Lifelong aerobic exercise protects against inflammaging and cancer
Authors: Michael Crozier
Ethan Adler
Adam L Bujak
Yifan Yang
Paul Azzopardi
Linda May
Jacqueline M Bourgeois
Jacob Young
Mark A Tarnopolsky
Daniel R Rusiecki
Chris Moffatt
Ethan Lin
Joshua P Nederveen
Mats I Nilsson
Bart P Hettinga
Lucy Lan
Marlon R Leite
Jenny Nguyen
Journal: PLoS ONE (2019)
Discipline: Multidisciplinary
Keywords: Biological aging; inflammation; aerobic exercise training; cancer; health
Affiliations: Department of Pathology and Molecular Medicine, McMaster University Medical Center (MUMC), Hamilton, Ontario, Canada., Exerkine Corporation, McMaster University Medical Center (MUMC), Hamilton, Ontario, Canada., Department of Pediatrics, McMaster University Medical Center (MUMC), Hamilton, Ontario, Canada.

Abstract:  Biological aging is associated with progressive damage accumulation, loss of organ reserves, and systemic inflammation ('inflammaging'), which predispose for a wide spectrum of chronic diseases, including several types of cancer. In contrast, aerobic exercise training (AET) reduces inflammation, lowers all-cause mortality, and enhances both health and lifespan. In this study, we examined the benefits of early-onset, lifelong AET on predictors of health, inflammation, and cancer incidence in a naturally aging mouse model (C57BL/J6). Lifelong, voluntary wheel-running (O-AET; 26-month-old) prevented age-related declines in aerobic fitness and motor coordination vs. age-matched, sedentary controls (O-SED). AET also provided partial protection against sarcopenia, dynapenia, testicular atrophy, and overall organ pathology, hence augmenting the 'physiologic reserve' of lifelong runners. Systemic inflammation, as evidenced by a chronic elevation in 17 of 18 pro- and anti-inflammatory cytokines and chemokines (P &lt; 0.05 O-SED vs. 2-month-old Y-CON), was potently mitigated by lifelong AET (P &lt; 0.05 O-AET vs. O-SED), including master regulators of the cytokine cascade and cancer progression (IL-1β, TNF-α, and IL-6). In addition, circulating SPARC, previously known to be upregulated in metabolic disease, was elevated in old, sedentary mice, but was normalized to young control levels in lifelong runners. Remarkably, malignant tumours were also completely absent in the O-AET group, whereas they were present in the brain (pituitary), liver, spleen, and intestines of sedentary mice. Collectively, our results indicate that early-onset, lifelong running dampens inflammaging, protects against multiple cancer types, and extends healthspan of naturally-aged mice. 

Retracted: No

PubPeer comments:
 GAPDH expression and activity changes with age and it is not a good internal control for aging studies. This has been shown numerous times using rat, mouse, and human models: PMID: 10795720 Glyceraldehyde-3-phosphate dehydrogenase varies with age in glycolytic muscles of rats. Lowe DA1, Degens H, Chen KD, Alway SE. J Gerontol A Biol Sci Med Sci. 2000 Mar;55(3):B160-4. PMID: 25429098 GAPDH and u03b2-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle. Vigelsu00f8 A1, Dybboe R2, Hansen CN2, Dela F2, Helge JW2, Guadalupe Grau A2. 2015 Feb 1;118(3):386-94. doi: 10.1152 japplphysiol.00840.2014. PMID: 26747222 Cell specific differences in the protein abundances of GAPDH and Na(+),K(+)-ATPase in skeletal muscle from aged individuals. Wyckelsma VL1, McKenna MJ2, Levinger I2, Petersen AC2, Lamboley CR2, Murphy RM3. J Mol Neurosci. 2009 Mar;37(3):238-53. doi: 10.1007 s12031-008-9128-9. PMID: 18607772 Selection of reference genes for quantitative real-time RT-PCR studies in mouse brain. Boda E1, Pini A, Hoxha E, Parolisi R, Tempia F. J Mol Neurosci. 2009 Mar;37(3):238-53. doi: 10.1007 s12031-008-9128-9. PMID: 28536183 The NLRP3 inflammasome contributes to sarcopenia and lower muscle glycolytic potential in old mice. McBride MJ1, Foley KP1,2, D Souza DM3, Li YE1, Lau TC1,2, Hawke TJ3, Schertzer JD4,2. Am J Physiol Endocrinol Metab. 2017 Aug 1;313(2):E222-E232. doi: 10.1152 ajpendo.00060.2017. This renders the results in fig. 4 suspect. Subsequently, the conclusion that aging affected the mRNA expression of genes in fig 4 is not believable (unless the authors can demonstrate that GAPDH did not change with aging in their study). Furthermore, current standards indicate qPCR data be normalized to at least 2 housekeeping genes. Additionally, the Livak method is: 2^(u2212u0394u0394CT) method. The authors used 2^(-u0394CT) which is not the Livak method as claimed in supplementary data. This is probably why the y-axis numbers are so small in fig. 4 and don t make much physiological sense. Finally, in the supplementary section the authors state the used 7900HT and attribute it as a BioRad machine. It is not. It s made by ThermoFisher. All together, this dampens confidence in the gene expression data. , 
 additionally, following references and many many other show that GAPDH expression and activity are both affected by aging. With the advent of sophisticated technologies like qPCR and digital-droplet PCR - and more sensitive ELISA protein quantification, in this day and age, a change in GAPDH is easily detected by researchers. Conclusions derived by authors from figure 4 are questionable. J Gerontol A Biol Sci Med Sci. 2000 Mar;55(3):B160-4. Glyceraldehyde-3-phosphate dehydrogenase varies with age in glycolytic muscles of rats. Lowe DA1, Degens H, Chen KD, Alway SE. Nutrition. 2003 May;19(5):438-40. Glyceraldehyde-3-phosphate dehydrogenase expression varies with age and nutrition status. Mozdziak PE1, Dibner JJ, McCoy DW. Mechanisms of Ageing and Development. Volume 53, Issue 3, 30 April 1990, Pages 243-257 Messenger RNA levels and methlation patterns of GAPDH and u03b2-actin genes in rat liver, spleen and brain in relation to aging Author links open overlay panelP.ElineSlagboomWiljo J.F.De LeeuwJanVijg Rydbirk, R. et al. Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases. Sci. Rep. 6, 1u201311 (2016). Touchberry, C. D., Wacker, M. J., Richmond, S. R., Whitman, S. A. Godard, M. P. Age-related changes in relative expression of real-time PCR housekeeping genes in human skeletal muscle. J. Biomol. Tech. 17, 157u201362 (2006). Zampieri, M. et al. Validation of suitable internal control genes for expression studies in aging. Mech. Ageing Dev. 131, 89u201395 (2010). Hernandez-Segura, A., Rubingh, R. Demaria, M. Identification of stable senescence-associated reference genes. Aging Cell 18, e12911 (2019). de Jonge, H. J. M. et al. Evidence Based Selection of Housekeeping Genes. PLoS One 2, e898 (2007). , 
 Limbodessus Micromatos and Aldrovandia Phalacra: Just some corrections and counterpoints to your original posts: 1.tGenerally speaking, GAPDH is acceptable as a housekeeper and loading control in many different types of studies. Broad generalizations regarding its lack of usefulness should be discouraged and recommendations should ideally be made on a case-by-case basis. In aging research, specifically, the suitability of GAPDH as a housekeeper depends on several factors, including the species, organ, and in this case even muscle fiber-type. 2.tIn Nilsson et al., the authors examined mRNA levels in cardiac muscle (main paper) and quadriceps muscle (supplements) of young and old C57 mice. a.tSince GAPDH expression in cardiac muscle does not appear to be affected by aging in the C57 strain(1,2), it was likely a suitable choice (assuming CT values were the same between groups). b.tSimilar observations have been made in slow-type muscles(3), which would make GAPDH an acceptable option for those tissues as well. c.tGoing by the reference lists supplied by the original posters, there appears to be several reports indicating that full-length GAPDH expression is unaffected by aging even in fast-type (FT) muscles of C57 mice, thus making GAPDH an appropriate housekeeper in FT muscles in this strain background(4,5). This is not to say that GAPDH activity is unaffected by aging in FT in these mice per se(5), but qPCR results are obviously not normalized to enzyme activity. So, there does not appear to be strong support against the use of GAPDH as a housekeeper in oxidative muscle tissues as a whole(1,2,3). Nor does there appear to be strong evidence against its use in quad muscles in the C57 strain(4). Thus, while I agree that it would be interesting to find out the authorsu2019 take on this, the discussion seems to be a u2018moot pointu2019 if the CT values indeed were the same between experimental conditions. As there appears to be no real reason to think they were different, I really donu2019t share the previous postersu2019 skepticism here. In the end, the qPCR data have no or little bearing on the main findings or take-home of the paper as well. Also, if you truly are interested in a response from the authors, I recommend that you post these questions on the PLOS website directly. The authors will then get an email notice and have the option to reply or not. The only crux is that the poster cannot be anonymous according to PLOS policy. 1)tBrink et al. 2009 (heart in C57): https: www.ncbi.nlm.nih.gov pmc articles PMC2730443 2)tYeh et al. 2019 (heart in C57): https: www.ncbi.nlm.nih.gov pmc articles PMC6799937 3)tLowe et al. 2000 (soleus in Fischer 344): https: www.ncbi.nlm.nih.gov pubmed 10795720 4)tMusaro et al. 1995 (quad in C57): https: www.ncbi.nlm.nih.gov pubmed 7589251 5)tMcBride at al. 2017 (TA in C57): https: www.ncbi.nlm.nih.gov pmc articles PMC5582883 , 
 Greetings, We appreciate the questions and answers provided by the previous posters. We want to echo that the appropriateness of the reference gene is dependent on the design of the study (mouse strain, tissue, muscle type). There were no differences in GAPDH expression between the groups in our study AND the Livak paper mentions 2^(-u0394CT) as an option for data presentation. It makes no difference for the relative group comparisons or the subsequent statistics. Cheers, Mats Nilsson, McMaster University ,