Overweight in the Elderly Induces a Switch in Energy Metabolism that Undermines Muscle Integrity
Potes Yaiza1,2, Pérez-Martinez Zulema3, Bermejo-Millo Juan C.1,2, Rubio-Gonzalez Adrian1, Fernandez-Fernández María4, Bermudez Manuel4, Arche Jose M.4, Solano Juan J.2,4, Boga Jose A.3, Oliván Mamen2,5, Caballero Beatriz1,2, Vega-Naredo Ignacio1,2, Coto-Montes Ana1,2,*
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.
Potes Yaiza,Pérez-Martinez Zulema,Bermejo-Millo Juan C., 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.
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