Ghiso JA, et al.. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 2012; 8:445-544; PMID:22966490; http://dx.doi.org/10.4161/auto.19496 PubMed DOI PMC

Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, Adachi H, Adams CM, Adams PD, Adeli K, et al.. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 2016; 12:1-222; PMID:26799652; http://dx.doi.org/10.1080/15548627.2015.1100356 PubMed DOI PMC

Rubinsztein DC, Cuervo AM, Ravikumar B, Sarkar S, Korolchuk V, Kaushik S, Klionsky DJ. In search of an "autophagomometer". Autophagy 2009; 5:585-9; PMID:19411822; http://dx.doi.org/10.4161/auto.5.5.8823 PubMed DOI

Ju JS, Varadhachary AS, Miller SE, Weihl CC. Quantitation of "autophagic flux" in mature skeletal muscle. Autophagy 2010; 6:929-35; PMID:20657169; http://dx.doi.org/10.4161/auto.6.7.12785 PubMed DOI PMC

Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 2004; 15:1101-11; PMID:14699058; http://dx.doi.org/10.1091/mbc.E03-09-0704 PubMed DOI PMC

Imai S, Guarente L. NAD+ and sirtuins in aging and disease. Trends Cell Biol 2014; 24:464-71; PMID:24786309; http://dx.doi.org/10.1016/j.tcb.2014.04.002 PubMed DOI PMC

Marino G, Pietrocola F, Eisenberg T, Kong Y, Malik SA, Andryushkova A, Schroeder S, Pendl T, Harger A, Niso-Santano M, et al.. Regulation of autophagy by cytosolic acetyl-coenzyme A. Mol Cell 2014; 53:710-25; PMID:24560926; http://dx.doi.org/10.1016/j.molcel.2014.01.016 PubMed DOI

Madeo F, Pietrocola F, Eisenberg T, Kroemer G. Caloric restriction mimetics: towards a molecular definition. Nat Rev Drug Discov 2014; 13:727-40; PMID:25212602; http://dx.doi.org/10.1038/nrd4391 PubMed DOI

Pietrocola F, Lachkar S, Enot DP, Niso-Santano M, Bravo-San Pedro JM, Sica V, Izzo V, Maiuri MC, Madeo F, Mariño G. Spermidine induces autophagy by inhibiting the acetyltransferase EP300. Cell Death Differ 2015; 22:509-16; PMID:25526088; http://dx.doi.org/10.1038/cdd.2014.215 PubMed DOI PMC

Kim H, Golub GH, Park H. Missing value estimation for DNA microarray gene expression data: local least squares imputation. Bioinformatics 2005; 21:187-98; PMID:15333461; http://dx.doi.org/10.1093/bioinformatics/bth499 PubMed DOI

Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 2015; 43:e47; PMID:25605792; http://dx.doi.org/10.1093/nar/gkv007 PubMed DOI PMC

Pietrocola F, Pol J, Vacchelli E, Baracco EE, Levesque S, Castoldi F, Maiuri MC, Madeo F, Kroemer G. Autophagy induction for the treatment of cancer. Autophagy 2016:12:1962-1964. PubMed PMC

Hariharan N, Maejima Y, Nakae J, Paik J, Depinho RA, Sadoshima J. Deacetylation of FoxO by Sirt1 Plays an Essential Role in Mediating Starvation-Induced Autophagy in Cardiac Myocytes. Circ Res 2010; 107:1470-82; PMID:20947830; http://dx.doi.org/10.1161/CIRCRESAHA.110.227371 PubMed DOI PMC

Longo VD. Linking sirtuins, IGF-I signaling, and starvation. Exp Gerontol 2009; 44:70; PMID:18638538; http://dx.doi.org/10.1016/j.exger.2008.06.005 PubMed DOI

Fendl B, Weiss R, Fischer MB, Spittler A, Weber V. Characterization of extracellular vesicles in whole blood: Influence of pre-analytical parameters and visualization of vesicle-cell interactions using imaging flow cytometry. Biochem Biophys Res Commun 2016; 478:168-73; PMID:27444383; http://dx.doi.org/10.1016/j.bbrc.2016.07.073 PubMed DOI

Samsel L, Dagur PK, Raghavachari N, Seamon C, Kato GJ, McCoy JP Jr.. Imaging flow cytometry for morphologic and phenotypic characterization of rare circulating endothelial cells. Cytometry B Clin Cytom 2013; 84:379-89; PMID:23554273; http://dx.doi.org/10.1002/cyto.b.21088 PubMed DOI PMC

Clark RT. Imaging flow cytometry enhances particle detection sensitivity for extracellular vesicle analysis; Nature Methods 2015; 12; PMID:27444383; http://dx.doi.org/2460690010.1038/nmeth.3321 DOI

Eisenberg T, Schroeder S, Andryushkova A, Pendl T, Kuttner V, Bhukel A, Mariño G, Pietrocola F, Harger A, Zimmermann A, et al.. Nucleocytosolic depletion of the energy metabolite acetyl-coenzyme a stimulates autophagy and prolongs lifespan. Cell Metab 2014; 19:431-44; PMID:24606900; http://dx.doi.org/10.1016/j.cmet.2014.02.010 PubMed DOI PMC

Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting. Autophagy 2007; 3:542-5; PMID:17611390; http://dx.doi.org/10.4161/auto.4600 PubMed DOI

Bjorkoy G, Lamark T, Pankiv S, Overvatn A, Brech A, Johansen T. Monitoring autophagic degradation of p62/SQSTM1. Methods Enzymol 2009; 452:181-97; PMID:19200883; http://dx.doi.org/10.1016/S0076-6879(08)03612-4 PubMed DOI

Komatsu M, Ichimura Y. Physiological significance of selective degradation of p62 by autophagy. FEBS Lett 2010; 584:1374-8; PMID:20153326; http://dx.doi.org/10.1016/j.febslet.2010.02.017 PubMed DOI

Sahani MH, Itakura E, Mizushima N. Expression of the autophagy substrate SQSTM1/p62 is restored during prolonged starvation depending on transcriptional upregulation and autophagy-derived amino acids. Autophagy 2014; 10:431-41; PMID:24394643; http://dx.doi.org/10.4161/auto.27344 PubMed DOI PMC

Mofarrahi M, Guo Y, Haspel JA, Choi AM, Davis EC, Gouspillou G, Hepple RT, Godin R, Burelle Y, Hussain SN. Autophagic flux and oxidative capacity of skeletal muscles during acute starvation. Autophagy 2013; 9:1604-20; PMID:23955121; http://dx.doi.org/10.4161/auto.25955 PubMed DOI

Esteban-Martinez L, Boya P. Autophagic flux determination in vivo and ex vivo. Methods 2015; 75:79-86; PMID:25644445; http://dx.doi.org/10.1016/j.ymeth.2015.01.008 PubMed DOI

Phadwal K, Alegre-Abarrategui J, Watson AS, Pike L, Anbalagan S, Hammond EM, Wade-Martins R, McMichael A, Klenerman P, Simon AK. A novel method for autophagy detection in primary cells: impaired levels of macroautophagy in immunosenescent T cells. Autophagy 2012; 8:677-89; PMID:22302009; http://dx.doi.org/10.4161/auto.18935 PubMed DOI PMC

Finn PF, Dice JF. Proteolytic and lipolytic responses to starvation. Nutrition 2006; 22:830-44; PMID:16815497; http://dx.doi.org/10.1016/j.nut.2006.04.008 PubMed DOI

GF Cahill., Jr Fuel metabolism in starvation. Annu Rev Nutr 2006; 26:1-22; PMID:16848698; http://dx.doi.org/10.1146/annurev.nutr.26.061505.111258 PubMed DOI

Bouwens M, Afman LA, Muller M. Fasting induces changes in peripheral blood mononuclear cell gene expression profiles related to increases in fatty acid beta-oxidation: functional role of peroxisome proliferator activated receptor alpha in human peripheral blood mononuclear cells. Am J Clin Nutr 2007; 86:1515-23; PMID:17991667 PubMed

Hildebrandt W, Kinscherf R, Hauer K, Holm E, Droge W. Plasma cystine concentration and redox state in aging and physical exercise. Mech Ageing Dev 2002; 123:1269-81; PMID:12020948; http://dx.doi.org/10.1016/S0047-6374(02)00013-1 PubMed DOI

Hine C, Harputlugil E, Zhang Y, Ruckenstuhl C, Lee BC, Brace L, Longchamp A, Treviño-Villarreal JH, Mejia P, Ozaki CK, et al.. Endogenous hydrogen sulfide production is essential for dietary restriction benefits. Cell 2015; 160:132-44; PMID:25542313; http://dx.doi.org/10.1016/j.cell.2014.11.048 PubMed DOI PMC

Green DR, Galluzzi L, Kroemer G. Cell biology. Metabolic control of cell death. Science 2014; 345:1250256. PubMed PMC

Galluzzi L, Pietrocola F, Levine B, Kroemer G. Metabolic control of autophagy. Cell 2014; 159:1263-76; PMID:25480292; http://dx.doi.org/10.1016/j.cell.2014.11.006 PubMed DOI PMC

Lee C, Safdie FM, Raffaghello L, Wei M, Madia F, Parrella E, Hwang D, Cohen P, Bianchi G, Longo VD. Reduced levels of IGF-I mediate differential protection of normal and cancer cells in response to fasting and improve chemotherapeutic index. Cancer Res 2010; 70:1564-72; PMID:20145127; http://dx.doi.org/10.1158/0008-5472.CAN-09-3228 PubMed DOI PMC

Longo VD, Fontana L. Calorie restriction and cancer prevention: metabolic and molecular mechanisms. Trends Pharmacol Sci 2010; 31:89-98; PMID:20097433; http://dx.doi.org/10.1016/j.tips.2009.11.004 PubMed DOI PMC

Lee JV, Carrer A, Shah S, Snyder NW, Wei S, Venneti S, Worth AJ, Yuan ZF, Lim HW, Liu S, et al.. Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation. Cell Metab 2014; 20:306-19; PMID:24998913; http://dx.doi.org/10.1016/j.cmet.2014.06.004 PubMed DOI PMC

Comerford SA, Huang Z, Du X, Wang Y, Cai L, Witkiewicz AK, Walters H, Tantawy MN, Fu A, Manning HC, et al.. Acetate dependence of tumors. Cell 2014; 159:1591-602; PMID:25525877; http://dx.doi.org/10.1016/j.cell.2014.11.020 PubMed DOI PMC

Mashimo T, Pichumani K, Vemireddy V, Hatanpaa KJ, Singh DK, Sirasanagandla S, Nannepaga S, Piccirillo SG, Kovacs Z, Foong C, et al.. Acetate is a bioenergetic substrate for human glioblastoma and brain metastases. Cell 2014; 159:1603-14; PMID:25525878; http://dx.doi.org/10.1016/j.cell.2014.11.025 PubMed DOI PMC

Wang Z, Cao L, Kang R, Yang M, Liu L, Zhao Y, Yu Y, Xie M, Yin X, Livesey KM, et al.. Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARalpha oncoprotein. Autophagy 2011; 7:401-11; PMID:21187718; http://dx.doi.org/10.4161/auto.7.4.14397 PubMed DOI PMC

Orfanelli T, Doulaveris G, Holcomb K, Jeong JM, Sisti G, Kanninen TT, Caputo TA, Gupta D, Witkin SS. Inhibition of autophagy in peripheral blood mononuclear cells by vaginal fluid from women with a malignant adnexal mass. Int J Cancer 2015; 137:2879-84; PMID:26132572; http://dx.doi.org/10.1002/ijc.29665 PubMed DOI

Leveque-El Mouttie L, Vu T, Lineburg KE, Kuns RD, Bagger FO, Teal BE, Lor M, Boyle GM, Bruedigam C, Mintern JD, et al.. Autophagy is required for stem cell mobilization by G-CSF. Blood 2015; 125:2933-6; PMID:25788702; http://dx.doi.org/10.1182/blood-2014-03-562660 PubMed DOI

Remijsen Q, Vanden Berghe T, Wirawan E, Asselbergh B, Parthoens E, De Rycke R, Noppen S, Delforge M, Willems J, Vandenabeele P. Neutrophil extracellular trap cell death requires both autophagy and superoxide generation. Cell Res 2011; 21:290-304; PMID:21060338; http://dx.doi.org/10.1038/cr.2010.150 PubMed DOI PMC

Francois A, Julian A, Ragot S, Dugast E, Blanchard L, Brishoual S, Terro F, Chassaing D, Page G, Paccalin M. Inflammatory Stress on Autophagy in Peripheral Blood Mononuclear Cells from Patients with Alzheimer's Disease during 24 Months of Follow-Up. PLoS One 2015; 10:e0138326; PMID:26393801; http://dx.doi.org/10.1371/journal.pone.0138326 PubMed DOI PMC

Compromised autophagy and mitophagy in brain ageing and Alzheimer's diseases