Overweight in the Elderly Induces a Switch in Energy Metabolism that Undermines Muscle Integrity
Yaiza Potes1,2, Zulema Pérez-Martinez3, Juan C. Bermejo-Millo1,2, Adrian Rubio-Gonzalez1, María Fernandez-Fernández4, Manuel Bermudez4, Jose M. Arche4, Juan J. Solano2,4, Jose A. Boga3, Mamen Oliván2,5, Beatriz Caballero1,2, Ignacio Vega-Naredo1,2, Ana Coto-Montes1,2,*
1Department of Morphology and Cell Biology, Faculty of Medicine, University of Oviedo, Asturias, Spain 2Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Spain 3Microbiology Service, Central University Hospital of Asturias, Asturias, Spain 4Geriatric Service, Monte Naranco Hospital, Asturias, Spain 5Servicio Regional de Investigación y Desarrollo Agroalimentario (SERIDA), Asturias, Spain 5Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Spain
Aging is characterized by a progressive loss of skeletal muscle mass and function (sarcopenia). Obesity exacerbates age-related decline and lead to frailty. Skeletal muscle fat infiltration increases with aging and seems to be crucial for the progression of sarcopenia. Additionally, skeletal muscle plasticity modulates metabolic adaptation to different pathophysiological situations. Thus, cellular bioenergetics and mitochondrial profile were studied in the skeletal muscle of overweight aged people without reaching obesity to prevent this extreme situation. Overweight aged muscle lacked ATP production, as indicated by defects in the phosphagen system, glycolysis and especially mostly by oxidative phosphorylation metabolic pathway. Overweight subjects exhibited an inhibition of mitophagy that was linked to an increase in mitochondrial biogenesis that underlies the accumulation of dysfunctional mitochondria and encourages the onset of sarcopenia. As a strategy to maintain cellular homeostasis, overweight subjects experienced a metabolic switch from oxidative to lactic acid fermentation metabolism, which allows continued ATP production under mitochondrial dysfunction, but without reaching physiological aged basal levels. This ATP depletion induced early signs of impaired contractile function and a decline in skeletal muscle structural integrity, evidenced by lower levels of filamin C. Our findings reveal the main effector pathways at an early stage of obesity and highlight the importance of mitochondrial metabolism in overweight and obese individuals. Exploiting mitochondrial profiles for therapeutic purposes in humans is an ambitious strategy for treating muscle impairment diseases.
Yaiza Potes,Zulema Pérez-Martinez,Juan C. Bermejo-Millo, et al. Overweight in the Elderly Induces a Switch in Energy Metabolism that Undermines Muscle Integrity[J]. Aging and disease,
2019, 10(2): 217-230.
Figure 1. Phosphagen pathway, glycogenolysis and glycolysis. Laser desorption/ionization-time of flight (MALDI-TOF/TOF) mass spectrometry analysis for studying the phosphagen system, glycogenolysis and glycolysis pathways in muscle tissues of normal weight and overweight patients. (A) Levels of proteins involved in the phosphagen system (creatine kinase and adenylate kinase). Bar charts show the means of semi-quantitative optical density (O.D.) ± SEM. (B) Levels of the main protein involved in the regulation of glycogenolysis (glycogen phosphorylase). Bar charts show the means of semi-quantitative optical density (O.D.) ± SEM. (C) Levels of proteins involved in glycolysis (fructose 1,6-bisphosphate aldolase A, glyceraldehyde 3-phosphate dehydrogenase, β-enolase and pyruvate kinase). Bar charts show the means of semi-quantitative optical density (O.D.) ± SEM. (D) Pyruvate kinase activity determination as the main enzyme involved in glycolysis regulation. The pyruvate kinase activity is presented as nmol pyruvate / min*mg protein. Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 2. Oxidative phosphorylation profile. Laser desorption/ionization-time of flight (MALDI-TOF/TOF) mass spectrometry, RT-qPCR and western blot analysis for studying the oxidative phosphorylation pathway in muscle tissues of normal weight (N) and overweight (OW) patients. (A) Levels of proteins implied in pyruvate dehydrogenase activity regulation (pyruvate dehydrogenase (PDH), phospho-pyruvate dehydrogenase (p-PDH) and its ratio (p-PDH/PDH)). Bar charts show means of semi-quantitative optical density (O.D.) ± SEM. Representative immunoblots of PDH and p-PDH. Ponceau staining was used as a loading control (B) The amount mitochondrial content is marked by the protein levels of TOM20. Bar charts show the means of semi-quantitative optical density (O.D.) ± SEM. (C) RT-qPCR was used to confirm mitochondrial DNA (mtDNA) content. mtDNA content was compared against genomic DNA (nDNA). Data are expressed as the mean ± SEM (D) Levels of subunits from the protein complexes of the mitochondrial electron transport chain (NADH dehydrogenase (ubiquitone) 1b subcomplex 8 (NDUFB8) from complex I (CI), iron sulfur subunit (SDHB) from complex II (CII), ubiquinolcytochrome c reductase core protein II (UQCRC2) subunit from complex III (CIII), cytochrome c oxidase subunit I (MTCO1) from complex IV (CIV), and ATP synthase subunit α (ATP5A) from complex V (CV), isoform c of the ATP synthase subunit α from complex V and the H+ transporting F1 complex subunit β from complex V). Bar charts show the means of semi-quantitative optical density (O.D.) ± SEM. Representative immunoblots of subunits from the protein complexes of the mitochondrial electron transport chain (CI, CII, CIII, CIV and CV). Ponceau staining was used as a loading control and TOM20 as a mitochondrial marker **, P < 0.01; ***, P < 0.001.
Figure 3. Fatty acid β-oxidation and fatty acid synthesis pathways. RT-qPCR and western blot analysis for studying the fatty acid β-oxidation and synthesis pathways in muscle tissues of normal weight (N) and overweight (OW) patients. (A) Relative mRNA expression of genes implicated in fatty acid β-oxidation (ADIPOR1 , ADIPOR2 and peroxisome proliferator-activated receptor alpha (PPARA )) and in fatty acid synthesis (acetyl-Coa carboxylase (ACACA )). Bar charts show the means of mRNA relative expression ± SEM. (B) Cyclophilin D protein levels. Bar charts show means of semi-quantitative optical density (O.D.) ± SEM. (C) A representative immunoblot of cyclophilin D. Ponceau staining was used as a loading control and TOM20 as a mitochondrial marker *, P < 0.05; **, P < 0.01.
Figure 4. Characterization of mitochondrial dynamics, biogenesis and mitophagy pathways. RT-qPCR and western blot analysis for studying mitochondrial dynamics, biogenesis and mitophagy in muscle tissues of normal weight (N) and overweight (OW) patients. (A) Levels of mRNA and proteins involved in mitochondrial dynamics. (mRNA levels of mitofusin 2 (MFN 2 ) and dynamin-related protein 1 (DRP1 ) and protein levels of mitofusin 2 (MFN 2), optin atrophy protein 1 (OPA1), dynamin-related protein 1 (DRP1) and mitochondrial fission 1 protein (FIS1). Bar charts show means of mRNA relative expression and semi-quantitative optical density (O.D.) ± SEM. (B) Levels of peroxisome proliferator-activated receptor-γ coactivator 1α (PPARGC1A ) involved in mitochondrial biogenesis. Bar charts show means of mRNA relative expression ± SEM. (C) Levels of proteins involved in mitophagy (Bcl-2 nineteen-kilodalton interacting protein 3 (BNIP3) and Bcl-2 nineteen-kilodalton interacting protein 3-like (BNIP3L/NIX)). Bar charts show means of semi-quantitative optical density (O.D.) ± SEM. (D) Representative immunoblots of mitochondrial dynamics and mitophagy markers. Ponceau staining was used as a loading control *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 5. Skeletal muscle ATP production, plasma lactate content, oxidative protein damage and characterization of excitation-contraction coupling mechanism. Luminometric and spectrophotometric analysis for ATP and oxidative protein damage in muscle tissue, lactate in plasma and RT-qPCR, western blot and laser desorption/ionization-time of flight (MALDI-TOF/TOF) mass spectrometry analysis for studying muscle contraction and integrity in muscle tissues of normal weight (N) and overweight (OW) patients. (A) Levels of ATP in skeletal muscle. The ATP content is presented as nmol ATP/g protein. Data are expressed as the mean ± SEM. (B) Levels of lactate in plasma. The lactate content is presented as mg lactate/dL. Data are expressed as the mean ± SEM. (C) Levels of protein oxidative damage in skeletal muscle. The protein oxidative damage is presented as nmol protein carbonyl/mg protein. Data are expressed as the mean ± SEM. (D) Levels of mRNA and proteins required for excitation-contraction coupling (Ryanodine receptor 1 (RYR1 ), phospho-RYR1 (p-RYR1), CALSTABIN , sarco/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1 ), Ca2+/Calmodulin dependent protein kinase II (CaMKII), phospho-CaMKII (p-CaMKII)). Bar charts show the means of mRNA relative expression and semi-quantitative optical density (O.D.) ± SEM. (E) Levels of filamin C protein involved in the maintenance of structural integrity. Bar charts show the means of semi-quantitative optical density (O.D.) ± SEM. (F) Representative immunoblots of muscle contraction markers. Ponceau staining was used as a loading control. *, P < 0.05; ***, P < 0.001.
Figure 6. Global effect of overweight during aging on energy metabolic systems. Overweight induces a defective phosphagen, glycolysis and oxidative phosphorylation metabolic pathways during aging, resulting in a switch from oxidative to lactic acid fermentation metabolism.
Kim TN, Choi KM (2013). Sarcopenia: definition, epidemiology, and pathophysiology. J Bone Metab, 20:1-10.
Di Monaco M, Castiglioni C, De Toma E, Gardin L, Giordano S, Di Monaco R, et al. (2015). Presarcopenia and sarcopenia in hip-fracture women: prevalence and association with ability to function in activities of daily living. Aging Clin Exp Res, 27:465-472.
Kirkland JL, Tchkonia T, Pirtskhalava T, Han J, Karagiannides I (2002). Adipogenesis and aging: does aging make fat go MAD? Exp Gerontol, 37:757-767.
Song MY, Ruts E, Kim J, Janumala I, Heymsfield S, Gallagher D (2004). Sarcopenia and increased adipose tissue infiltration of muscle in elderly African American women. Am J Clin Nutr, 79:874-880.
Sinha I, Sakthivel D, Varon DE (2017). Systemic Regulators of Skeletal Muscle Regeneration in Obesity. Front Endocrinol (Lausanne), 8:29.
Woo M, Isganaitis E, Cerletti M, Fitzpatrick C, Wagers AJ, Jimenez-Chillaron J, et al. (2011). Early life nutrition modulates muscle stem cell number: implications for muscle mass and repair. Stem Cells Dev, 20:1763-1769.
Atkins JL, Whincup PH, Morris RW, Lennon LT, Papacosta O, Wannamethee SG (2014). Sarcopenic obesity and risk of cardiovascular disease and mortality: a population-based cohort study of older men. J Am Geriatr Soc, 62:253-260.
Pugh TD, Conklin MW, Evans TD, Polewski MA, Barbian HJ, Pass R, et al. (2013). A shift in energy metabolism anticipates the onset of sarcopenia in rhesus monkeys. Aging Cell, 12:672-681.
Chanseaume E, Barquissau V, Salles J, Aucouturier J, Patrac V, Giraudet C, et al. (2010). Muscle mitochondrial oxidative phosphorylation activity, but not content, is altered with abdominal obesity in sedentary men: synergism with changes in insulin sensitivity. J Clin Endocrinol Metab, 95:2948-2956.
Cerletti M, Jang YC, Finley LW, Haigis MC, Wagers AJ (2012). Short-term calorie restriction enhances skeletal muscle stem cell function. Cell Stem Cell, 10:515-519.
Fontes-Oliveira CC, Steinz M, Schneiderat P, Mulder H, Durbeej M (2017). Bioenergetic Impairment in Congenital Muscular Dystrophy Type 1A and Leigh Syndrome Muscle Cells. Sci Rep, 7:45272.
Koopman R, Ly CH, Ryall JG (2014). A metabolic link to skeletal muscle wasting and regeneration. Front Physiol, 5:32.
Baraibar MA, Hyzewicz J, Rogowska-Wrzesinska A, Bulteau AL, Prip-Buus C, Butler-Browne G, et al. (2016). Impaired energy metabolism of senescent muscle satellite cells is associated with oxidative modifications of glycolytic enzymes. Aging (Albany NY), 8:3375-3389.
Ni HM, Williams JA, Ding WX (2015). Mitochondrial dynamics and mitochondrial quality control. Redox Biol, 4:6-13.
Romanello V, Guadagnin E, Gomes L, Roder I, Sandri C, Petersen Y, et al. (2010). Mitochondrial fission and remodelling contributes to muscle atrophy. EMBO J, 29:1774-1785.
Eisner V, Lenaers G, Hajnoczky G (2014). Mitochondrial fusion is frequent in skeletal muscle and supports excitation-contraction coupling. J Cell Biol, 205:179-195.
Bellinger AM, Mongillo M, Marks AR (2008). Stressed out: the skeletal muscle ryanodine receptor as a target of stress. J Clin Invest, 118:445-453.
Hwang H, Bowen BP, Lefort N, Flynn CR, De Filippis EA, Roberts C, et al. (2010). Proteomics analysis of human skeletal muscle reveals novel abnormalities in obesity and type 2 diabetes. Diabetes, 59:33-42.
Charlson ME, Pompei P, Ales KL, MacKenzie CR (1987). A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J Chronic Dis, 40:373-383.
Torres Moreno B, Nunez Gonzalez E, Perez Hernandez Dde G, Simon Turriate JP, Alastuey Gimenez C, Diaz Melian J, et al. (2009). [Barthel and Charlson indexes for the prognosis of mortality and institutionalization in hospitalized geriatric patients]. Rev Esp Geriatr Gerontol, 44:209-212.
Bermúdez M, Caballero B, de Luxán-Delgado B, Potes Y, Fernández-Fernández M, Coto-Montes A, et al. (2018). Physical performance drops after hip fracture surgery. HIPA study. J Gerontol Aging Res, In press.
Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72:248-254.
Oliván M, Fernández-Suárez V, Díaz-Martínez F, Sierra V, Coto-Montes A, de Luxan-Delgado B, et al. (2016). Identification of biomarkers of stress in meat of pigs managed under different mixing treatments. Br Biotechnol J, 11:1-13.
Potes Y, de Luxan-Delgado B, Rodriguez-Gonzalez S, Guimaraes MRM, Solano JJ, Fernandez-Fernandez M, et al. (2017). Overweight in elderly people induces impaired autophagy in skeletal muscle. Free Radic Biol Med, 110:31-41.
Fortes MA, Marzuca-Nassr GN, Vitzel KF, da Justa Pinheiro CH, Newsholme P, Curi R (2016). Housekeeping proteins: How useful are they in skeletal muscle diabetes studies and muscle hypertrophy models? Anal Biochem, 504:38-40.
Livak KJ, Schmittgen TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25:402-408.
Venegas V, Halberg MC (2012). Measurement of mitochondrial DNA copy number. Methods Mol Biol, 837:327-335.
de Gonzalo-Calvo D, de Luxan-Delgado B, Martinez-Camblor P, Rodriguez-Gonzalez S, Garcia-Macia M, Suarez FM, et al. (2012). Chronic inflammation as predictor of 1-year hospitalization and mortality in elderly population. Eur J Clin Invest, 42:1037-1046.
de Gonzalo-Calvo D, de Luxan-Delgado B, Rodriguez-Gonzalez S, Garcia-Macia M, Suarez FM, Solano JJ, et al. (2012). Interleukin 6, soluble tumor necrosis factor receptor I and red blood cell distribution width as biological markers of functional dependence in an elderly population: a translational approach. Cytokine, 58:193-198.
de Gonzalo-Calvo D, de Luxan-Delgado B, Rodriguez-Gonzalez S, Garcia-Macia M, Suarez FM, Solano JJ, et al. (2012). Oxidative protein damage is associated with severe functional dependence among the elderly population: a principal component analysis approach. J Gerontol A Biol Sci Med Sci, 67:663-670.
Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, et al. (1990). Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol, 186:464-478.
Coto-Montes A, Hardeland R (1999). Antioxidative effects of melatonin in Drosophila melanogaster: antagonization of damage induced by the inhibition of catalase. J Pineal Res, 27:154-158.
Loson OC, Song Z, Chen H, Chan DC (2013). Fis1, Mff, MiD49, and MiD51 mediate Drp1 recruitment in mitochondrial fission. Mol Biol Cell, 24:659-667.
Lawrenson L, Hoff J, Richardson RS (2004). Aging attenuates vascular and metabolic plasticity but does not limit improvement in muscle VO(2) max. Am J Physiol Heart Circ Physiol, 286:H1565-1572.
Jeong HJ, Lee HJ, Vuong TA, Choi KS, Choi D, Koo SH, et al. (2016). Prmt7 Deficiency Causes Reduced Skeletal Muscle Oxidative Metabolism and Age-Related Obesity. Diabetes, 65:1868-1882.
van Deursen J, Ruitenbeek W, Heerschap A, Jap P, ter Laak H, Wieringa B (1994). Creatine kinase (CK) in skeletal muscle energy metabolism: a study of mouse mutants with graded reduction in muscle CK expression. Proc Natl Acad Sci U S A, 91:9091-9095.
Nuss JE, Amaning JK, Bailey CE, DeFord JH, Dimayuga VL, Rabek JP, et al. (2009). Oxidative modification and aggregation of creatine kinase from aged mouse skeletal muscle. Aging (Albany NY), 1:557-572.
Gelfi C, Vigano A, Ripamonti M, Pontoglio A, Begum S, Pellegrino MA, et al. (2006). The human muscle proteome in aging. J Proteome Res, 5:1344-1353.
O'Connell K, Gannon J, Doran P, Ohlendieck K (2007). Proteomic profiling reveals a severely perturbed protein expression pattern in aged skeletal muscle. Int J Mol Med, 20:145-153.
Nakai N, Sato Y, Oshida Y, Yoshimura A, Fujitsuka N, Sugiyama S, et al. (1997). Effects of aging on the activities of pyruvate dehydrogenase complex and its kinase in rat heart. Life Sci, 60:2309-2314.
Porter C, Hurren NM, Cotter MV, Bhattarai N, Reidy PT, Dillon EL, et al. (2015). Mitochondrial respiratory capacity and coupling control decline with age in human skeletal muscle. Am J Physiol Endocrinol Metab, 309:E224-232.
Hawkins BJ, Levin MD, Doonan PJ, Petrenko NB, Davis CW, Patel VV, et al. (2010). Mitochondrial complex II prevents hypoxic but not calcium- and proapoptotic Bcl-2 protein-induced mitochondrial membrane potential loss. J Biol Chem, 285:26494-26505.
Pfleger J, He M, Abdellatif M (2015). Mitochondrial complex II is a source of the reserve respiratory capacity that is regulated by metabolic sensors and promotes cell survival. Cell Death Dis, 6:e1835.
Choi CS, Savage DB, Abu-Elheiga L, Liu ZX, Kim S, Kulkarni A, et al. (2007). Continuous fat oxidation in acetyl-CoA carboxylase 2 knockout mice increases total energy expenditure, reduces fat mass, and improves insulin sensitivity. Proc Natl Acad Sci U S A, 104:16480-16485.
Penzo D, Tagliapietra C, Colonna R, Petronilli V, Bernardi P (2002). Effects of fatty acids on mitochondria: implications for cell death. Biochim Biophys Acta, 1555:160-165.
Devalaraja-Narashimha K, Diener AM, Padanilam BJ (2011). Cyclophilin D deficiency prevents diet-induced obesity in mice. FEBS Lett, 585:677-682.
Gineste C, Hernandez A, Ivarsson N, Cheng AJ, Naess K, Wibom R, et al. (2015). Cyclophilin D, a target for counteracting skeletal muscle dysfunction in mitochondrial myopathy. Hum Mol Genet, 24:6580-6587.
Liesa M, Shirihai OS (2013). Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure. Cell Metab, 17:491-506.
Molina AJ, Wikstrom JD, Stiles L, Las G, Mohamed H, Elorza A, et al. (2009). Mitochondrial networking protects beta-cells from nutrient-induced apoptosis. Diabetes, 58:2303-2315.
Liu R, Jin P, Yu L, Wang Y, Han L, Shi T, et al. (2014). Impaired mitochondrial dynamics and bioenergetics in diabetic skeletal muscle. PLoS One, 9:e92810.
Mao K, Wang K, Liu X, Klionsky DJ (2013). The scaffold protein Atg11 recruits fission machinery to drive selective mitochondria degradation by autophagy. Dev Cell, 26:9-18.
Palikaras K, Lionaki E, Tavernarakis N (2015). Balancing mitochondrial biogenesis and mitophagy to maintain energy metabolism homeostasis. Cell Death Differ, 22:1399-1401.
Palikaras K, Lionaki E, Tavernarakis N (2015). Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans. Nature, 521:525-528.
Green DR, Galluzzi L, Kroemer G (2011). Mitochondria and the autophagy-inflammation-cell death axis in organismal aging. Science, 333:1109-1112.
Calvani R, Joseph AM, Adhihetty PJ, Miccheli A, Bossola M, Leeuwenburgh C, et al. (2013). Mitochondrial pathways in sarcopenia of aging and disuse muscle atrophy. Biol Chem, 394:393-414.
Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, Lesnefsky EJ (2003). Production of reactive oxygen species by mitochondria: central role of complex III. J Biol Chem, 278:36027-36031.
Lourenco dos Santos S, Baraibar MA, Lundberg S, Eeg-Olofsson O, Larsson L, Friguet B (2015). Oxidative proteome alterations during skeletal muscle ageing. Redox Biol, 5:267-274.
Dorn GW2nd, Maack C (2013). SR and mitochondria: calcium cross-talk between kissing cousins. J Mol Cell Cardiol, 55:42-49.