Life-long spontaneous exercise does not prolong lifespan but improves health span in mice
Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Av. Blasco Ibañez, 15, Valencia 46010, Spain
Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF), Instituto de Salud Carlos III, Servicio de Geriatría, Hospital Universitario de Getafe, Ministerio de Sanidad y Consumo, Madrid, Spain
Virgen del Valle Geriatric Hospital, Toledo, Spain
UCIM, University of Valencia, Valencia, Spain
Abstract
Background
Life expectancy at birth in the first world has increased from 35 years at the beginning of the 20th century to more than 80 years now. The increase in life expectancy has resulted in an increase in age-related diseases and larger numbers of frail and dependent people. The aim of our study was to determine whether life-long spontaneous aerobic exercise affects lifespan and healthspan in mice.
Results
Male C57Bl/6J mice, individually caged, were randomly assigned to one of two groups: sedentary (n = 72) or spontaneous wheel-runners (n = 72). We evaluated longevity and several health parameters including grip strength, motor coordination, exercise capacity (VO2max) and skeletal muscle mitochondrial biogenesis. We also measured the cortical levels of the brain-derived neurotrophic factor (BDNF), a neurotrophin associated with brain plasticity. In addition, we measured systemic oxidative stress (malondialdehyde and protein carbonyl plasma levels) and the expression and activity of two genes involved in antioxidant defense in the liver (that is, glutathione peroxidase (GPx) and manganese superoxide dismutase (Mn-SOD)). Genes that encode antioxidant enzymes are considered longevity genes because their over-expression may modulate lifespan. Aging was associated with an increase in oxidative stress biomarkers and in the activity of the antioxidant enzymes, GPx and Mn-SOD, in the liver in mice. Life-long spontaneous exercise did not prolong longevity but prevented several signs of frailty (that is, decrease in strength, endurance and motor coordination). This improvement was accompanied by a significant increase in the mitochondrial biogenesis in skeletal muscle and in the cortical BDNF levels.
Conclusion
Life-long spontaneous exercise does not prolong lifespan but improves healthspan in mice. Exercise is an intervention that delays age-associated frailty, enhances function and can be translated into the clinic.
Background
World life expectancy has more than doubled over the past two centuries
Limits to healthspan include disability, frailty, chronic diseases and, of course, lifespan
Thus, the primary aim of our study was to develop an intervention (that is, spontaneous exercise) that could increase survival but that could also enhance function, delay frailty and be easily translated into the clinic. Due to the current lack of a test for frailty in rodents we performed four different physiological measurements: grip strength, motor coordination, exercise capacity and skeletal muscle mitochondrial biogenesis, which have been linked to clinically relevant age-related frailty. We also evaluated brain-derived neurotrophic factor (BDNF) as an indicator of brain plasticity, in addition to oxidative stress markers (malondialdehyde and carbonylated protein plasma levels) and the expression and activity of two genes involved in the antioxidant defense (that is, glutathione peroxidase (GPx) and manganese superoxide dismutase (Mn-SOD).
Results
Longevity curve and running wheel activity
Figure
Figure 1
Survival curves of cohorts of sedentary (n = 72) and spontaneous wheel-running mice (n = 72)
Survival curves of cohorts of sedentary (n = 72) and spontaneous wheel-running mice (n = 72). The Kaplan-Meier representation of the two groups is shown. The median lifespan of the sedentary group was 750 days and it was 770 days in the wheel-runners. Maximal lifespan in both groups was 950 days. The functional tests were performed at different survival time points (3, 17, 20, 23, 26, and 29 months old) as shown by arrows in the longevity curve.
Grip strength
Loss of grip strength is strongly associated with increasing chronological age
Figure 2
Functional tests performed at different survival time points (3, 17, 20, 23, 26 and 29 months old) in the longevity curve
Functional tests performed at different survival time points (3, 17, 20, 23, 26 and 29 months old) in the longevity curve. A) shows the mouse grip strength values in grams. All the available animals were tested at the different ages. B) shows motor coordination. It was determined as the percentage of animals that successfully passed the tightrope test. The fraction of mice passing the test is indicated above the bars. The number of animals tested (n = 12 to 24) varied at the different ages. C) shows the maximal running speed achieved in a VO2max test and that was considered the maximal aerobic workload capacity of the animal (n = 20). Values are shown as mean ± SD. (*) indicates
Motor coordination
Neuromuscular coordination declines with aging
VO2max test
Large-scale epidemiological studies of subjects with and without cardiovascular disease demonstrate that low aerobic exercise capacity (VO2max) is a stronger predictor of mortality than other established risk factors, such as diabetes, smoking, body mass index (BMI) >30, hypertension and chronic obstructive pulmonary disease (COPD)
Mitochondrial biogenesis in skeletal muscle
Aging causes a decrease in mitochondrial content and activity
Figure 3
Exercise-induced activation of the mitochondrial biogenesis pathway in mice skeletal muscle during aging
Exercise-induced activation of the mitochondrial biogenesis pathway in mice skeletal muscle during aging. Western blotting analysis to detect (A) Peroxisome Proliferator-Activated Receptor-γ Coactivator 1α (PGC-1α) and (B) cytochrome C at different survival time points. Representative blots are shown. For the densitometric analysis of the results, values are shown as mean (± SD). The content of α-actin, a housekeeping protein marker in skeletal muscle, was determined in all the experimental groups. (*) indicates
Mitochondrial content was estimated measuring cytochrome C protein levels in skeletal muscle
Oxidative stress and antioxidant enzymes
We did not find any change in plasma protein oxidation (Figure
Figure 4
Plasma oxidative stress biomarkers at different survival time points (3, 20, 26 and 29 months old) in the longevity curve
Plasma oxidative stress biomarkers at different survival time points (3, 20, 26 and 29 months old) in the longevity curve. A) shows a representative Western blot and the densitometric quantification of protein carbonyls in plasma. Values were normalized to those observed in the samples obtained from the three-month-old group, which was assigned a value of 100%. B) shows plasma lipid peroxidation determined as malondialdehyde {MDA). Values are shown as mean ± SD. (*) indicates
We also determined the expression and the activity of the antioxidant enzymes MnSOD and GPx. We found no major changes in the mRNA levels of the enzymes (See Figure
Figure 5
Expression and activity of GPx and MnSOD in the liver at different survival time points (3, 20, 26 and 29 months old) in the longevity curve
Expression and activity of GPx and MnSOD in the liver at different survival time points (3, 20, 26 and 29 months old) in the longevity curve. A) and C) show the expression of GPx and MnSOD studied by real time RT-PCR. Values were normalized to those observed in the samples obtained from the three-month-old group, which was assigned a value of 100%. B) and D) show GPx and MnSOD activity. Values are shown as mean ± SD. (*) indicates
Cortical BDNF
Protein levels of BDNF were quantified in the cortex of the animals by ELISA. We found a significant increase in the neurotrophin in the active animals. Thus, spontaneous wheel-running significantly prevented the age-associated fall in BDNF in mice 20- and 26-months old.
Discussion
Most of the interventions devised to understand the mechanisms of aging have been focused on survival
We have recently proposed that ‘exercise acts as a drug’
We chose the C57BL/6J mouse as the strain for our model for three main reasons. First, this strain has been shown to perform well in spontaneous exercise studies
To support our results we determined oxidative stress parameters and the activity of the antioxidant enzymes in our study. The free radical theory of aging is one of the most prominent theories to explain aging. This theory, although recently questioned
The negative results in terms of lifespan led us to focus our study on healthspan. Ignatz Nascher, who coined the term ‘geriatrics’ and who founded this clinical field in the US, described the concept of healthspan (without using the term) as a goal of being productive and happy for an individual’s entire lifespan, rather than seeking longevity despite severely hindering impairments of body and mind
Frailty is a geriatric syndrome with a tremendous impact on the older individual, their family, and society as a whole. The components of frailty are a mixture of physiological, psychological, social and environmental factors (for example, sarcopenia, functional impairment, cognitive impairment, and depression). Physical exercise may affect all these factors. Thus, we aimed to determine whether life-long spontaneous exercise was a good strategy to prevent frailty in a mouse model. Our major problem was the lack of a test for frailty in experimental animals. Although the clinical interest in frailty has grown in recent years
The beneficial effects of exercise on cognitive
Figure 6
Cortex BDNF levels, determined by ELISA, in sedentary and wheel-running mice at different survival time points (3, 20, 26 and 29 months old) in the longevity curve
Cortex BDNF levels, determined by ELISA, in sedentary and wheel-running mice at different survival time points (3, 20, 26 and 29 months old) in the longevity curve. Values are shown as mean ± SD. (*) indicates
A functional muscle that has not lost the capacity to synthesize healthy mitochondria is an important contributor in the prevention of frailty
Conclusion
Lifelong spontaneous exercise does not prolong lifespan but improves healthspan in mice. Exercise is an intervention that enhances function and delays frailty in experimental animals. These results stress the importance of this intervention to prevent human frailty and dependency.
Methods
Experimental animals
Adult male C57BL/6J mice, three-months old, were randomly assigned to one of two groups: sedentary control (n = 72) or spontaneous exercise (n = 72). The animals were housed in individual cages. The mice in the exercise group had 24-hour access to a 11.5 cm diameter running wheel connected to an electronic wheel-revolution counter located at the top of the cage. The sedentary mice were free to move around their cage but did not have access to a running wheel. We chose to wait until three months of age to allow our animals access to exercise wheels, as this is the age at which mice reach musculoskeletal maturity, and we estimated that our animals would achieve maximal performance if they were exposed to running wheels at this age
The average temperature in the animal house was 23 ± 1°C, relative humidity was 60%, and 12 hour day/night cycles were maintained. The mice were checked daily. Water and food were available
The values obtained in the longevity curve were registered for as long as the experiment lasted, sacrificing four mice from each group at specific survival points: 3, 20, 26 and 29 months of age, to obtain data for subsequent analysis. Liver, skeletal muscle and brain were immediately dissected and stored at −80°C for further analysis.
The experimental protocol was approved by the Committee of Ethics in Research of the Faculty of Medicine, University of Valencia.
Wheel running activity
The activity of the mice on the running wheels was monitored by a magnetic switch affixed to each wheel, which recorded the number of revolutions completed. Physical activity was recorded continuously and added up each week for analysis. Free open-field locomotor activity of mice within cages was not measured.
Motor coordination test (tight-rope test)
The tight-rope test was based on the method previously described by Miquel
Incremental treadmill test (VO2max test)
The animals were submitted to a graded intensity treadmill test (Model 1050 LS Exer3/6; Columbus Instruments, Columbus, OH, USA) to determine their endurance and ‘slowness’ along the longevity curve. We followed a modification of the protocol of Davidson and co-workers
Grip strength test
A grip strength meter (Panlab, Harvard Apparatus. Barcelone. Spain) was employed in assessing neuromuscular function by sensing the peak amount of force that the mice applied in grasping specially designed pull bar assemblies. Metering was performed with precision force gauges in such a manner as to retain the peak force applied on a digital display. Mice were randomly chosen to grasp the pull-bar with their forelimb for a few seconds. The animals were then drawn along a straight line leading away from the sensor. The animals released at some point and the maximum force attained was stored on the display. Peak force was automatically registered in grams-force by the apparatus. Data were recorded, and four additional trials were immediately given
SDS-PAGE and western blotting
Aliquots of muscle lysate were separated by SDS-PAGE. Proteins were then transferred to nitrocellulose membranes, which were incubated overnight at 4°C with appropriate primary antibodies: anti-PGC-1α (1:1000, Cayman. Ann Arbor. Michigan. USA), anti-cytochrome C (1:1000, Santa Cruz Biotechnology Inc. Dallas. Texas. USA), and anti-α-actin (1:700, Sigma Aldrich. St. Louis. Missouri. USA). Thereafter, membranes were incubated with a secondary antibody for one hour at room temperature. Specific proteins were visualized by using the enhanced chemiluminescence procedure, as specified by the manufacturer (Amersham Biosciences, Piscataway, NJ, USA). Autoradiographic signals were assessed by using a scanning densitometer (BioRad, Hercules, CA, USA). The densitometry analysis was carried out immediately before saturation of the immunosignal. Data were represented as arbitrary units of immunostaining. To check for differences in loading and transfer efficiency across membranes, an antibody directed against α-actin was used to hybridize with all the membranes previously incubated with the respective antibodies.
Determination of plasma MDA and protein carbonyls
MDA was determined in plasma by an HPLC method as described in
RNA isolation, reverse transcription and PCR
Total RNA was extracted from liver tissue with Trizol™ (Invitrogen. Madrid. Spain) according to the manufacturer’s protocol. The purity of the samples was assessed by determining the 260 nm/280nm ratio, which was always above 1.9, and total RNA was quantified from the absorbance at 260 nm. We synthesized cDNA from 1 μg of RNA using random hexamer primers and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Madrid, Spain). Reverse transcription conditions comprised an initial incubation step at 25°C for 10 minutes to allow random hexamers to anneal, followed by cDNA synthesis at 37°C for 120 minutes and the final inactivation step for 5 minutes at 95°C. Real-time PCR was performed with an ABI 7900 sequence-detection system (Applied Biosystems). Primers for amplifying specific fragments of the genes were obtained from Thermo Fisher Scientific GmbH (Ulm, Germany). Real-time PCR was performed in duplicate in a total reaction volume of 20 μL using Maxima™ SYBR green/ROX qPCR Master Mix (Fermentas, Madrid, Spain). The thermal cycling protocol was as follows: initial denaturation for 10 minutes at 95°C followed by 40 cycles of 10 seconds at 95°C, 10 seconds at 62°C, and 10 seconds at 72°C. The fluorescence signal was measured at the end of each extension step at 72°C. At the end of each reaction, a melting curve analysis was performed to confirm that only the specific products were amplified. The threshold cycle (Ct) was converted to a relative gene expression by the use of a standard curve. For each sample, the expression of the target gene mRNA was normalized with the GAPDH mRNA content. The specific primers used for GPx were: 5’-GAC ATC AGG AGA ATG GCA AG-3’ (forward) and 5’- CAT CAC CAA GCC AAT ACC AC-3’ (reverse); for MnSOD they were: 5’-CGT GCT CCC ACA CAT CAA TG-3’ (forward) and 5’-TGA ACG TCA CCG AGG AGA AG-3’ (reverse); and for the housekeeping gene GAPDH they were: 5’- CCT GGA GAA ACC TGC CAA GTA TG-3’ (forward) and 5’-GGT CCT CAG TGT AGC CCA AGA TG-3’ (reverse).
Enzyme activities
GPx activity was measured as described by Flohe
ELISA analysis
Protein levels of BDNF were quantified in the cortex by ELISA (CYT306 Millipore, Bedford, MA, USA), following the manufacturer’s instructions. The samples were measured at 450 nm using a plate reader (iEMS Reader MF; Labsystems, Vantaa, Finland).
Data analysis
Mean values and standard deviation were considered for descriptive statistics. To estimate lifespan differences between groups, a Kaplan-Meier curve was performed. Differences in maximal running time and speed, grip strength test and motor coordination were tested using Fisher’s exact test for each age group and parameter. To determine the effect of spontaneous exercise on BDNF, PGC-1α and cytochome C protein expression in skeletal muscle we performed a two-tailed Student’s t-test for unpaired samples. Differences were considered significant at
Abbreviations
BDNF: Brain-derived neurotrophic factor; ELISA: Enzyme-linked immunosorbent assay; GPx: Glutathione peroxidase; HPLC: High performance liquid chromatography; MDA: Malondialdehyde; Mn-SOD: Manganese superoxide dismutase; PCR: Polymerase chain reaction; VO2max: exercise capacity.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RG-V and GO-G performed the experimental work; MCG-C, FJG-G and LR-M analyzed data and assisted in editing and writing the paper; DA, and NI supervised all the animal work and designed research; and J V wrote the paper and directed the project. All authors read and approved the final manuscript.
Acknowledgements
We thank Mrs Marilyn Noyes for her kind help in reviewing the manuscript. This work was supported by grants SAF2010-19498,from the Spanish Ministry of Education and Science (MEC); ISCIII2006-RED13-027 from the Red Temática de investigación cooperativa en envejecimiento y fragilidad (RETICEF); P2011/02 RM Cátedra Real Madrid-UEM; PROMETEO2010/074 from ‘Conselleria de Sanitat de la Generalitat Valenciana’; 35NEURO GentxGent from ‘Fundació Gent Per Gent de la Comunitat Valenciana’; PI11/01068 del ISCIII, and EU Funded COSTB35 and CM1001. This study has been co-financed by FEDER funds from the European Union.
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Lipoperoxides in plasma as measured by liquid-chromatographic separation of malondialdehyde-thiobarbituric acid adduct
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Life-long spontaneous exercise does not prolong lifespan but improves health span in mice
Department of Physiology, Faculty of Medicine, University of Valencia, Fundacion Investigacion Hospital Clinico Universitario/INCLIVA, Av. Blasco Ibañez, 15, Valencia 46010, Spain
Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF), Instituto de Salud Carlos III, Servicio de Geriatría, Hospital Universitario de Getafe, Ministerio de Sanidad y Consumo, Madrid, Spain
Virgen del Valle Geriatric Hospital, Toledo, Spain
UCIM, University of Valencia, Valencia, Spain
Abstract
Background
Life expectancy at birth in the first world has increased from 35 years at the beginning of the 20th century to more than 80 years now. The increase in life expectancy has resulted in an increase in age-related diseases and larger numbers of frail and dependent people. The aim of our study was to determine whether life-long spontaneous aerobic exercise affects lifespan and healthspan in mice.
Results
Male C57Bl/6J mice, individually caged, were randomly assigned to one of two groups: sedentary (n = 72) or spontaneous wheel-runners (n = 72). We evaluated longevity and several health parameters including grip strength, motor coordination, exercise capacity (VO2max) and skeletal muscle mitochondrial biogenesis. We also measured the cortical levels of the brain-derived neurotrophic factor (BDNF), a neurotrophin associated with brain plasticity. In addition, we measured systemic oxidative stress (malondialdehyde and protein carbonyl plasma levels) and the expression and activity of two genes involved in antioxidant defense in the liver (that is, glutathione peroxidase (GPx) and manganese superoxide dismutase (Mn-SOD)). Genes that encode antioxidant enzymes are considered longevity genes because their over-expression may modulate lifespan. Aging was associated with an increase in oxidative stress biomarkers and in the activity of the antioxidant enzymes, GPx and Mn-SOD, in the liver in mice. Life-long spontaneous exercise did not prolong longevity but prevented several signs of frailty (that is, decrease in strength, endurance and motor coordination). This improvement was accompanied by a significant increase in the mitochondrial biogenesis in skeletal muscle and in the cortical BDNF levels.
Conclusion
Life-long spontaneous exercise does not prolong lifespan but improves healthspan in mice. Exercise is an intervention that delays age-associated frailty, enhances function and can be translated into the clinic.
Background
World life expectancy has more than doubled over the past two centuries
Limits to healthspan include disability, frailty, chronic diseases and, of course, lifespan
Thus, the primary aim of our study was to develop an intervention (that is, spontaneous exercise) that could increase survival but that could also enhance function, delay frailty and be easily translated into the clinic. Due to the current lack of a test for frailty in rodents we performed four different physiological measurements: grip strength, motor coordination, exercise capacity and skeletal muscle mitochondrial biogenesis, which have been linked to clinically relevant age-related frailty. We also evaluated brain-derived neurotrophic factor (BDNF) as an indicator of brain plasticity, in addition to oxidative stress markers (malondialdehyde and carbonylated protein plasma levels) and the expression and activity of two genes involved in the antioxidant defense (that is, glutathione peroxidase (GPx) and manganese superoxide dismutase (Mn-SOD).
Results
Longevity curve and running wheel activity
Figure
Figure 1
Survival curves of cohorts of sedentary (n = 72) and spontaneous wheel-running mice (n = 72)
Survival curves of cohorts of sedentary (n = 72) and spontaneous wheel-running mice (n = 72). The Kaplan-Meier representation of the two groups is shown. The median lifespan of the sedentary group was 750 days and it was 770 days in the wheel-runners. Maximal lifespan in both groups was 950 days. The functional tests were performed at different survival time points (3, 17, 20, 23, 26, and 29 months old) as shown by arrows in the longevity curve.
Grip strength
Loss of grip strength is strongly associated with increasing chronological age
Figure 2
Functional tests performed at different survival time points (3, 17, 20, 23, 26 and 29 months old) in the longevity curve
Functional tests performed at different survival time points (3, 17, 20, 23, 26 and 29 months old) in the longevity curve. A) shows the mouse grip strength values in grams. All the available animals were tested at the different ages. B) shows motor coordination. It was determined as the percentage of animals that successfully passed the tightrope test. The fraction of mice passing the test is indicated above the bars. The number of animals tested (n = 12 to 24) varied at the different ages. C) shows the maximal running speed achieved in a VO2max test and that was considered the maximal aerobic workload capacity of the animal (n = 20). Values are shown as mean ± SD. (*) indicates
Motor coordination
Neuromuscular coordination declines with aging
VO2max test
Large-scale epidemiological studies of subjects with and without cardiovascular disease demonstrate that low aerobic exercise capacity (VO2max) is a stronger predictor of mortality than other established risk factors, such as diabetes, smoking, body mass index (BMI) >30, hypertension and chronic obstructive pulmonary disease (COPD)
Mitochondrial biogenesis in skeletal muscle
Aging causes a decrease in mitochondrial content and activity
Figure 3
Exercise-induced activation of the mitochondrial biogenesis pathway in mice skeletal muscle during aging
Exercise-induced activation of the mitochondrial biogenesis pathway in mice skeletal muscle during aging. Western blotting analysis to detect (A) Peroxisome Proliferator-Activated Receptor-γ Coactivator 1α (PGC-1α) and (B) cytochrome C at different survival time points. Representative blots are shown. For the densitometric analysis of the results, values are shown as mean (± SD). The content of α-actin, a housekeeping protein marker in skeletal muscle, was determined in all the experimental groups. (*) indicates
Mitochondrial content was estimated measuring cytochrome C protein levels in skeletal muscle
Oxidative stress and antioxidant enzymes
We did not find any change in plasma protein oxidation (Figure
Figure 4
Plasma oxidative stress biomarkers at different survival time points (3, 20, 26 and 29 months old) in the longevity curve
Plasma oxidative stress biomarkers at different survival time points (3, 20, 26 and 29 months old) in the longevity curve. A) shows a representative Western blot and the densitometric quantification of protein carbonyls in plasma. Values were normalized to those observed in the samples obtained from the three-month-old group, which was assigned a value of 100%. B) shows plasma lipid peroxidation determined as malondialdehyde {MDA). Values are shown as mean ± SD. (*) indicates
We also determined the expression and the activity of the antioxidant enzymes MnSOD and GPx. We found no major changes in the mRNA levels of the enzymes (See Figure
Figure 5
Expression and activity of GPx and MnSOD in the liver at different survival time points (3, 20, 26 and 29 months old) in the longevity curve
Expression and activity of GPx and MnSOD in the liver at different survival time points (3, 20, 26 and 29 months old) in the longevity curve. A) and C) show the expression of GPx and MnSOD studied by real time RT-PCR. Values were normalized to those observed in the samples obtained from the three-month-old group, which was assigned a value of 100%. B) and D) show GPx and MnSOD activity. Values are shown as mean ± SD. (*) indicates
Cortical BDNF
Protein levels of BDNF were quantified in the cortex of the animals by ELISA. We found a significant increase in the neurotrophin in the active animals. Thus, spontaneous wheel-running significantly prevented the age-associated fall in BDNF in mice 20- and 26-months old.
Discussion
Most of the interventions devised to understand the mechanisms of aging have been focused on survival
We have recently proposed that ‘exercise acts as a drug’
We chose the C57BL/6J mouse as the strain for our model for three main reasons. First, this strain has been shown to perform well in spontaneous exercise studies
To support our results we determined oxidative stress parameters and the activity of the antioxidant enzymes in our study. The free radical theory of aging is one of the most prominent theories to explain aging. This theory, although recently questioned
The negative results in terms of lifespan led us to focus our study on healthspan. Ignatz Nascher, who coined the term ‘geriatrics’ and who founded this clinical field in the US, described the concept of healthspan (without using the term) as a goal of being productive and happy for an individual’s entire lifespan, rather than seeking longevity despite severely hindering impairments of body and mind
Frailty is a geriatric syndrome with a tremendous impact on the older individual, their family, and society as a whole. The components of frailty are a mixture of physiological, psychological, social and environmental factors (for example, sarcopenia, functional impairment, cognitive impairment, and depression). Physical exercise may affect all these factors. Thus, we aimed to determine whether life-long spontaneous exercise was a good strategy to prevent frailty in a mouse model. Our major problem was the lack of a test for frailty in experimental animals. Although the clinical interest in frailty has grown in recent years
The beneficial effects of exercise on cognitive
Figure 6
Cortex BDNF levels, determined by ELISA, in sedentary and wheel-running mice at different survival time points (3, 20, 26 and 29 months old) in the longevity curve
Cortex BDNF levels, determined by ELISA, in sedentary and wheel-running mice at different survival time points (3, 20, 26 and 29 months old) in the longevity curve. Values are shown as mean ± SD. (*) indicates
A functional muscle that has not lost the capacity to synthesize healthy mitochondria is an important contributor in the prevention of frailty
Conclusion
Lifelong spontaneous exercise does not prolong lifespan but improves healthspan in mice. Exercise is an intervention that enhances function and delays frailty in experimental animals. These results stress the importance of this intervention to prevent human frailty and dependency.
Methods
Experimental animals
Adult male C57BL/6J mice, three-months old, were randomly assigned to one of two groups: sedentary control (n = 72) or spontaneous exercise (n = 72). The animals were housed in individual cages. The mice in the exercise group had 24-hour access to a 11.5 cm diameter running wheel connected to an electronic wheel-revolution counter located at the top of the cage. The sedentary mice were free to move around their cage but did not have access to a running wheel. We chose to wait until three months of age to allow our animals access to exercise wheels, as this is the age at which mice reach musculoskeletal maturity, and we estimated that our animals would achieve maximal performance if they were exposed to running wheels at this age
The average temperature in the animal house was 23 ± 1°C, relative humidity was 60%, and 12 hour day/night cycles were maintained. The mice were checked daily. Water and food were available
The values obtained in the longevity curve were registered for as long as the experiment lasted, sacrificing four mice from each group at specific survival points: 3, 20, 26 and 29 months of age, to obtain data for subsequent analysis. Liver, skeletal muscle and brain were immediately dissected and stored at −80°C for further analysis.
The experimental protocol was approved by the Committee of Ethics in Research of the Faculty of Medicine, University of Valencia.
Wheel running activity
The activity of the mice on the running wheels was monitored by a magnetic switch affixed to each wheel, which recorded the number of revolutions completed. Physical activity was recorded continuously and added up each week for analysis. Free open-field locomotor activity of mice within cages was not measured.
Motor coordination test (tight-rope test)
The tight-rope test was based on the method previously described by Miquel
Incremental treadmill test (VO2max test)
The animals were submitted to a graded intensity treadmill test (Model 1050 LS Exer3/6; Columbus Instruments, Columbus, OH, USA) to determine their endurance and ‘slowness’ along the longevity curve. We followed a modification of the protocol of Davidson and co-workers
Grip strength test
A grip strength meter (Panlab, Harvard Apparatus. Barcelone. Spain) was employed in assessing neuromuscular function by sensing the peak amount of force that the mice applied in grasping specially designed pull bar assemblies. Metering was performed with precision force gauges in such a manner as to retain the peak force applied on a digital display. Mice were randomly chosen to grasp the pull-bar with their forelimb for a few seconds. The animals were then drawn along a straight line leading away from the sensor. The animals released at some point and the maximum force attained was stored on the display. Peak force was automatically registered in grams-force by the apparatus. Data were recorded, and four additional trials were immediately given
SDS-PAGE and western blotting
Aliquots of muscle lysate were separated by SDS-PAGE. Proteins were then transferred to nitrocellulose membranes, which were incubated overnight at 4°C with appropriate primary antibodies: anti-PGC-1α (1:1000, Cayman. Ann Arbor. Michigan. USA), anti-cytochrome C (1:1000, Santa Cruz Biotechnology Inc. Dallas. Texas. USA), and anti-α-actin (1:700, Sigma Aldrich. St. Louis. Missouri. USA). Thereafter, membranes were incubated with a secondary antibody for one hour at room temperature. Specific proteins were visualized by using the enhanced chemiluminescence procedure, as specified by the manufacturer (Amersham Biosciences, Piscataway, NJ, USA). Autoradiographic signals were assessed by using a scanning densitometer (BioRad, Hercules, CA, USA). The densitometry analysis was carried out immediately before saturation of the immunosignal. Data were represented as arbitrary units of immunostaining. To check for differences in loading and transfer efficiency across membranes, an antibody directed against α-actin was used to hybridize with all the membranes previously incubated with the respective antibodies.
Determination of plasma MDA and protein carbonyls
MDA was determined in plasma by an HPLC method as described in
RNA isolation, reverse transcription and PCR
Total RNA was extracted from liver tissue with Trizol™ (Invitrogen. Madrid. Spain) according to the manufacturer’s protocol. The purity of the samples was assessed by determining the 260 nm/280nm ratio, which was always above 1.9, and total RNA was quantified from the absorbance at 260 nm. We synthesized cDNA from 1 μg of RNA using random hexamer primers and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Madrid, Spain). Reverse transcription conditions comprised an initial incubation step at 25°C for 10 minutes to allow random hexamers to anneal, followed by cDNA synthesis at 37°C for 120 minutes and the final inactivation step for 5 minutes at 95°C. Real-time PCR was performed with an ABI 7900 sequence-detection system (Applied Biosystems). Primers for amplifying specific fragments of the genes were obtained from Thermo Fisher Scientific GmbH (Ulm, Germany). Real-time PCR was performed in duplicate in a total reaction volume of 20 μL using Maxima™ SYBR green/ROX qPCR Master Mix (Fermentas, Madrid, Spain). The thermal cycling protocol was as follows: initial denaturation for 10 minutes at 95°C followed by 40 cycles of 10 seconds at 95°C, 10 seconds at 62°C, and 10 seconds at 72°C. The fluorescence signal was measured at the end of each extension step at 72°C. At the end of each reaction, a melting curve analysis was performed to confirm that only the specific products were amplified. The threshold cycle (Ct) was converted to a relative gene expression by the use of a standard curve. For each sample, the expression of the target gene mRNA was normalized with the GAPDH mRNA content. The specific primers used for GPx were: 5’-GAC ATC AGG AGA ATG GCA AG-3’ (forward) and 5’- CAT CAC CAA GCC AAT ACC AC-3’ (reverse); for MnSOD they were: 5’-CGT GCT CCC ACA CAT CAA TG-3’ (forward) and 5’-TGA ACG TCA CCG AGG AGA AG-3’ (reverse); and for the housekeeping gene GAPDH they were: 5’- CCT GGA GAA ACC TGC CAA GTA TG-3’ (forward) and 5’-GGT CCT CAG TGT AGC CCA AGA TG-3’ (reverse).
Enzyme activities
GPx activity was measured as described by Flohe
ELISA analysis
Protein levels of BDNF were quantified in the cortex by ELISA (CYT306 Millipore, Bedford, MA, USA), following the manufacturer’s instructions. The samples were measured at 450 nm using a plate reader (iEMS Reader MF; Labsystems, Vantaa, Finland).
Data analysis
Mean values and standard deviation were considered for descriptive statistics. To estimate lifespan differences between groups, a Kaplan-Meier curve was performed. Differences in maximal running time and speed, grip strength test and motor coordination were tested using Fisher’s exact test for each age group and parameter. To determine the effect of spontaneous exercise on BDNF, PGC-1α and cytochome C protein expression in skeletal muscle we performed a two-tailed Student’s t-test for unpaired samples. Differences were considered significant at
Abbreviations
BDNF: Brain-derived neurotrophic factor; ELISA: Enzyme-linked immunosorbent assay; GPx: Glutathione peroxidase; HPLC: High performance liquid chromatography; MDA: Malondialdehyde; Mn-SOD: Manganese superoxide dismutase; PCR: Polymerase chain reaction; VO2max: exercise capacity.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RG-V and GO-G performed the experimental work; MCG-C, FJG-G and LR-M analyzed data and assisted in editing and writing the paper; DA, and NI supervised all the animal work and designed research; and J V wrote the paper and directed the project. All authors read and approved the final manuscript.
Acknowledgements
We thank Mrs Marilyn Noyes for her kind help in reviewing the manuscript. This work was supported by grants SAF2010-19498,from the Spanish Ministry of Education and Science (MEC); ISCIII2006-RED13-027 from the Red Temática de investigación cooperativa en envejecimiento y fragilidad (RETICEF); P2011/02 RM Cátedra Real Madrid-UEM; PROMETEO2010/074 from ‘Conselleria de Sanitat de la Generalitat Valenciana’; 35NEURO GentxGent from ‘Fundació Gent Per Gent de la Comunitat Valenciana’; PI11/01068 del ISCIII, and EU Funded COSTB35 and CM1001. This study has been co-financed by FEDER funds from the European Union.
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