Effects of different modes of exercise on skeletal muscle mass and function and IGF-1 signaling during early aging in mice

Aging, a natural and irreversible process, significantly reduces health and quality of life (Tieland et al., 2018). The process of aging is often accompanied by many physiological degenerative changes, such as cardiovascular function decline, skeletal muscle atrophy and body fat storage (Tieland et al., 2018; da Costa et al., 2016). Skeletal muscle atrophy increases the incidence of chronic metabolic diseases in older individuals, which is a major public health issue (da Costa et al., 2016; Keng et al., 2019; Kim and Won, 2022). Promoting muscle hypertrophy in middle age and maintaining skeletal muscle mass and function could effectively reduce the risk of skeletal muscle atrophy in later old age (Kukuljan et al., 2009). There are many factors that affect skeletal muscle mass and exercise capacity, including levels of various hormones and metabolic enzymes (Brioche and Lemoine-Morel, 2016; Tallis et al., 2017; Barone et al., 2022; Martin et al., 2021). It has been reported that improving antioxidant capacity, inhibiting excessive apoptosis and enhancing the efficiency of protein synthesis in skeletal muscle could effectively promote skeletal muscle hypertrophy (Brioche and Lemoine-Morel, 2016; Barclay et al., 2019; Dethlefsen et al., 2018; Fernando et al., 2019; Jackson et al., 2022).

Insulin-like growth factor-1 (IGF-1) is an effective mediator in alleviating disease-induced organ dysfunction by promoting cell proliferation and differentiation (Ahmad et al., 2020). Moreover, IGF-1 is closely associated with the development of muscle mass and strength and the regulation of skeletal muscle metabolism and regeneration (Yoshida and Delafontaine (2020). Previous studies have demonstrated that exercise could upregulate IGF-1 expression in skeletal muscle under pathological conditions, improving muscle mass and function (Ribeiro et al., 2017; Sellami et al., 2019; Feng et al., 2022). A recent study has shown that healthy individuals can secrete sufficient IGF-1 and stimulate phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling to promote muscle hypertrophy (Yoshida and Delafontaine, 2020). In aging models, decreased IGF-1 expression in skeletal muscle was observed, and inactivation of the IGF-1/PI3K/Akt signaling pathway reduced protein synthesis (Barclay et al., 2019). Therefore, it is worth investigating whether the secretion of endogenous IGF-1 can be stimulated by exercise intervention, to promote skeletal muscle hypertrophy and improve exercise and metabolic capacities in early aging mice.

Exercise effectively promotes skeletal muscle hypertrophy in middle-aged and aged individuals, improves muscle mass and exercise capacity, and reduces the incidence of diabetes, obesity and sarcopenia in old age (Colleluori et al., 2019; Schumann et al., 2022; Yasuda, 2022). The efficiency and benefit of improving skeletal muscle mass through exercise in early old age is higher than that of relieving skeletal muscle atrophy in late old age (Anton et al., 2018; Tucker et al., 2018; Mende et al., 2022). Therefore, we need adequate exercise to maintain physical health and life quality in early old age. Both traditional aerobic exercise (AE) and resistance exercise (RE) were found to promote muscle hypertrophy and improve energy metabolism. Of these, RE was more effective than AE, although the mechanism of action of the two types of exercise differed (Egan and Zierath, 2013; Foure and Gondin, 2021): AE improved skeletal muscle mass mainly by increasing the number and volume of mitochondria, promoting angiogenesis, enhancing antioxidant capacity and inhibiting excessive cell apoptosis (Egan and Zierath, 2013), whereas RE improved skeletal muscle mass mainly by promoting protein synthesis, increasing non-contractile tissue and contractile tissue volume, and reducing protein degradation (Egan and Zierath, 2013). In previous work, we confirmed both AE and RE could increase the protein expression of IGF-1 and promote skeletal muscle hypertrophy in mice with myocardial infarction (Feng et al., 2022).

However, AE and RE might induce muscle and joint stiffness, acute inflammation and swelling, delayed-onset muscle soreness and even muscle damage (Kosar et al., 2012). In contrast, whole-body vibration (WBV) and electrical stimulation (ES) increased skeletal muscle mass, physical fitness and quality of life, and were less likely to cause muscle damage (Chang et al., 2018; Paillard, 2018; Balke et al., 2022). WBV stimulated muscle tension and relaxation in a short period of time, increased muscle spindle activity and muscle recruitment, and in turn promoted capillary angiogenesis, mitochondrial generation and protein synthesis, which were similar to the effects of combined AE and RE (Kosar et al., 2012; Kaneguchi et al., 2014). ES – passive stimulation with electrodes – activated fast muscle fibers, promoted the synthesis of type I and type III collagen in myofibrils, activated muscle satellite cells and further promoted myogenesis (Foure and Gondin, 2021).

Thus, evidence suggests that AE, RE, WBV and ES stimulate muscle hypertrophy; however, the following points have not been addressed in the early aging population: (1) as active forms of exercise, moderate and high-intensity AE and RE both promote muscle hypertrophy and improve the ability to exercise and metabolize energy, but which works better?; (2) does short-term low-intensity passive stimulation by WBV and ES achieve similar effects to AE and RE in promoting muscle hypertrophy?; and (3) are the effects of the above four types of exercise on muscle hypertrophy all related to the activation of IGF-1/IGF-1R–PI3K/Akt signaling? The aim of this study was to confirm the effects of various exercise forms on skeletal muscle hypertrophy, exercise capacity, indicators related to metabolism and activation of IGF-1 signaling in mice undergoing early aging. Importantly, in recombinant human IGF-1 (rhIGF-1)-treated aging myotubes, we further investigated whether the beneficial effects of exercise on promoting skeletal muscle hypertrophy occur through activation of the IGF-1/IGF-1R–PI3K/Akt pathway. These results would provide a new perspective on the effects of different exercises on promoting muscle hypertrophy, exercise capacity and energy metabolism, in early aging individuals. We also assessed whether WBV and ES, as types of efficient passive stimulation, can exert similar effects to traditional exercise.

Male C57/BL6 mice were purchased from the laboratory animal center of the Xi'an Jiaotong University [Xi'an, China; no. SCXK (Shan) 2017-003]. All mice were housed in a constant-temperature (20–22°C) and humidity (50–60%) animal room with a 12 h light/dark cycle and free access to standard rodent chow and water. All surgical procedures were performed according the Guide for the Care and Use of Laboratory Animals (Washington DC, The National Academies Press, 2011), and were approved by the ethical committee of Shaanxi Normal University.

Mice were grouped (n=8 per group) by a stratified randomization method based on the body mass of each 12 month old mouse (Sayed et al., 2016): control group, aerobic exercise group (AE), resistance exercise group (RE), whole-body vibration group (WBV) and electrical stimulation group (ES). After grouping, mouse body mass, maximum carrying capacity and endurance exercise capacity were measured. There was no significant difference in the above indicators among the groups. The reason for choosing 12 month old mice is that the mass and function of skeletal muscle in early aging (12 months) mice are good, but the skeletal muscle begins to show significant changes in gastrocnemius muscle morphology and ultrastructure, suggesting that skeletal muscle is beginning to be lost. Exercise at this time can effectively prevent the later occurrence of skeletal muscle loss (Sayed et al., 2016).

To allow mice to adapt to the exercise stress, 3 days of low-intensity adaptation training before the start of formal training was carried out. The training program for each group was as follows. After 4 weeks of intervention, each group of mice was assessed for endurance exercise capacity (total running distance) by an incremental running test and for maximal carrying capacity by a ladder-climbing test (Wang et al., 2019; Horii et al., 2018). After the training and testing period, mice were anesthetized with isoflurane. Blood and gastrocnemius muscle were rapidly removed, and stored in formaldehyde or liquid nitrogen for subsequent experiments.

Adaptive treadmill (DSPT-202, Li Tai Technology, Hangzhou, China) training was performed for 3 days at a speed of 5 m min⁻¹ at a 5 deg incline for 30 min per day. After 3 days of adaptive training, regular exercise training was performed at 12 m min⁻¹ and a 5 deg incline for 60 min per day at 16:00–18:00 h, equal to 76% ⁠, for 5 days per week for 4 weeks (Sonobe et al., 2015; Schefer and Talan, 1996) (Fig. 1A).

RE was carried out through ladder-climbing training. Adaptive ladder-climbing training was performed for 3 days. The ladder (custom-made) was set at a height of 1 m with 1 cm steps and an 85 deg incline. The maximal carrying capacity of mice was measured before and after the whole RE process for each mouse. Each mouse climbed the ladder carrying a load equivalent to 75% of its body weight. Then, the load was increased by 15% of body weight for each climb until the mouse failed to climb the entire ladder. The maximum carrying capacity was considered as the heaviest load a mouse successfully carried to climb the entire length of the ladder (Gomes et al., 2020). On the first day, mice trained without weight bearing. Then, the mice were offered a load comparable to 25% of their maximal carrying capacity on the second day, and 50% of their maximal carrying capacity on the third day. The adaptive training comprised 6 sets per day, three times per set, with 1 min rest between each set. After 3 days of adaptive training, loads of 75% of maximal carrying capacity were affixed to the mouse's tail for ladder-climbing training. The formal RE training comprised 9 sets, with three times per set, for approximately 60 min per day at 16:00–18:00 h, 5 days per week for 4 weeks, with 1–2 min rest between each set (Horii et al., 2018) (Fig. 1A).

Adaptive training was performed for 3 days for 10 min at 16:00–18:00 h, 13 Hz frequency and 1 mm amplitude using a vertical vibration machine (LD-P, Li Hui Technology, Beijing, China). The mice in the WBV group were put in an empty cage which was directly and tightly attached to the top surface of a vibrating platform. After 3 days of adaptive training, the mice underwent vibration exercise training daily for 15 min at 16:00–18:00 h, 13 Hz frequency and 2 mm amplitude, 5 days per week for 4 weeks (Lin et al., 2015) (Fig. 1A).

Sterile needles with a diameter of 0.25 mm and a length of 25 mm were directly inserted 3-4 mm, with the positive electrode at the Yang Ling Quan acupuncture point and the negative electrode at the Zu San Li acupuncture point on the same side. The needles were connected to an electronic stimulator (Electronic Acupuncture Instrument SDZ-II, Suzhou Medical Technology, Suzhou, China) using a consistent pulse with a frequency of 20 Hz and current of 0.5 mA. The ES was administered for 10 min per day for 3 days as the adaptive training. After 3 days of adaptive training, the mice were trained for 15 min per day at 16:00–18:00 h with a frequency of 20 Hz and electric current of 1 mA, 5 days per week for 4 weeks as the formal training (Hu et al., 2015) (Fig. 1A).

An aged-cell model was established using C2C12 cells (Procell Life Science & Technology Co., Ltd, Wuhan, China) by multiple population doublings (Sharples et al., 2011) in an incubator with 5% CO₂ at 37°C. The complete medium consisted of high-sugar DMEM, 10% FBS, 1% penicillin (100 U ml⁻¹) and streptomycin (0.1 mg ml⁻¹). C2C12 cells were divided into two groups: control and rhIGF-1 group. When cells reached 70–80% confluence, the medium was replaced with differentiation medium containing DMEM and 2% horse serum. The rhIGF-1 group was additionally supplemented with recombinant human IGF-1 protein (rhIGF-1, 100 ng ml⁻¹, 18 h, Abcam, Cambridge, MA, USA). After 4 days incubation, the differentiated myotubes were ready for experiments (Fig. 1B). Cell viability was measured (Cell Counting Kit-8, Beyotime, Shanghai, China) with a wavelength of 450nm using a Microplate Reader (Bio-Rad, Hercules, CA, USA) (Sun et al., 2020). Photos of the myotubes (×100 magnification) were taken with an optical microscope (Olympus, Tokyo, Japan). Each group had four parallel wells, and five fields in each well were randomly selected. The myotube diameter and length in each visual field were calculated using Media Cybernetics^® IPP 6.0.

Cell apoptosis was assessed by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL). Myotube cells were permeated with 0.03% Triton X-100 at room temperature for 10 min. Then, cells were incubated with TdT and fluorescein (FITC)-dUTP solution at 37°C for 1 h. After washing, TUNEL positive particles were observed with a fluorescence microscope (×400 magnification). Four sections from each group were scanned, with five fields per section viewed, and the of cell apoptosis count was calculated by IPP 6.0 (Feng et al., 2022).

Laminin immunostaining of gastrocnemius muscle was used to assess the cross-sectional area (CSA) of skeletal muscle fibers (Siles et al., 2019). Frozen sections were incubated with rabbit polyclonal anti-Laminin primary antibody (1:1000 dilution, Abcam), overnight at 4°C. After washing with PBS, the sections were incubated with fluorescein-conjugated goat anti-rabbit IgG (1:100 dilution), at room temperature for 1 h in the dark. After washing with PBS, anti-fluorescence quenching agent was used to seal sections to keep the sample moist. Images were visualized with a Nikon fluorescence microscope (×200 magnification; Nikon Eclipse 55i, Tokyo, Japan). Four samples of gastrocnemius muscle from each group were selected, and three sections of each sample were scanned and five regions of each section were observed under the microscope. The CSA of skeletal muscle fiber was assessed by calculating 40 fibers per field with IPP 6.0. Cells were washed with PBS at 37°C, fixed in 4% paraformaldehyde for 15 min, and subjected to immunofluorescence staining. Then, the cells were incubated using primary antibody against major histocompatibility complex (MHC; 1:50 dilution, Affinity Biosciences, Cincinnati, OH, USA), followed by incubation with fluorescence-conjugated goat anti-rabbit IgG secondary antibody (1:100). The stained cells were examined under a fluorescence microscope (×400 magnification). Each group had four parallel wells, and five fields in each well were randomly selected. The positive area for MHC was calculated by IPP 6.0 (Luo et al., 2019).

The total protein content of gastrocnemius muscle tissues was assessed by western blotting. Samples were lysed in lysis buffer (100:1:1 RIPA:PMSF:phosphatase inhibitor). The supernatant was collected after homogenization and centrifugation and the protein concentration of the supernatant was determined using a bicinchoninic acid protein quantitative kit (GSEBIO, Xi'an, China). Then, 20 µg protein was separated by 8%, 10% or 12% SDS-PAGE and transferred onto nitrocellulose membrane (Millipore). After blocking with 5% bovine serum albumin (BSA, Amresco) at room temperature for 1 h, membranes were incubated with the following primary antibodies: anti-IGF-1 (1:800 dilution, Bioss, Beijing, China); anti-IGF-1R (1:1000 dilution, Abcam); anti-p-PI3K, anti-PI3K, anti-p-Akt, anti-Akt, anti-p-mTOR, anti-mTOR, anti-p-P70S6K, anti-P70S6K [1:800 dilution, Cell Signaling Technology (CST), Boston, MA, USA], anti-Bax, anti-Bcl-2, anti-Caspase3 (1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA); anti-MuRF1, anti-MAFbx, anti-SOD1, anti-SOD2 (1:1000 dilution, Abcam); anti-GAPDH and anti-β-tubulin (1:5000, ZhuangZhi Biotech, Xi'an, China), at 4°C overnight. On the second day, after washing with Tris-buffered saline Tween (TBST) (5 min, 3 times), the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5000, ZhuangZhi Biotech) for 2 h at room temperature. Protein bands were then enhanced by chemiluminescence liquid (ECL, Bio-Rad) and detected by the Bio-Rad ChemiDoc™ MP Imaging System. Image processing and analysis was carried out in Java (ImageJ, version 1.48, National Institutes of Health, Bethesda, MD, USA) and results were normalized with respect to GAPDH or β-tubulin.

RNA was extracted using Trizol reagent (Invitrogen, Sao Paulo, Brazil). First-strand cDNA was synthesized using Revertaid First Strand cDNA synthesis kit (Takara, Kusatsu, Japan). SYBR Green PCR Master Mix (Beyotime, Shanghai, China) and CFX96™ Real-Time PCR Detection System (Bio-Rad) were used for qPCR, with 30 cycles at 58°C. The relative gene expression level of Igf-1 was determined by the 2^−ΔΔCt method using the following primers (Sangon Biotech Co., Ltd, Shanghai, China): Igf-1 F: 5′-CACTCATCCACAATGCCTGT-3′; R: 5′-TGGATGCTCTTCAGTTCGTG-3′; Gapdh F: 5′-ACCACAGTCCATGCCATCAC-3′; R: 5′-TCCACCACCCTGTTGCTGTA-3′.

The effect of exercise on levels of malondialdehyde (MDA), total antioxidant capacity (T-AOC), succinate dehydrogenase (SDH), lactate dehydrogenase (LDH) and adenosine triphosphate (ATP) in gastrocnemius muscle was assessed (Beyotime Biotechnology, Shanghai, China).

Histograms were prepared using GraphPad Prism 8 analysis software. SPSS (version 19.0, IBM Corp., Armonk, NY, USA) was used to verify the normality of the data. One-way ANOVA was used for group comparison. Tukey post hoc tests were conducted to complement ANOVA. P<0.05 was considered significant. All data were analyzed prior to statistical analysis to meet the homoscedasticity and normality assumptions of parametric tests. If there was a significant difference in variances, the effect size was further calculated. We extracted the sample size, mean and s.d. for each group and calculated the effect sizes for the four intervention groups versus the control group using an effect size calculator (https://www.campbellcollaboration.org/escalc/html/EffectSizeCalculator-Home.php). Effect sizes (Cohen’s d) are presented, with d≥0.20 indicating a small effect size, d≥0.50 indicating a medium effect size, and d≥0.80 indicating a large effect size.

Compared with the control, the relative mass (gastrocnemius mass/body mass) was significantly increased after AE (P<0.01, Cohen’s d=0.76), RE (P<0.01, Cohen’s d=2.08), WBV (P<0.01, Cohen’s d=3.73) and ES (P<0.01, Cohen’s d=1.18), and RE resulted in the greatest increase (Table 1).

Compared with the control group, the CSA of gastrocnemius muscle fibers was significantly increased after RE (P<0.01, Cohen’s d=4.96) and WBV (P<0.01, Cohen’s d=4.28), with the greatest increase following RE (Fig. 2A,B).

Compared with the control group, AE (P<0.01, Cohen’s d=3.31), RE (P<0.01, Cohen’s d=12.71), WBV (P<0.01, Cohen’s d=6.20) and ES (P<0.01, Cohen’s d=5.06) significantly increased maximum carrying capacity, and RE resulted in the greatest increase. Compared with the control group, AE (P<0.01, Cohen’s d=9.88), RE (P<0.01, Cohen’s d=6.08) and WBV (P<0.01, Cohen’s d=4.82) significantly increased running distance, and AE caused the greatest increase (Fig. 2C,D).

Compared with the control group, AE (P<0.01, Cohen’s d=10.62), RE (P<0.01, Cohen’s d=5.57) and WBV (P<0.01, Cohen’s d=3.92) significantly increased SDH levels, and levels following AE increased the most. Compared with the control group, RE increased the level of LDH (P<0.01, Cohen’s d=3.39). Compared with the control group, AE (P<0.01, Cohen’s d=6.75), RE (P<0.01, Cohen’s d=6.75) and WBV (P<0.01, Cohen’s d=5.28) significantly increased the ATP level, and AE increased it the most. (Fig. 2E–G).

Although there was a different trend in the four interventions for the above-mentioned indicators, overall, the results showed that AE, RE, WBV and ES were effective at improving muscle mass and function of skeletal muscle. After training, relative muscle mass, muscle CSA, maximum carrying capacity, and levels of LDH and ATP increased the most in the RE group, and endurance exercise capacity and SDH levels increased the most in the AE group, versus the other groups.

IGF-1 is an important growth factor, which plays a critical role in muscle fiber myogenesis and hypertrophy through the IGF-1R–PI3K/Akt signaling pathway (Yoshida and Delafontaine, 2020). As shown in Fig. 3, compared with the control group, the levels of Igf-1 mRNA (all P<0.01), IGF-1 protein (all P<0.01), IGF-1R (all P<0.01), phosphorylated PI3K (p-PI3K; all P<0.01) and p-Akt (P<0.01) were significantly upregulated after the four types of exercise training (Fig. 3A–C).

The balance between protein synthesis and degradation determines the mass of skeletal muscle (Phillips, 2014; Lee and Neppl, 2021). Moreover, Akt-stimulated mTOR/P70S6k signaling promotes protein synthesis and muscle fiber growth (Barclay et al., 2019; Timmer et al., 2018). Compared with the control group, the levels of p-mTOR/mTOR (all P<0.01) and p-p70S6K/p70S6K (all P<0.01) were significantly increased in the AE, RE, WBV and ES groups (Fig. 4).

Protein degradation in skeletal muscle is caused by the activation of the ubiquitin–proteasome system. The muscle-specific E3 ubiquitin ligases Muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx)/atrogin-1, used as muscle atrophy markers, were upregulated under skeletal muscle atrophy conditions (Bodine and Baehr, 2014). In this study, we found the expression levels of MuRF1 (all P<0.01) and MAFbx (all P<0.01) were significantly decreased in AE, RE, WBV and ES groups, when compared with the control group (Fig. 4). These results indicate that exercise upregulated the protein expression of mTOR/P70S6k and inhibited the expression of MuRF1 and MAFbx.

With the decrease of antioxidant capacity, the increase in oxidative stress can induce apoptosis (Wang et al., 2020). The results showed that levels of T-AOC (all P<0.01), SOD1 (all P<0.01) and SOD2 (all P<0.01) were significantly increased in all types of exercise training when compared with the control group. The level of MDA was significantly decreased in the AE, RE, WBV and ES groups (all P<0.01) (Fig. 5). The data indicate that exercise effectively improved the level of antioxidant enzymes in skeletal muscle.

The results showed that compared with the control group, the levels of Bax/Bcl-2 (all P<0.01) and caspase-3 (all P<0.01) in the four exercise groups were significantly decreased (Fig. 5A,D). Collectively, these data indicate that exercise effectively reduced the expression of Bax/Bcl-2 and caspase-3 in skeletal muscle.

The IGF-1R–PI3K/Akt pathway plays an important role in skeletal muscle growth (Guan et al., 2022). We confirmed that our exercise interventions activated this signaling pathway and promoted skeletal muscle hypertrophy during early aging in mice. However, whether there is a direct link between activation of IGF-1 signaling and skeletal muscle hypertrophy in early aging mice was not fully presented in our animal experiments. Thus, we further explored the molecular mechanism by performing an in vitro experiment. Here, an aging cell model was established by the multiple population doubling method to simulate the skeletal muscle cells of mice. C2C12 cells were treated with rhIGF-1. The results showed that compared with the control group, rhIGF-1 intervention increased the levels of IGF-1R protein (P<0.01), p-PI3 K (P<0.01) and p-Akt (P<0.01) (Fig. 6).

The rhIGF-1 intervention also increased myotube diameter (P<0.01) and length (P<0.01), and the MHC positive area (P<0.01) (Fig. 7A–D,F). In addition, cell viability was significantly increased by rhIGF-1 (P<0.01) (Fig. 7E).

These data indicate that IGF-1 efficiently promoted the differentiation and growth of C2C12 myoblasts through the IGF-1R–PI3K/Akt signaling pathway.

We found that rhIGF-1 increased the levels of mTOR (P<0.01) and P70S6K (P<0.01) protein, and reduced the levels of MuRF1 (P<0.01) and MAFbx (P<0.01) (Fig. 8). Protein expression levels of SOD1 (P<0.01) and SOD2 (P<0.01) were significantly increased by rhIGF-1 intervention. Moreover, rhIGF-1 intervention significantly decreased the level of TUNEL-positive particles (P<0.01) and the level of Bax/Bcl-2 (P<0.01) and Caspase-3 (P<0.01) protein (Fig. 9).

These data indicate that IGF-1 improved protein synthesis and anti-oxidant capacity and inhibited expression of proteins associated with degradation and apoptosis in myotube cells through the IGF-1R–PI3K/Akt pathway.

In the present study, the results demonstrated that AE, RE, WBV and ES activate the IGF-1/IGF-1R–PI3K/Akt pathway, and improve skeletal muscle mass and function in mice undergoing early aging. After training, skeletal muscle mass, CSA, maximal carrying capacity and levels of LDH and ATP increased the most in the RE group versus the other groups; and endurance exercise capacity and SDH levels increased the most in the AE group. We found that WBV promoted muscle hypertrophy better than traditional AE. Moreover, ES only increased maximal carrying capacity and skeletal muscle mass in these mice. In addition, rhIGF-1 upregulated the expression level of proteins relevant to protein synthesis and reduced the expression of markers for protein degradation and apoptosis in myotubes through IGF-1R–PI3K/Akt signaling.

Muscle mass and function are key factors in improving quality of life (Tieland et al., 2018). Exercise, in middle age or early old age, could efficiently promote muscle hypertrophy, and improve exercise and metabolic capacity (Lewsey et al., 2020). Exercise in early old age could also effectively reduce the incidence of skeletal muscle atrophy and complications in later old age, providing a basic guarantee for maintaining physical health. Moreover, traditional AE and RE effectively promote muscle hypertrophy and alleviate disuse and pathological muscle atrophy (Yin et al., 2020). AE improves skeletal muscle mass and function mainly through increasing the density of capillaries and mitochondria, and by increasing (to a small extent) the amount of type I muscle fibers in skeletal muscle; RE promotes the hypertrophy of type IIa muscle fibers and an increase of non-contractile tissue (such as collagen) by increasing the rate of myofibrillar protein synthesis. However, there are some disadvantages of AE and RE. First, AE and RE have certain requirements in terms of cardiopulmonary function and exercise capacity, and are not suitable for people with cardiovascular disease and mobility impairment (Del Buono et al., 2019). Second, AE and RE might induce muscle and joint stiffness, acute inflammation and swelling, delayed-onset muscle soreness and even muscle damage (Kosar et al., 2012).

WBV stimulates muscle tension and relaxation in a short period of time, increases muscle spindle activity and muscle recruitment, and in turn promotes capillary angiogenesis, mitochondrial generation and protein synthesis (Chang et al., 2018; Sayed et al., 2016). Long-term ES activates muscle satellite cell proliferation (Kosar et al., 2012). WBV and ES are conducive to avoiding the risk of injury during exercise training, meanwhile improving muscle mass and function by inducing passive contraction (Rittweger, 2010; Guo et al., 2021). Thus, WBV and ES were chosen in this study, in addition to AE and RE. In this study, we confirmed that these four types of intervention promoted skeletal muscle hypertrophy, increased the activity of enzymes related to energy metabolism, and improved exercise capacity, similar to a previous study using a model of aging (Zampieri et al., 2015).

After the same duration and intensity of AE and RE intervention, skeletal muscle mass, CSA, maximal carrying capacity and levels of LDH and ATP increased the most in the RE group versus the other groups, which is consistent with previous reports (Egan and Zierath, 2013; Shirai et al., 2021). In contrast, endurance exercise capacity and SDH level increased the most in the AE group, which is related to the improvement of oxidative efficiency by AE through increased mitochondrial biogenesis and vascular density (Egan and Zierath, 2013; Heo et al., 2021). We found that short-term WBV promoted muscle hypertrophy better than traditional AE. Surprisingly, in addition to AE and RE, we found that WBV, as a passive exercise training model, also effectively increased skeletal muscle mass, CSA, and indicators related to the metabolism of SDH, LDH and ATP, and exercise capacity. The underlying reason may be that WBV can activate and enhance muscle fiber recruitment and, secondly, promote capillary angiogenesis and protein synthesis, while improving aerobic oxidation and glycolysis. WBV combined some of the benefits of AE and RE, whereas short-term ES only promoted muscle fiber recruitment, and thus only increased maximal carrying capacity in mice, without promoting muscle fiber hypertrophy. Overall, for traditional exercises at the same intensity, RE was more effective in promoting muscle hypertrophy. However, it is undeniable that short-term low-intensity WBV could also achieve intervention effects similar to those of AE and RE. WBV and ES are expected to be used as efficient auxiliary exercise methods to improve skeletal muscle mass and strength in the future.

Several studies have explored possible target factors for exercise-induced muscle hypertrophy, including increasing IGF-1 levels, protein synthesis and angiogenesis, and activating muscle satellite cell proliferation (Keng et al., 2019; Larsson et al., 2019). IGF-1 could also improve the structure and strength of muscle fibers (Yoshida and Delafontaine, 2020; Ascenzi et al., 2019). Moreover, IGF-1 alleviates the myotube cell atrophy and C2C12 cell apoptosis induced by drug interventions (Feng et al., 2022; Nakamura et al., 2019). It has been reported that IGF-1/IGF-1R mediates growth effects of skeletal muscle through various pathways such as the PI3K/Akt signaling pathway (Yoshida and Delafontaine, 2020). Exercise improves muscle mass via activation of the IGF-1R−PI3K/Akt pathway in skeletal muscle (Ascenzi et al., 2019). In this study, we demonstrated that all four types of exercise intervention significantly upregulated the expression of IGF-1 and IGF-1R, and activated the PI3K/Akt signaling pathway in gastrocnemius muscle in mice undergoing early aging. The results also suggested that WBV and ES can activate the canonical pathway of IGF-1 to promote muscle hypertrophy as for traditional exercise.

To explore the downstream activation followed IGF-1 signaling, we detected the expression levels of indicators of protein synthesis and degradation, and apoptosis. Skeletal muscle mass is directly determined by the balance between protein synthesis and degradation. Anabolic/catabolic balance in skeletal muscle was closely associated with the activation of mTOR and P70S6K. IGF-1 activates PI3K/Akt/mTOR signaling to promote muscle protein synthesis (Barclay et al., 2019; Yoshida and Delafontaine, 2020; Li et al., 2022). It has also been confirmed that the IGF-1/Akt/mTOR/P70S6K pathway plays an important role in promoting muscle hypertrophy (Timmer et al., 2018). In this study, we found AE, RE, WBV and ES upregulated the expression of p-mTOR and p-P70S6K in gastrocnemius muscle. These results indicate that exercise can promote protein synthesis and muscle hypertrophy by upregulating mTOR/P70S6K signaling.

The ubiquitin proteasome system is one of the main proteolytic systems (Khalil, 2018). MAFbx and MuRF-1 are important regulators of ubiquitin-mediated protein degradation in skeletal muscle (Bodine and Baehr, 2014). IGF-1 can suppress levels of MAFbx and MuRF1, and alleviate muscle atrophy through activation of the IGF-1R–PI3K/Akt signaling pathway (Glass, 2005). In this study, AE, RE, WBV and ES downregulated the expression of MAFbx and MuRF1 in gastrocnemius muscle. The results showed that the promotion of muscle hypertrophy by different types exercise may be related to the activation of the IGF-1 pathway and the reduction of proteins related to ubiquitination and degradation. In addition, oxidative stress and apoptosis can also affect the mass of skeletal muscle (Glass, 2005). The oxidation–reduction reaction imbalance induces oxidative stress and apoptosis in skeletal muscle in aged rat and rabbit (Li et al., 2016). IGF-1 can inhibit pro-apoptotic proteins to prevent apoptosis in skeletal muscle of aged rats (Marzetti et al., 2008). We detected apoptosis-related proteins in skeletal muscle after training, and found that AE, RE, WBV and ES upregulated the expression of skeletal muscle antioxidant enzymes and the anti-apoptosis protein Bcl-2, and reduced the levels of pro-apoptosis proteins Bax and Caspase-3. We speculate that, on the one hand, the four types of exercise exerted positive regulatory effects by improving the efficiency of protein synthesis and the level of antioxidant enzymes, and, on the other hand, skeletal muscle hypertrophy is associated with low levels of MuRF1, MAFbx, Caspase-3 and Bax/Bcl-2 after exercise, while exercise prevents protein degradation and reduces the possibility of apoptosis to some extent (Feng et al., 2022).

In our animal experiments, we found that skeletal muscle mass and function were significantly improved after exercise training, which is closely related to the activation of the IGF-1/IGF-1R–PI3K/Akt signaling pathway. However, it remained to be determined whether activation of IGF-1 signaling was primarily responsible for the effect of exercise on promoting muscle hypertrophy. Therefore, we further explored the molecular mechanism by performing in vitro experiments. Studies have demonstrated that IGF-1 treatment prompts differentiation and growth of C2C12 cells under pathological or normal conditions. Currently, no study has reported whether IGF-1 can promote differentiation and growth in aged C2C12 cells. Thus, in vitro experiments, C2C12 cells were induced to age by multiple population doublings to mimic the skeletal muscle microenvironment in mice undergoing early aging. We confirmed that rhIGF-1 improved anti-oxidant capacity and differentiation, and inhibited protein degradation and apoptosis in vitro. Combining the results of our animal and cellular experiments, the exercise-activated IGF-1/IGF-1R–PI3K/Akt pathway plays a key role in promoting muscle hypertrophy in early aging mice.

Unfortunately, we did not use gene editing or injection of pathway inhibitors into mice to confirm that exercise-induced upregulation of IGF-1 exerts its protective effects through IGF-1R–PI3K/Akt signaling. Our experiments were performed on the gastrocnemius muscle of male mice and did not include other muscles. In addition, we could not compare AE, RE, WBV and ES at uniform intensity and time because of their different principles of motion. At present, it can only be concluded that of the two active exercises, AE and RE, the effect of RE intervention is better, while short-term low-intensity WBV and ES can achieve the same effect as AE, but cannot be directly compared with AE. Therefore, the outcomes obtained from this experiment should be interpreted with some caution.

To sum up, we have shown that AE, RE, WBV and ES are effective in mice in terms of improving muscle mass, increasing levels of SDH, LDH and ATP and exercise capacity, upregulating IGF-1 and IGF-1R levels, and activating the PI3K/Akt pathway, to promote protein synthesis and anti-oxidant capacity, reduce protein degradation and apoptosis, and thus improve muscle hypertrophy in mice undergoing early aging. After training, skeletal muscle mass, CSA, maximal carrying capacity and levels of LDH and ATP increased the most in the RE group, and endurance exercise capacity and SDH levels increased the most in the AE group. Moreover, we confirmed that WBV could also achieve significant effects similar to traditional exercise (AE and RE), and ES exerted significant effects on increasing maximal carrying capacity in mice. WBV and ES might be effective strategies to improve skeletal muscle mass and function in people with limited mobility. In addition, we further confirmed by in vitro experiments that the activation of IGF-1 signaling is the main reason for the protective effects of exercise on aging skeletal muscle cells. These results provide new sight into the positive effects of exercise on improving skeletal muscle mass and function.

We thank the experimental platform provided by the Institute of Sports Biology, College of Physical Education, Shaanxi Normal University. We also would like to thank the editors for supporting open science and hard work during the COVID-19 pandemic.