Dietary restriction and aerobic exercise alleviate obesity-related skeletal muscle impairment via the miR-130/PPARγ axis and IGF-1/Akt/mTOR signaling activation

Dietary restriction and aerobic exercise alleviate obesity-related skeletal muscle impairment via the miR-130/PPARγ axis and IGF-1/Akt/mTOR signaling activation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dietary restriction and aerobic exercise alleviate obesity-related skeletal muscle impairment via the miR-130/PPARγ axis and IGF-1/Akt/mTOR signaling activation Qiyi Zhang, Tianqi Liu, Xiaoxiong He This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8116495/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract This study investigated the effects of dietary restriction (DR) combined with aerobic exercise training (ET) on high-fat diet (HFD)-induced obesity and associated skeletal muscle impairment in male Sprague-Dawley rats, as well as the underlying mechanisms. An 8-week DR + ET intervention effectively increased the cross-sectional area and protein content of the soleus muscle. Both DR and ET alone promoted the activation of the IGF-1/Akt/mTOR signaling pathway and reduced the protein expression of peroxisome proliferator-activated receptor γ (PPARγ), a target of miR-130 in adipocytes, in HFD-induced obese rats. However, no additional benefit was observed with combined DR + ET treatment. Consistent with these findings, miR-130 expression was upregulated in the skeletal muscle of obese rats compared with those fed a normal diet, and this increase was significantly attenuated by DR, ET, or DR + ET, with DR + ET showing the strongest inhibitory effect. To further explore the role of miR-130 in skeletal muscle cells, L6 and C2C12 cells were transfected with miR-130 mimics. Overexpression of miR-130 markedly suppressed proliferation and differentiation in these cells, accompanied by reduced creatine kinase activity and decreased myogenin expression—both key markers of myogenic differentiation. Moreover, miR-130 overexpression inhibited the luciferase activity of a reporter vector containing the PPARγ-3′-UTR, and this inhibition was abolished by mutation of the PPARγ-3′-UTR, indicating a direct regulatory mechanism affecting protein synthesis in skeletal muscle. In summary, DR and ET each alleviated obesity-related skeletal muscle impairment by activating IGF-1/Akt/mTOR signaling and suppressing miR-130 expression, but their combination did not produce synergistic effects. Biological sciences/Cell biology Health sciences/Diseases Health sciences/Endocrinology Biological sciences/Molecular biology Biological sciences/Physiology Obesity Dietary restriction Aerobic exercise Skeletal muscle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Obesity emerges a worldwide health problem and has been associated with several chronic diseases, e.g. diabetes and certain cancers [ 1 , 2 ]. Skeletal muscle comprises 30–40% of body mass in humans. It is the predominant part for glucose disposal and fatty acid consumption and is an important determinant of human health and diseases[ 3 – 5 ]. Emerging data have revealed deleterious effects of diet-induced over weight on skeletal muscle fitness and plasticity[ 4 , 6 – 8 ]. Specifically, obesity may negatively influence muscle protein turnover, or the breaking down and rebuilding of functional proteins, with the myofibrillar proteins being particularly susceptible to anabolic resistance. Obesity-induced reduction in protein synthesis of skeletal muscle has shown to cause sarcopenia and skeletal muscle strength loss, thereafter impairing self-care ability and quality of life particularly in elderly[ 6 , 9 ]. Improving the health of skeletal muscle is an important component of obesity treatment and is imperative to improve public health. Insulin-like growth factor-1 (IGF-1) is a key growth factor regulating both anabolic and catabolic pathways in skeletal muscle[ 10 – 12 ]. IGF-1 increases skeletal muscle protein synthesis via multiple signaling pathways, including PI3K/Akt/mTOR activation[ 13 ]. IGF-1 also potentiates skeletal muscle regeneration via activating skeletal muscle stem cells, which may contribute to muscle hypertrophy and/or inhibit atrophy. IGF-1 levels are often suppressed in many chronic disease conditions [ 14 – 16 ]. A recent study showed that increased serum IGF-1 was inversely associated with total body fat and trunk fat, while positively associated with lean mass in 15 abdominally obese men who were treated for 12 weeks with recombinant human GH[ 15 ]. Stimulating the IGF-1/Akt/mTOR pathway is thus critical for muscle mass maintenance, muscle hypertrophy, and muscle protein regulation, and ultimately for treating obesity. Apart from the suppressive effect on IGF signaling, obesity alters microRNAs expression in metabolically important organs, including skeletal muscles[ 17 – 19 ]. MicroRNAs are 19–22 nucleotides in length, and modulate gene expression on post-arrangements extent by targeting the 3' untranslated region (3'UTR), ultimately leading to reduced protein translation of miRNA targets. MicroRNAs regulate multiple pathways including adipogenesis, insulin signaling, immune-mediated inflammation and lipid metabolism, which play an important role in the pathophysiology of metabolic diseases [ 20 – 22 ]. Plasma miR-130 level was found to be lower in obese humans or in patients with coronary artery disease relative to lean people[ 23 ]. The muscle-specific miR-130 has shown to suppress adipogenesis by targeting of peroxisome proliferator-activated receptor gamma (PPARγ) in adipocytes in mouse models [ 19 , 22 , 24 ]. Accumulating evidence highlight the benefits of exercise and (or) dietary restriction (DR) on prevention and treatment of obesity [ 9 , 25 ]. Skeletal muscle is a highly adaptable tissue and remodels in response to life-style modifications including exercise training and dietary factors. Both strategies have shown to effectively increase the expression of IGF-1 in skeletal muscle and stimulate skeletal muscle hypertrophy [ 15 , 26 – 30 ]. Moreover, aerobic exercise training has shown to remodel miRNA expression in skeletal muscle[ 31 – 33 ]. However, little data exist regarding whether and how DR could impact skeletal muscle mass and protein synthesis. In our previous study, DR failed to effectively improve metabolic homeostasis and biological dysfunction in obese mice. However, by exploring the potential inter organ communication, the causal relationship between genes Gdf15, Tfrc, Cdv3, Map2k4 and Nqo1 and metabolic disorders induced by high-fat diet was determined[ 34 ]. Here, we explored effects of calorie restriction, aerobic exercise and their potential synergetic effect on skeletal muscle metabolism in high-fat diet induced obese mouse models, with specific focus on skeletal muscle protein synthesis, IGF-1/Akt/mTOR signal pathway and miR130 expression. Moreover, L6 and C2C12 skeletal muscle cells were transfected with miR-130 mimics and expressions of miR-130 and PPARγ, as well as activity of creatine kinase and expression of muscle-specific protein, i.e. myogenin, were measured to elucidate the role of miR-130 in the function of skeletal muscle cells. 2. Materials and Methods 2.1. Animals and study design Fourty 3-month old healthy male Sprague Dawley rats were purchased from Experimental Animal Center of Xi'an Jiaotong University (Xi’an, China). The rats were fed with normal chow or with the high fat diet. The ingredients of normal chow were 40% corn flour, 26% wheat flour, 10% bran, 10% fish meal, 10% bean cake, 2% mineral, 1% coarse grains, and 1% vitamin. The high fat diet contained 60% fat, 20% carbohydrates and 20% protein. All animals were maintained under controlled environmental conditions (room temperature and 40%-50% relative humidity). Rats were housed in cages and had free access to food and tap water. After 12 weeks high-fat diet, obese rats were determined according to Lee’s index [body weight (g)1/3 ×1000/body length(cm)]. Obese rats were then divided into four groups: obesity group (O group, n = 8), dietary restriction group (DR group, n = 8), exercise training group (ET group, n = 8) and the group treated by DR and ET (DR + ET group, n = 8). Rats in obesity group were continually fed with high-fat diet for additional 5 weeks. In the DR and DR + ET group, rats were given 60% food intake relative to the O group. In the ET and DR + ET group, rats were subjected to swimming training for 5 weeks. Sedentary rats were handled in a similar manner to the exercised rats, but were not exercised. Swimming training is conducted according to the established plan[ 35 ] (80% maximum intensity).The training was carried out between 09:00 am and 12.00 am. The training program consisted in swimming 6 days/week, 50 min/day, during 5 weeks. Rats in exercise groups were adapted to swimming training f4`or 5 days before starting the protocol to reduce stress in the animals. The water temperature was maintained at 31 ± 1°C. Body weight of animals was recorded every week. After deep anesthesia with isoflurane (maintained at 2–3% in oxygen), the rats were sacrificed by cervical dislocation, a method that ensures rapid and painless death, consistent with the ethical guidelines for animal experimentation approved by our Institutional Animal Care and Use Committee (IACUC). All animal experiments were conducted in accordance with the ARRIVE guidelines (PLoS Biol. 8 (6), e1000412, 2010) and approved by the (Institutional Animal Care and Use Committee of Shaanxi Normal University) under approval number No. 202216012. All efforts were made to minimize animal suffering and adhere to veterinary best practices. 2.2. Skeletal Muscle processing and measurement of proteins Rats were fasted but allowed free access to water, and their 12 h urine and feces were collected. All animals were anesthetized by of isoflurane inhalation, and then were sacrificed to obtain soleus muscle. Muscle samples were fixed by 10% neutral formaldehyde solution for histological studies or homogenized for total protein content measurement. Muscle samples from the left foot were used for measurement of protein levels and were stored at − 80°C until analysis. Total protein was extracted after homogenization in RIPA buffer (pH 7.2–7.4) and centrifuged for 20 min at 1000g at 4°C. The main components of RIPA cracking solution are 50 mM Tris (pH 7.4), 150mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, And various inhibitors such as sodium orthovanadate, sodium fluoride, EDTA, leupeptin, etc.The resulting supernatants were removed and target proteins extracted. BCA method was used for protein quantification. 2.3. Histological Analysis and Morphometry The soleus muscle tissue of rats were fixed with 4% paraformaldehyde, and were performed for histopathological analysis. The fixed tissues were embedded in paraffin, sectioned (5–6 µm) and stained with H&E. These slides were analyzed under the Olympus light microscope for observations and photograph. We systematically selected five visual fields per tissue section, with four sections analyzed for each biological sample and three samples per experimental group. The CSA of soleus muscle fibers in each visual field was quantitatively analyzed using Image-Pro Plus 7.0. 2.4. Western Blotting The muscle sample (50 mg) was added into pre-cooled protein extraction reagents (Beyotime Biotechnology, Haimen, China) and was homogenized with electric homogenizer (Thermo Fisher Scientific, Waltham, MA, USA). The samples were then centrifuged at 12000 rpm for 15 minutes at 4°C. The proteins were separated by SDS-PAGE in Tris-glycine buffer and transferred under constant current. After transferring, the PVDF membrane (Millipore, Burlington, MA, USA) was blocked by 3% bovine serum albumin (BSA) (Beyotime Biotechnology, Haimen, China) at room temperature. Primary antibodies were incubated at 4°C overnight with different dilutions: IGF-1 (1:500, Beyotime Biotechnology, Haimen, China), GAPDH (1:10000), AKT, p-Akt, mTOR, p-mTOR (1:1000, Cell Signaling, Danvers, MA, USA), myogenin (1:500) and PPARγ (1:500, Beyotime Biotechnology, Haimen, China). The membrane was then washed with TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20). Secondary antibody of goat anti-rabbit HRP (Thermo Fisher Scientific, Waltham, MA, USA) was incubated for 1.5 hours at room temperature. After washing with TBST, membranes were developed with ECL (Beyotime Biotechnology, Haimen, China) according to the manufacturer’s instructions. For western blot with protein extracts from L6 and C2C12 cells, primary antibodies against PPARγ and myogenin were used. Universal Blocking and Antibody Dilution Buffer were purchased from Beyotime (Shanghai, China). The results of Western blotting were analyzed using Image Processing and Analysis in Java (ImageJ, v. 1.48, National Institutes of Health, Bethesda). 2.5. Cell Culture The L6 and C2C12 cells were purchased from ATCC (Manassas, VA, USA). L6 cells were cultured in α-minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, St Louis, MO, USA) at 37°C and 5% CO2. On reaching 90% confluence, L6 cells were induced to differentiate by reducing the serum concentration from 10% to 2% in the culture medium. C2C12 cells were maintained in DMEM-F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) with 10% FBS (Sigma Aldrich, St Louis, MO, USA). Serum concentration was decreased from 10% to 2% to initiate myogenic differentiation. 2.6. RNA Extraction and Real Time qPCR Total RNA was extracted from soleus muscle samples, L6 and C2C12 cells, respectively by using the miRNA isolation kit (Sigma Aldrich, St Louis, MO, USA) according to the manufacturer’s protocol. The level of miR-130 was assessed by real time qRT-PCR by using a TaqMan® MicroRNA Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The miR-130a primers were purchased from Sigma and U6 small nuclear RNA (Sigma) served as internal control. The miR-130a mimic primer species was from rats, and the specific sequence was GCUCUUUUCACAUUGUGCUACU. To determine the expression of PPARγ mRNA in the muscle tissues and cell lines, total RNA was extracted by using RNeasy kit (Qiagen, Gaithersburg, MD, USA), and cDNA was synthesized from RNA using the reverse transcription system (Promega, USA). GAPDH was used as internal control for qPCR. The primers used in qRT-PCR are as follows: forward 5’-TGATATCGACCAGCTGAACC-3’ and reverse 5’-GTCCTCCAGCTGTTCGCCA-3’ for PPARγ (rat); forward 5’-TCGCTGATGCACTGCCTATG-3’ and reverse 5’-GAGAGGTCCACAGAGCTGATT-3’ for PPARγ; forward 5’-AATGCATCCTGCACCACCAA-3’ and reverse 5’-GATGCCATATTCATTGTCATA-3’ for GAPDH; forward 5’-AGGTCGGTGTGAACGGATTTG-3’ and reverse 5’-TGTAGACCATGTAGTTGAGGTCA-3’ for GAPDH. 2.7. Transfection The miR-130 mimics (50 nM) and the negative control (ABM, BC, Canada) were transfected into L6 and C2C12 cells with Lipofectamine® RNAiMAX transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The pCDNA3-1-PPARγ plasmid was transfected into L6 or C2C12 cells with Lipofectamine 2000 transfection reagent. Plasmid extraction was performed using the Shanghai Sangong 48-Channel Prefilled Plate Mag-MK Plasmid Extraction Kit (B614801). 2.8. Proliferation Assay Cell proliferation was assessed using a CCK8 cell counting kit (Sigma, St Louis, MO, USA), according to the manufacturer’s instructions. Briefly, 24 hours after transfection, L6 and C2C12 cells transfected with miR130 mimics or PPARγ overexpressing plasmids were seeded at a density of 1×104 cells/well in Corning 96-well culture plate (Sigma Aldrich, St Louis, MO, USA). The medium was removed at 72 or 96 hours later, and the cells were washed with PBS twice followed by addition of 10 µl of tetrazolium substrate. The optical density (OD) at 450 nm was measured using a BioTek ELISA microplate reader (BioTek, Winooski, VT, USA) at 37°C for an additional hour. 2.9. The PPARγ 3'UTR Reporter Activity Assay The PPARγ 3'UTR reporter plasmids (pRL-PPARγ) were kept in our lab. Mutation in the miR-130 seed regions of the PPARγ 3'UTR was generated using a QuikChange lightning site directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). The wild type or mutated vector were co-transfected with miR-130 mimics into L6 and C2C12 cells by using Lipofectamine® RNAiMAX or lipfectamine 2000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). Reporter gene assays were conducted 72 h post-transfection by using the Dual luciferase Reporter assay system (Promega, Fitchburg, WI, USA) according to the manufacturer’s protocol. 2.10. Statistical Analysis All experiments were performed in triplicate and the continuous variables were expressed as means ± SD (standard deviation). Data were analyzed using one-way analysis of variance (ANOVA) and followed by a post-hoc Turkey test in SPSS 26.0. For all analysis, the p-value of 0.05 or less was considered statistically significant. 3. Results and Discussions After 8 weeks of high-fat diet, both the body weight (high-fat groups: 398.7 ± 18.5 g v.s. C group: 338.2 ± 10.3 g) and Lee's index (high-fat groups: 30.5 ± 0.4 vs. C group: 29.1 ± 0.2) of high-fat diet-fed rats (O, DR, ET, ET + DR) were significantly higher than those of the normal chow control group (all p < 0.001). Following 5 weeks of intervention, the O group maintained elevated weight and index (483.6 ± 16.7 g, 31.6 ± 0.4), while the DR, ET, and ET + DR groups showed significant reductions in both parameters (weight: 23.3%, 16.0%, 30.2% decrease; Lee's index: 3.9%, 1.3%, 6.9% decrease, all p < 0.05 v.s. Week 8), with the ET + DR group exhibiting the most pronounced effects ( p < 0.05 vs. DR and ET groups) (Supplementary Table 1–2). The obesity epidemic continues its relentless advance and is often accompanied with skeletal muscle remodeling that presented with impairments of muscle structure and function, deleteriously affecting metabolic homeostasis [ 1 , 4 , 6 , 7 ]. In line with previous studies, compared with the rats fed with normal chow, we found that the cross-sectional area of soleus muscle fiber was significantly lower in high-fat diet induced obese rats (Fig. 1 A, p < 0.01), which was whereas increased in ET and DR + ET groups (p < 0.05). Rats in ET and DR + ET group had higher skeletal muscle protein contents than obese rats (p < 0.05) (Fig. 1 B). Yet no effect was seen for dietary restriction. Our data support benefical effects of exercise on skeletal muscle structure morphology and increased muscle mass. Several mechanisms have been proposed to be involved in obesity-induced skeletal muscle remodeling, like inflammatory cytokines, endoplasmic reticulum stress, and autophagy of skeletal muscle cell[ 36 ]. IGF-1 is an important modulator of skeletal muscle growth and could functionally promote muscle protein synthesis via stimulating muscle cell proliferation and differentiation. IGF-1/PI3K/AKT/mTOR signaling pathway is one of major mechanisms linking obesity with impaired muscle regeneration, which critically involves in regulating protein synthesis in skeletal muscle. In obese mice and Zucker rats, muscle growth in response to mechanical loading was reduced due to decreased activation of Akt, p70S6 kinase and mTOR[ 37 ]. Obesity also led to increased levels of pro-inflammatory cytokines, e.g.TNF-α, IL-1β and IL-6, which could inhibit the activation of IGF-1/AKT/mTOR signals, and subsequently impair normal muscle physiological maintenance, cell growth and regeneration function[ 4 , 38 ]. In the present study, as shown in Fig. 2 , feeding high-fat diet for 12 weeks led to a sharp decrease in the IGF-1 level in skeletal muscles compared with rats fed with normal chow (p < 0.05). DR, ET or DR + ET effectively inhibited the obesity caused reduction in skeletal muscles IGF-1, respectively (p < 0.01) (Fig. 2 A), while increased Akt phosphorylation compared with sedentary rats fed with high-fat diet. Opposite regulating effects were observed for DR, ET or DR + ET, respectively, on the level of mTOR phosphorylation in obese rats. We also examined levels of myogenin, a downstream target of IGF-1/Akt/mTOR. We found that DR, ET or DR + ET reduced high-fat diet induced increase in myogenin in skeletal muscle, resulting in the level of myogenin comparable to that of normal chow -fed rats (p < 0.01) (Fig. 2 B). In agreement with our findings, Ochi et al. reported that exercise activate the IGF1/Akt/mTOR signaling pathway to promote muscle hypertrophy[ 14 ]. Calorie restriction has also shown to inhibit energy-dependent pathways including IGF-1/PI3K/Akt/mTOR and to activate AMP-dependent protein kinase activity in cancer cells [ 39 ]. Notably, although both dietary restriction and exercise per se has exhibited promoting effects on activation of IGF1/Akt/mTOR signaling, no significant improvement was observed for DR + EX. Skeletal muscle is one of the main end organs of insulin resistance, and its damage is manifested as mitochondrial dysfunction. However, the combined intervention program including endurance training and dietary restriction effectively restored mitochondrial function, which showed the reduction of mitochondrial reactive oxygen species (ROS), the stability of membrane potential and the reduction of mitochondrial swelling. In this study, the reason why DR + ET did not improve significantly may be due to the low intensity of swimming and the short duration of intervention, which failed to effectively restore mitochondrial function. MicroRNAs critically involved in the regulation of gene expression programs, emerging role as biomarkers and therapeutics of metabolic diseases[ 17 ], and have been shown to play an important role in cell proliferation and differentiation[ 17 – 23 , 40 , 41 ]. The miR-130a is highly expressed in human endothelial cells and has shown to strongly affect adipocyte differentiation that is linked to human obesity, as overexpressing miR-130 impaired adipogenesis and reducing miR-130 enhanced adipogenesis[ 21 ]. The miR-130 also aggravated acute myocardial infarction-induced myocardial injury in H9c2 cells by targeting PPARγ [ 42 ]. More and more evidences show that PPAR γ was also important for the maturation and function of various immune system related cell types, such as monocytes/macrophages, dendritic cells and lymphocytes. In addition, PPAR γ controls cell proliferation in other tissues and organs (including colon, breast, prostate and bladder), and the disorder of PPAR γ signal transduction is related to tumor development in these organs[ 43 ]. Yuan et al., recently identified circulating miR-130 (miR-130a and miR-130b) and its target PPARγ as potential biomarkers in patients of coronary artery disease with type 2 diabetes mellitus[ 22 ]. Yet little evidence regarding the functional role of miR-130 in skeletal muscle exists[ 44 ]. We found that the miR-130 level in skeletal muscle was upregulated in obese rats than in rats fed with normal chow, which was significantly abrogated by DR, ET or DR + ET, with DR + ET exhibiting the most pronounced inhibitory ability. Similarly, high-fat diet suppressed levels of PPARγ, a target of miR-130 in adipocytes, obese rats were beneficially reversed by DR, ET or DR + ET, respectively (Fig. 3 B and Fig. 3 C). To further elucidate the role of miR-130 in the function of skeletal muscle cells, L6 and C2C12 skeletal muscle cells were transfected with miR-130 mimics. The miR-130 levels were significantly promoted by transfection of the mimics (Fig. 4 A) and dramatically reduced proliferation rate in both L6 and C2C12 cells (Fig. 4 B). Besides, increased activity of creatine kinase (Fig. 4 C) and expression of muscle-specific protein, i.e. myogenin (Fig. 4 D), are main characteristics of muscle differentiation. PPARγ expressed in skeletal muscle cells can protect against high-fat diet induced insulin resistance and muscle dysfunction [ 45 – 47 ]. In this study, we found that transfection of miR-130 mimics significantly inhibited luciferase activity of pRL-PPARγ-3’-UTR, but failed to alter the activity of the mutated version in both L6 and C2C12 cells (Fig. 5 A). The inhibitory effect of miR-130 on PPARγ expression was also indicated by the reduction of PPARγ mRNA expression in L6 and C2C12 cells transfected with miR-130 mimics (Fig. 5 B). We then sought to investigate the role of PPARγ in proliferation and differentiation of skeletal muscle cells. Specifically, the proliferation rates of L6 and C2C12 cells were dramatically increased in cells with forced expression of PPARγ. Moreover, the improving effect of PPARγ on myogenic differentiation was evidenced by elevated creatine kinases activity and upregulated myogenin expression in L6 and C2C12 cells transfected with plasmid expressing PPARγ (Fig. 6 B and 6 C). These findings collectively suggest that miR-130 inhibited PPARγ expression, potentially through binding to PPARγ 3’-UTR in L6 and C2C12 cells, mechanistically influencing skeletal muscle protein synthesis. 4. Conclusions This study systematically investigated the protective effects and underlying mechanisms of dietary restriction (DR) and aerobic exercise training (ET) against high-fat diet (HFD)-induced obesity and associated skeletal muscle impairment through in vivo and in vitro models. Our results demonstrate that both DR and ET, as independent interventions, significantly improved the cross-sectional area and protein content of the soleus muscle, activated the IGF-1/Akt/mTOR signaling pathway, and suppressed miR-130 expression in skeletal muscle, thereby alleviating muscle atrophy and functional decline. Notably, although DR and ET each produced significant benefits, their combination (DR + ET) did not yield synergistic or additive effects, suggesting possible shared signaling pathways or cellular mechanisms leading to effect saturation. At the mechanistic level, this study provides the first evidence in skeletal muscle cells that miR-130 inhibits PPARγ expression by directly targeting its 3′-UTR, thereby impairing muscle cell proliferation and differentiation. Overexpression of miR-130 significantly suppressed proliferation and differentiation in L6 and C2C12 cells, accompanied by reduced expression of the myogenic marker myogenin and decreased creatine kinase activity. These effects were reversed by restoring PPARγ expression. In conclusion, DR and ET play important roles in ameliorating obesity-induced skeletal muscle injury by modulating the IGF-1/Akt/mTOR/miR-130/PPARγ axis. These findings provide new theoretical insights and potential therapeutic targets for managing obesity-related sarcopenia. Future studies should further elucidate the mechanisms underlying the lack of synergistic effects between DR and ET and explore optimized strategies for clinical application. Declarations Conflict of interest There are no conflicts of interest to declare. Funding: This work was supported by the Fundamental Research Funds for the Central Universities of Shaanxi Normal University (2019TS085 and TD2020043Y). Author Contribution **Qiyi Zhang:** Conceptualization, Data curation, Writing - Original Draft, Writing - Review & Editing, Software, Formal analysis, Visualization., **Tianqi Liu:** Investigation, Validation, Visualization, Writing - Original Draft., **Xiaoxiong He** : Conceptualization, Supervision, Funding acquisition, Writing - Original Draft, Writing - Review & Editing. Acknowledgement This work was supported by the Fundamental Research Funds for the Central Universities of Shaanxi Nor-mal University (2019TS085 and TD2020043Y). Data Availability The datasets used and analyzed during the current study available from the corresponding author on reasonable request. References Wang, Y., Zhao, L., Gao, L., Pan, A. & Xue, H. Health policy and public health implications of obesity in China. Lancet Diabetes Endocrinol. 9 , 446–461 (2021). Bristow, C., Meurer, C., Simmonds, J. & Snell, T. Anti-obesity public health messages and risk factors for disordered eating: a systematic review. Health Promot. Int. 35 , 1551–1569 (2020). Richter, E. A., Hargreaves, M. & Exercise GLUT4, and Skeletal Muscle Glucose Uptake. Physiol. Rev. 93 , 993–1017 (2013). Wu, H. & Ballantyne, C. M. Skeletal muscle inflammation and insulin resistance in obesity. J. Clin. Invest. 127 , 43–54 (2017). Cokorinos, E. C. et al. Activation of Skeletal Muscle AMPK Promotes Glucose Disposal and Glucose Lowering in Non-human Primates and Mice. Cell Metabol. 25 , 1147–1159e10 (2017). Akhmedov, D. & Berdeaux, R. The effects of obesity on skeletal muscle regeneration. Front. Physiol. 4 , 371–371 (2013). Beals, J. W., Burd, N. A., Moore, D. R. & van Vliet Obesity Alters the Muscle Protein Synthetic Response to Nutrition and Exercise. Front. Nutr. 6 , 87 (2019). Costa, R. R. S. et al. High fat diet induces central obesity, insulin resistance and microvascular dysfunction in hamsters. Microvasc. Res. 82 , 416–422 (2011). Colleluori, G. et al. Aerobic Plus Resistance Exercise in Obese Older Adults Improves Muscle Protein Synthesis and Preserves Myocellular Quality Despite Weight Loss. Cell Metabol. 30 , 261–273e6 (2019). Ahmad, S. S., Ahmad, K., Lee, E. J., Lee, Y. H. & Choi, I. Implications of Insulin-Like Growth Factor-1 in Skeletal Muscle and Various Diseases. Cells 9 , 1773 (2020). Christoffolete, M. A. et al. Muscle IGF-1-induced skeletal muscle hypertrophy evokes higher insulin sensitivity and carbohydrate use as preferential energy substrate. Biomed Res Int 282984–282984 (2015). (2015). Yoshida, T. & Delafontaine, P. Mechanisms of IGF-1-Mediated Regulation of Skeletal Muscle Hypertrophy and Atrophy. Cells 9, (2020). Rommel, C. et al. Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat. Cell Biol. 3 , 1009–1013 (2001). Ochi, E., Ishii, N. & Nakazato, K. Time Course Change of IGF1/Akt/mTOR/p70s6k Pathway Activation in Rat Gastrocnemius Muscle During Repeated Bouts of Eccentric Exercise. J. Sports Sci. Med. 9 , 170–175 (2010). Sullivan, B. P. et al. Skeletal muscle IGF-1 is lower at rest and after resistance exercise in humans with obesity. Eur. J. Appl. Physiol. 120 , 2835–2846 (2020). Hamarneh, S. R. et al. Relationship Between Serum IGF-1 and Skeletal Muscle IGF-1 mRNA Expression to Phosphocreatine Recovery After Exercise in Obese Men With Reduced GH. J. Clin. Endocrinol. Metabolism . 100 , 617–625 (2015). Deiuliis, J. A. MicroRNAs as regulators of metabolic disease: pathophysiologic significance and emerging role as biomarkers and therapeutics. Int. J. Obes. 40 , 88–101 (2016). Güller, I. & Russell, A. P. MicroRNAs in skeletal muscle: their role and regulation in development, disease and function. J. Physiol. 588 , 4075–4087 (2010). Horak, M., Novak, J. & Bienertova-Vasku, J. Muscle-specific microRNAs in skeletal muscle development. Dev. Biol. 410 , 1–13 (2016). Chu, X. et al. miR-130 aggravates acute myocardial infarction-induced myocardial injury by targeting PPAR-γ. J. Cell. Biochem. 119 , 7235–7244 (2018). Lee Eun et al. miR-130 Suppresses Adipogenesis by Inhibiting Peroxisome Proliferator-Activated Receptor γ Expression. Mol. Cell. Biol. 31 , 626–638 (2011). Yuan, Y., Peng, W., Liu, Y. & Xu, Z. Circulating miR-130 and its target PPAR-γ may be potential biomarkers in patients of coronary artery disease with type 2 diabetes mellitus. Mol. Genet. Genomic Med. 7 , e909–e909 (2019). Al-Rawaf, H. A. Circulating microRNAs and adipokines as markers of metabolic syndrome in adolescents with obesity. Clin. Nutr. 38 , 2231–2238 (2019). Wang, Y. et al. Circulating miR-130b mediates metabolic crosstalk between fat and muscle in overweight/obesity. Diabetologia 56 , 2275–2285 (2013). Hall, K. D. et al. Calorie for Calorie, Dietary Fat Restriction Results in More Body Fat Loss than Carbohydrate Restriction in People with Obesity. Cell Metabol. 22 , 427–436 (2015). Schoenfeld, B. J. Does Exercise-Induced Muscle Damage Play a Role in Skeletal Muscle Hypertrophy? The J. Strength. & Conditioning Research 26 , (2012). Pedersen, B. K. & Febbraio, M. A. Muscles, exercise and obesity: skeletal muscle as a secretory organ. Nat. Reviews Endocrinol. 8 , 457–465 (2012). Chen, H. T., Chung, Y. C., Chen, Y. J., Ho, S. Y. & Wu, H. J. Effects of Different Types of Exercise on Body Composition, Muscle Strength, and IGF-1 in the Elderly with Sarcopenic Obesity. J. Am. Geriatr. Soc. 65 , 827–832 (2017). López-Lluch, G. & Navas, P. Calorie restriction as an intervention in ageing. J. Physiol. 594 , 2043–2060 (2016). Sharples, A. P. et al. Longevity and skeletal muscle mass: the role of IGF signalling, the sirtuins, dietary restriction and protein intake. Aging Cell. 14 , 511–523 (2015). Massart, J. et al. Endurance exercise training-responsive miR-19b-3p improves skeletal muscle glucose metabolism. Nat. Commun. 12 , 5948–5948 (2021). Keller, P. et al. A transcriptional map of the impact of endurance exercise training on skeletal muscle phenotype. J. Appl. Physiol. (1985) . 110 , 46–59 (2011). Nielsen, S. et al. Muscle specific microRNAs are regulated by endurance exercise in human skeletal muscle. J. Physiol. 588 , 4029–4037 (2010). Liu, T. et al. Intermittent fasting, exercise, and dietary modification induce unique transcriptomic signatures of multiple tissues governing metabolic homeostasis during weight loss and rebound weight gain. J. Nutr. Biochem. 130 , 109649 (2024). Duan, Y. C. et al. Swimming Exercise Ameliorates Hypertension-Induced Kidney Dysfunction via Alleviating Renal Interstitial Fibrosis and Apoptosis. Kidney Blood Press. Res. 46 , 219–228 (2021). Ahmad, K., Choi, I. & Lee, Y. H. Implications of Skeletal Muscle Extracellular Matrix Remodeling in Metabolic Disorders: Diabetes Perspective. International J. Mol. Sciences 21 , (2020). Argilés, J. M., Busquets, S., Alvarez, B. & López-Soriano, F. J. Mechanism for the increased skeletal muscle protein degradation in the obese zucker rat. J. Nutr. Biochem. 10 , 244–248 (1999). Bassel-Duby, R. & Olson, E. N. Signaling Pathways in Skeletal Muscle Remodeling. Annu. Rev. Biochem. 75 , 19–37 (2006). Speakman, J. R. & Mitchell, S. E. Caloric restriction. Mol. Aspects Med. 32 , 159–221 (2011). Bartel, D. P. & MicroRNAs Genomics, Biogenesis, Mechanism, and Function. Cell 116 , 281–297 (2004). Chen, D., Hu, S., Wu, Z., Liu, J. & Li, S. The Role of MiR-132 in Regulating Neural Stem Cell Proliferation, Differentiation and Neuronal Maturation. Cell. Physiol. Biochem. 47 , 2319–2330 (2018). Pan, X., He, Y., Chen, Z., Yan, G. & Ma, G. Circulating miR-130 is a potential bio signature for early prognosis of acute myocardial infarction. Journal of Thoracic Disease; Vol 12, No 12 (December : Journal of Thoracic Disease (2020). https://jtd.amegroups.com/article/view/47461 (2020). Hernandez-Quiles, M., Broekema, M. F. & Kalkhoven, E. PPARgamma in metabolism, immunity, and cancer: unified and diverse mechanisms of action. Front. Endocrinol. 12 , 624112 (2021). Wang, Y. et al. Specific muscle targeted delivery of miR-130a loaded lipid nanoparticles: a novel approach to inhibit lipid accumulation in skeletal muscle and obesity. J. Nanobiotechnol. 23 , 159 (2025). Amin, R. H., Mathews, S. T., Camp, H. S., Ding, L. & Leff, T. Selective activation of PPARγ in skeletal muscle induces endogenous production of adiponectin and protects mice from diet-induced insulin resistance. Am. J. Physiology-Endocrinology Metabolism . 298 , E28–E37 (2010). Grygiel-Górniak, B. Peroxisome proliferator-activated receptors and their ligands: nutritional and clinical implications - a review. Nutr. J. 13 , 17 (2014). Xu, P., Zhai, Y. & Wang, J. The Role of PPAR and Its Cross-Talk with CAR and LXR in Obesity and Atherosclerosis. International J. Mol. Sciences 19 , (2018). Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable.xlsx Cite Share Download PDF Status: Published Journal Publication published 01 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 30 Dec, 2025 Reviews received at journal 23 Dec, 2025 Reviews received at journal 12 Dec, 2025 Reviewers agreed at journal 06 Dec, 2025 Reviewers agreed at journal 05 Dec, 2025 Reviewers agreed at journal 01 Dec, 2025 Reviewers invited by journal 01 Dec, 2025 Editor assigned by journal 01 Dec, 2025 Editor invited by journal 25 Nov, 2025 Submission checks completed at journal 22 Nov, 2025 First submitted to journal 22 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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08:17:04","extension":"xml","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":101312,"visible":true,"origin":"","legend":"","description":"","filename":"8fd04d0a2db74e0b93da85fd0f018aee1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8116495/v1/721ea9b9bf5d721c8fb79b7c.xml"},{"id":97323122,"identity":"b9e8607a-2250-42c5-8523-ca5aefda98e3","added_by":"auto","created_at":"2025-12-03 08:17:04","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":111557,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8116495/v1/c74552d35c6d0fbe401a2cab.html"},{"id":97323102,"identity":"4c8d9ba6-3078-4b50-8bc0-ea0776d1b9a8","added_by":"auto","created_at":"2025-12-03 08:17:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":722029,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of skeletal muscle fiber cross-sectional area and protein content. (A) Comparison of skeletal muscle fiber cross-sectional area (scale=100µm). (B) Comparison of protein content in soleus muscle. \u0026amp;p \u0026lt; 0.05 and \u0026amp;\u0026amp;p \u0026lt; 0.01, compared with the C group.#p \u0026lt; 0.05 and ##p \u0026lt; 0.01, compared with the O group. ※p \u0026lt; 0.05 and ※※p \u0026lt; 0.01, compared with the DR group. *p \u0026lt; 0.05 and **p \u0026lt; 0.01, compared with the ET group. Control group (C group, n=8), Obesity group (O group, n=8), Dietary Restriction group (DR group, n=8), Exercise Training group (ET group, n=8) and the group treated by DR and ET (DR+ET group, n=8).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8116495/v1/7fe651894b0e9456752c7121.png"},{"id":97370933,"identity":"8604c597-70d2-480b-94cd-9b77bf3dd861","added_by":"auto","created_at":"2025-12-03 16:28:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":210815,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of protein content of IGF-1/Akt/mTOR signal pathway and myogenin. (A) IGF-1 level and activation of Akt and mTOR. (B) Protein expression of myogenin. GAPDH was used as an internal control for western blot analysis. \u0026amp;p \u0026lt; 0.05 and \u0026amp;\u0026amp;p \u0026lt; 0.01, compared with the C group. #p \u0026lt; 0.05 and ##p \u0026lt; 0.01, compared with the O group. Control group (C group, n=3), Obesity group (O group, n=3), Dietary Restriction group (DR group, n=3), Exercise Training group (ET group, n=3) and the group treated by DR and ET (DR+ET group, n=3).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8116495/v1/5f809fd405ccc92ceb087f40.png"},{"id":97323105,"identity":"125d9ee8-c4d4-4fbd-bafd-b277af2d4136","added_by":"auto","created_at":"2025-12-03 08:17:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72706,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of expressions of miR-130 and PPARγ. (A) Expression of miR-130. (B) Expression of PPARγ mRNA. (C) Expression of PPARγ protein, GAPDH was used as an internal control for western blot analysis. \u0026amp;p \u0026lt; 0.05 and \u0026amp;\u0026amp;p \u0026lt; 0.01, compared with the C group. #p \u0026lt; 0.05 and ##p \u0026lt; 0.01, compared with the O group. Control group (C group, n=3), Obesity group (O group, n=3), Dietary Restriction group (DR group, n=3), Exercise Training group (ET group, n=3) and the group treated by DR and ET (DR+ET group, n=3).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8116495/v1/632e1bcc862e0494bc49b55b.png"},{"id":97323108,"identity":"48f93da7-f846-4f1a-b84f-e82dce074960","added_by":"auto","created_at":"2025-12-03 08:17:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":97876,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of miR-130 overexpression on proliferation, differentiation and expression of myogenin in L6 and C2C12 skeleton muscle cells. (A) miR-130 mimics or control miR were transfected into L6 cells or C2C12 cells, miR-130 level was detected by RT-qPCR. *p \u0026lt; 0.05 and **p \u0026lt; 0.01, compared with the Negative control. (B) Cell proliferation rate at 0h, 72h and 96h after transfection in L6 cells and C2C12 cells. (C) Creatine kinase activity at 0d, 3d and 6d after transfection in L6 cells and C2C12 cells. (D) Expression of myogenin at 0d, 3d and 6d after transfection in L6 cells and C2C12 cells. GAPDH was used as an internal control for western blot analysis. *p \u0026lt; 0.05 and **p \u0026lt; 0.01, compared with the 0 h or 0 d. #p \u0026lt; 0.05 and ##p \u0026lt; 0.01, compared with the Negative control. Control group (C group, n=3), Obesity group (O group, n=3), Dietary Restriction group (DR group, n=3), Exercise Training group (ET group, n=3) and the group treated by DR and ET (DR+ET group, n=3).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8116495/v1/172acf76cfd62e3284ba4bc8.png"},{"id":97370989,"identity":"73884486-2385-41d0-bd23-a2fc0cc5959c","added_by":"auto","created_at":"2025-12-03 16:28:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":32153,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of miR-130 on PPARγ expression in L6 and C2C12 cells. (A) The pRL reporter plasmids (pRL-PPARγ-3’UTR, pRL-mutated PPARγ 3’-UTR) and miR-130 mimics or control miR were co-transfected into L6 and C2C12 cells. Luciferase activities were measured after 48 h. MiR-130 mimics downregulated luciferase activity controlled by wild-type PPARγ-3′-UTR but did not affect luciferase activity driven by mutant PPARγ-3′-UTR. (B) PPARγ mRNA in L6 and C2C12 cells was measured by RT-qPCR at 48 h post-transfection. *p \u0026lt; 0.05 and **p \u0026lt; 0.01, compared with Negative control. Control group (C group, n=3), Obesity group (O group, n=3), Dietary Restriction group (DR group, n=3), Exercise Training group (ET group, n=3) and the group treated by DR and ET (DR+ET group, n=3).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8116495/v1/d6087bb15b8e485c40354155.png"},{"id":97369859,"identity":"a9fb5d90-0168-4a9d-9b2e-3c8f912b2d2c","added_by":"auto","created_at":"2025-12-03 16:25:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":83352,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of PPARγ overexpression on proliferation, differentiation and expression of myogenin in L6 and C2C12 skeleton muscle cells. (A) Cell proliferation rate at 0h, 72h and 96h after PPARγ overexpression in L6 cells and C2C12 cells. (B) Creatine kinase activity at 0d, 3d and 6d after PPARγ overexpression in L6 cells and C2C12 cells. (C) Expression of myogenin at 0d, 3d and 6d after PPARγ overexpression in L6 cells and C2C12 cells. GAPDH was used as an internal control for western blot analysis. *p \u0026lt; 0.05 and **p \u0026lt; 0.01, compared with the 0 h or 0 d. #p \u0026lt; 0.05 and ##p \u0026lt; 0.01, compared with the Negative control. Control group (C group, n=3), Obesity group (O group, n=3), Dietary Restriction group (DR group, n=3), Exercise Training group (ET group, n=3) and the group treated by DR and ET (DR+ET group, n=3).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8116495/v1/545b98f7a9a09beb5f180060.png"},{"id":106343345,"identity":"ecb79e80-84de-4c29-8102-82d02e3612d2","added_by":"auto","created_at":"2026-04-07 16:02:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1825141,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8116495/v1/a285c99b-8015-46dc-a81b-22fd8771d7fc.pdf"},{"id":97323104,"identity":"e3e67859-934c-41d1-946e-17ee23147abf","added_by":"auto","created_at":"2025-12-03 08:17:04","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16013,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8116495/v1/648a332fca523db83275164a.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dietary restriction and aerobic exercise alleviate obesity-related skeletal muscle impairment via the miR-130/PPARγ axis and IGF-1/Akt/mTOR signaling activation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eObesity emerges a worldwide health problem and has been associated with several chronic diseases, e.g. diabetes and certain cancers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Skeletal muscle comprises 30\u0026ndash;40% of body mass in humans. It is the predominant part for glucose disposal and fatty acid consumption and is an important determinant of human health and diseases[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Emerging data have revealed deleterious effects of diet-induced over weight on skeletal muscle fitness and plasticity[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Specifically, obesity may negatively influence muscle protein turnover, or the breaking down and rebuilding of functional proteins, with the myofibrillar proteins being particularly susceptible to anabolic resistance. Obesity-induced reduction in protein synthesis of skeletal muscle has shown to cause sarcopenia and skeletal muscle strength loss, thereafter impairing self-care ability and quality of life particularly in elderly[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Improving the health of skeletal muscle is an important component of obesity treatment and is imperative to improve public health.\u003c/p\u003e\u003cp\u003eInsulin-like growth factor-1 (IGF-1) is a key growth factor regulating both anabolic and catabolic pathways in skeletal muscle[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. IGF-1 increases skeletal muscle protein synthesis via multiple signaling pathways, including PI3K/Akt/mTOR activation[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. IGF-1 also potentiates skeletal muscle regeneration via activating skeletal muscle stem cells, which may contribute to muscle hypertrophy and/or inhibit atrophy. IGF-1 levels are often suppressed in many chronic disease conditions [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A recent study showed that increased serum IGF-1 was inversely associated with total body fat and trunk fat, while positively associated with lean mass in 15 abdominally obese men who were treated for 12 weeks with recombinant human GH[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Stimulating the IGF-1/Akt/mTOR pathway is thus critical for muscle mass maintenance, muscle hypertrophy, and muscle protein regulation, and ultimately for treating obesity.\u003c/p\u003e\u003cp\u003eApart from the suppressive effect on IGF signaling, obesity alters microRNAs expression in metabolically important organs, including skeletal muscles[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. MicroRNAs are 19\u0026ndash;22 nucleotides in length, and modulate gene expression on post-arrangements extent by targeting the 3' untranslated region (3'UTR), ultimately leading to reduced protein translation of miRNA targets. MicroRNAs regulate multiple pathways including adipogenesis, insulin signaling, immune-mediated inflammation and lipid metabolism, which play an important role in the pathophysiology of metabolic diseases [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Plasma miR-130 level was found to be lower in obese humans or in patients with coronary artery disease relative to lean people[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The muscle-specific miR-130 has shown to suppress adipogenesis by targeting of peroxisome proliferator-activated receptor gamma (PPARγ) in adipocytes in mouse models [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAccumulating evidence highlight the benefits of exercise and (or) dietary restriction (DR) on prevention and treatment of obesity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Skeletal muscle is a highly adaptable tissue and remodels in response to life-style modifications including exercise training and dietary factors. Both strategies have shown to effectively increase the expression of IGF-1 in skeletal muscle and stimulate skeletal muscle hypertrophy [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Moreover, aerobic exercise training has shown to remodel miRNA expression in skeletal muscle[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, little data exist regarding whether and how DR could impact skeletal muscle mass and protein synthesis. In our previous study, DR failed to effectively improve metabolic homeostasis and biological dysfunction in obese mice. However, by exploring the potential inter organ communication, the causal relationship between genes Gdf15, Tfrc, Cdv3, Map2k4 and Nqo1 and metabolic disorders induced by high-fat diet was determined[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Here, we explored effects of calorie restriction, aerobic exercise and their potential synergetic effect on skeletal muscle metabolism in high-fat diet induced obese mouse models, with specific focus on skeletal muscle protein synthesis, IGF-1/Akt/mTOR signal pathway and miR130 expression. Moreover, L6 and C2C12 skeletal muscle cells were transfected with miR-130 mimics and expressions of miR-130 and PPARγ, as well as activity of creatine kinase and expression of muscle-specific protein, i.e. myogenin, were measured to elucidate the role of miR-130 in the function of skeletal muscle cells.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Animals and study design\u003c/h2\u003e\u003cp\u003eFourty 3-month old healthy male Sprague Dawley rats were purchased from Experimental Animal Center of Xi'an Jiaotong University (Xi\u0026rsquo;an, China). The rats were fed with normal chow or with the high fat diet. The ingredients of normal chow were 40% corn flour, 26% wheat flour, 10% bran, 10% fish meal, 10% bean cake, 2% mineral, 1% coarse grains, and 1% vitamin. The high fat diet contained 60% fat, 20% carbohydrates and 20% protein. All animals were maintained under controlled environmental conditions (room temperature and 40%-50% relative humidity). Rats were housed in cages and had free access to food and tap water. After 12 weeks high-fat diet, obese rats were determined according to Lee\u0026rsquo;s index [body weight (g)1/3 \u0026times;1000/body length(cm)]. Obese rats were then divided into four groups: obesity group (O group, n\u0026thinsp;=\u0026thinsp;8), dietary restriction group (DR group, n\u0026thinsp;=\u0026thinsp;8), exercise training group (ET group, n\u0026thinsp;=\u0026thinsp;8) and the group treated by DR and ET (DR\u0026thinsp;+\u0026thinsp;ET group, n\u0026thinsp;=\u0026thinsp;8). Rats in obesity group were continually fed with high-fat diet for additional 5 weeks. In the DR and DR\u0026thinsp;+\u0026thinsp;ET group, rats were given 60% food intake relative to the O group. In the ET and DR\u0026thinsp;+\u0026thinsp;ET group, rats were subjected to swimming training for 5 weeks. Sedentary rats were handled in a similar manner to the exercised rats, but were not exercised. Swimming training is conducted according to the established plan[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] (80% maximum intensity).The training was carried out between 09:00 am and 12.00 am. The training program consisted in swimming 6 days/week, 50 min/day, during 5 weeks. Rats in exercise groups were adapted to swimming training f4`or 5 days before starting the protocol to reduce stress in the animals. The water temperature was maintained at 31\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. Body weight of animals was recorded every week. After deep anesthesia with isoflurane (maintained at 2\u0026ndash;3% in oxygen), the rats were sacrificed by cervical dislocation, a method that ensures rapid and painless death, consistent with the ethical guidelines for animal experimentation approved by our Institutional Animal Care and Use Committee (IACUC). All animal experiments were conducted in accordance with the ARRIVE guidelines (PLoS Biol. 8 (6), e1000412, 2010) and approved by the (Institutional Animal Care and Use Committee of Shaanxi Normal University) under approval number No. 202216012. All efforts were made to minimize animal suffering and adhere to veterinary best practices.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Skeletal Muscle processing and measurement of proteins\u003c/h2\u003e\u003cp\u003eRats were fasted but allowed free access to water, and their 12 h urine and feces were collected. All animals were anesthetized by of isoflurane inhalation, and then were sacrificed to obtain soleus muscle. Muscle samples were fixed by 10% neutral formaldehyde solution for histological studies or homogenized for total protein content measurement. Muscle samples from the left foot were used for measurement of protein levels and were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis. Total protein was extracted after homogenization in RIPA buffer (pH 7.2\u0026ndash;7.4) and centrifuged for 20 min at 1000g at 4\u0026deg;C. The main components of RIPA cracking solution are 50 mM Tris (pH 7.4), 150mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, And various inhibitors such as sodium orthovanadate, sodium fluoride, EDTA, leupeptin, etc.The resulting supernatants were removed and target proteins extracted. BCA method was used for protein quantification.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Histological Analysis and Morphometry\u003c/h2\u003e\u003cp\u003eThe soleus muscle tissue of rats were fixed with 4% paraformaldehyde, and were performed for histopathological analysis. The fixed tissues were embedded in paraffin, sectioned (5\u0026ndash;6 \u0026micro;m) and stained with H\u0026amp;E. These slides were analyzed under the Olympus light microscope for observations and photograph. We systematically selected five visual fields per tissue section, with four sections analyzed for each biological sample and three samples per experimental group. The CSA of soleus muscle fibers in each visual field was quantitatively analyzed using Image-Pro Plus 7.0.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Western Blotting\u003c/h2\u003e\u003cp\u003eThe muscle sample (50 mg) was added into pre-cooled protein extraction reagents (Beyotime Biotechnology, Haimen, China) and was homogenized with electric homogenizer (Thermo Fisher Scientific, Waltham, MA, USA). The samples were then centrifuged at 12000 rpm for 15 minutes at 4\u0026deg;C. The proteins were separated by SDS-PAGE in Tris-glycine buffer and transferred under constant current. After transferring, the PVDF membrane (Millipore, Burlington, MA, USA) was blocked by 3% bovine serum albumin (BSA) (Beyotime Biotechnology, Haimen, China) at room temperature. Primary antibodies were incubated at 4\u0026deg;C overnight with different dilutions: IGF-1 (1:500, Beyotime Biotechnology, Haimen, China), GAPDH (1:10000), AKT, p-Akt, mTOR, p-mTOR (1:1000, Cell Signaling, Danvers, MA, USA), myogenin (1:500) and PPARγ (1:500, Beyotime Biotechnology, Haimen, China). The membrane was then washed with TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20). Secondary antibody of goat anti-rabbit HRP (Thermo Fisher Scientific, Waltham, MA, USA) was incubated for 1.5 hours at room temperature. After washing with TBST, membranes were developed with ECL (Beyotime Biotechnology, Haimen, China) according to the manufacturer\u0026rsquo;s instructions. For western blot with protein extracts from L6 and C2C12 cells, primary antibodies against PPARγ and myogenin were used. Universal Blocking and Antibody Dilution Buffer were purchased from Beyotime (Shanghai, China). The results of Western blotting were analyzed using Image Processing and Analysis in Java (ImageJ, v. 1.48, National Institutes of Health, Bethesda).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Cell Culture\u003c/h2\u003e\u003cp\u003eThe L6 and C2C12 cells were purchased from ATCC (Manassas, VA, USA). L6 cells were cultured in α-minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, St Louis, MO, USA) at 37\u0026deg;C and 5% CO2. On reaching 90% confluence, L6 cells were induced to differentiate by reducing the serum concentration from 10% to 2% in the culture medium. C2C12 cells were maintained in DMEM-F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) with 10% FBS (Sigma Aldrich, St Louis, MO, USA). Serum concentration was decreased from 10% to 2% to initiate myogenic differentiation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. RNA Extraction and Real Time qPCR\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from soleus muscle samples, L6 and C2C12 cells, respectively by using the miRNA isolation kit (Sigma Aldrich, St Louis, MO, USA) according to the manufacturer\u0026rsquo;s protocol. The level of miR-130 was assessed by real time qRT-PCR by using a TaqMan\u0026reg; MicroRNA Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The miR-130a primers were purchased from Sigma and U6 small nuclear RNA (Sigma) served as internal control. The miR-130a mimic primer species was from rats, and the specific sequence was GCUCUUUUCACAUUGUGCUACU. To determine the expression of PPARγ mRNA in the muscle tissues and cell lines, total RNA was extracted by using RNeasy kit (Qiagen, Gaithersburg, MD, USA), and cDNA was synthesized from RNA using the reverse transcription system (Promega, USA). GAPDH was used as internal control for qPCR. The primers used in qRT-PCR are as follows: forward 5\u0026rsquo;-TGATATCGACCAGCTGAACC-3\u0026rsquo; and reverse 5\u0026rsquo;-GTCCTCCAGCTGTTCGCCA-3\u0026rsquo; for PPARγ (rat); forward 5\u0026rsquo;-TCGCTGATGCACTGCCTATG-3\u0026rsquo; and reverse 5\u0026rsquo;-GAGAGGTCCACAGAGCTGATT-3\u0026rsquo; for PPARγ; forward 5\u0026rsquo;-AATGCATCCTGCACCACCAA-3\u0026rsquo; and reverse 5\u0026rsquo;-GATGCCATATTCATTGTCATA-3\u0026rsquo; for GAPDH; forward 5\u0026rsquo;-AGGTCGGTGTGAACGGATTTG-3\u0026rsquo; and reverse 5\u0026rsquo;-TGTAGACCATGTAGTTGAGGTCA-3\u0026rsquo; for GAPDH.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Transfection\u003c/h2\u003e\u003cp\u003eThe miR-130 mimics (50 nM) and the negative control (ABM, BC, Canada) were transfected into L6 and C2C12 cells with Lipofectamine\u0026reg; RNAiMAX transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The pCDNA3-1-PPARγ plasmid was transfected into L6 or C2C12 cells with Lipofectamine 2000 transfection reagent. Plasmid extraction was performed using the Shanghai Sangong 48-Channel Prefilled Plate Mag-MK Plasmid Extraction Kit (B614801).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Proliferation Assay\u003c/h2\u003e\u003cp\u003eCell proliferation was assessed using a CCK8 cell counting kit (Sigma, St Louis, MO, USA), according to the manufacturer\u0026rsquo;s instructions. Briefly, 24 hours after transfection, L6 and C2C12 cells transfected with miR130 mimics or PPARγ overexpressing plasmids were seeded at a density of 1\u0026times;104 cells/well in Corning 96-well culture plate (Sigma Aldrich, St Louis, MO, USA). The medium was removed at 72 or 96 hours later, and the cells were washed with PBS twice followed by addition of 10 \u0026micro;l of tetrazolium substrate. The optical density (OD) at 450 nm was measured using a BioTek ELISA microplate reader (BioTek, Winooski, VT, USA) at 37\u0026deg;C for an additional hour.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. The PPARγ 3'UTR Reporter Activity Assay\u003c/h2\u003e\u003cp\u003eThe PPARγ 3'UTR reporter plasmids (pRL-PPARγ) were kept in our lab. Mutation in the miR-130 seed regions of the PPARγ 3'UTR was generated using a QuikChange lightning site directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). The wild type or mutated vector were co-transfected with miR-130 mimics into L6 and C2C12 cells by using Lipofectamine\u0026reg; RNAiMAX or lipfectamine 2000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). Reporter gene assays were conducted 72 h post-transfection by using the Dual luciferase Reporter assay system (Promega, Fitchburg, WI, USA) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll experiments were performed in triplicate and the continuous variables were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (standard deviation). Data were analyzed using one-way analysis of variance (ANOVA) and followed by a post-hoc Turkey test in SPSS 26.0. For all analysis, the p-value of 0.05 or less was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussions","content":"\u003cp\u003eAfter 8 weeks of high-fat diet, both the body weight (high-fat groups: 398.7\u0026thinsp;\u0026plusmn;\u0026thinsp;18.5 g v.s. C group: 338.2\u0026thinsp;\u0026plusmn;\u0026thinsp;10.3 g) and Lee's index (high-fat groups: 30.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 vs. C group: 29.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2) of high-fat diet-fed rats (O, DR, ET, ET\u0026thinsp;+\u0026thinsp;DR) were significantly higher than those of the normal chow control group (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Following 5 weeks of intervention, the O group maintained elevated weight and index (483.6\u0026thinsp;\u0026plusmn;\u0026thinsp;16.7 g, 31.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4), while the DR, ET, and ET\u0026thinsp;+\u0026thinsp;DR groups showed significant reductions in both parameters (weight: 23.3%, 16.0%, 30.2% decrease; Lee's index: 3.9%, 1.3%, 6.9% decrease, all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 v.s. Week 8), with the ET\u0026thinsp;+\u0026thinsp;DR group exhibiting the most pronounced effects (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. DR and ET groups) (Supplementary Table\u0026nbsp;1\u0026ndash;2). The obesity epidemic continues its relentless advance and is often accompanied with skeletal muscle remodeling that presented with impairments of muscle structure and function, deleteriously affecting metabolic homeostasis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In line with previous studies, compared with the rats fed with normal chow, we found that the cross-sectional area of soleus muscle fiber was significantly lower in high-fat diet induced obese rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), which was whereas increased in ET and DR\u0026thinsp;+\u0026thinsp;ET groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Rats in ET and DR\u0026thinsp;+\u0026thinsp;ET group had higher skeletal muscle protein contents than obese rats (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Yet no effect was seen for dietary restriction. Our data support benefical effects of exercise on skeletal muscle structure morphology and increased muscle mass.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSeveral mechanisms have been proposed to be involved in obesity-induced skeletal muscle remodeling, like inflammatory cytokines, endoplasmic reticulum stress, and autophagy of skeletal muscle cell[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. IGF-1 is an important modulator of skeletal muscle growth and could functionally promote muscle protein synthesis via stimulating muscle cell proliferation and differentiation. IGF-1/PI3K/AKT/mTOR signaling pathway is one of major mechanisms linking obesity with impaired muscle regeneration, which critically involves in regulating protein synthesis in skeletal muscle. In obese mice and Zucker rats, muscle growth in response to mechanical loading was reduced due to decreased activation of Akt, p70S6 kinase and mTOR[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Obesity also led to increased levels of pro-inflammatory cytokines, e.g.TNF-α, IL-1β and IL-6, which could inhibit the activation of IGF-1/AKT/mTOR signals, and subsequently impair normal muscle physiological maintenance, cell growth and regeneration function[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In the present study, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, feeding high-fat diet for 12 weeks led to a sharp decrease in the IGF-1 level in skeletal muscles compared with rats fed with normal chow (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). DR, ET or DR\u0026thinsp;+\u0026thinsp;ET effectively inhibited the obesity caused reduction in skeletal muscles IGF-1, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), while increased Akt phosphorylation compared with sedentary rats fed with high-fat diet. Opposite regulating effects were observed for DR, ET or DR\u0026thinsp;+\u0026thinsp;ET, respectively, on the level of mTOR phosphorylation in obese rats. We also examined levels of myogenin, a downstream target of IGF-1/Akt/mTOR. We found that DR, ET or DR\u0026thinsp;+\u0026thinsp;ET reduced high-fat diet induced increase in myogenin in skeletal muscle, resulting in the level of myogenin comparable to that of normal chow -fed rats (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In agreement with our findings, Ochi et al. reported that exercise activate the IGF1/Akt/mTOR signaling pathway to promote muscle hypertrophy[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Calorie restriction has also shown to inhibit energy-dependent pathways including IGF-1/PI3K/Akt/mTOR and to activate AMP-dependent protein kinase activity in cancer cells [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Notably, although both dietary restriction and exercise per se has exhibited promoting effects on activation of IGF1/Akt/mTOR signaling, no significant improvement was observed for DR\u0026thinsp;+\u0026thinsp;EX. Skeletal muscle is one of the main end organs of insulin resistance, and its damage is manifested as mitochondrial dysfunction. However, the combined intervention program including endurance training and dietary restriction effectively restored mitochondrial function, which showed the reduction of mitochondrial reactive oxygen species (ROS), the stability of membrane potential and the reduction of mitochondrial swelling. In this study, the reason why DR\u0026thinsp;+\u0026thinsp;ET did not improve significantly may be due to the low intensity of swimming and the short duration of intervention, which failed to effectively restore mitochondrial function.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMicroRNAs critically involved in the regulation of gene expression programs, emerging role as biomarkers and therapeutics of metabolic diseases[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and have been shown to play an important role in cell proliferation and differentiation[\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The miR-130a is highly expressed in human endothelial cells and has shown to strongly affect adipocyte differentiation that is linked to human obesity, as overexpressing miR-130 impaired adipogenesis and reducing miR-130 enhanced adipogenesis[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The miR-130 also aggravated acute myocardial infarction-induced myocardial injury in H9c2 cells by targeting PPARγ [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. More and more evidences show that PPAR γ was also important for the maturation and function of various immune system related cell types, such as monocytes/macrophages, dendritic cells and lymphocytes. In addition, PPAR γ controls cell proliferation in other tissues and organs (including colon, breast, prostate and bladder), and the disorder of PPAR γ signal transduction is related to tumor development in these organs[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Yuan et al., recently identified circulating miR-130 (miR-130a and miR-130b) and its target PPARγ as potential biomarkers in patients of coronary artery disease with type 2 diabetes mellitus[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Yet little evidence regarding the functional role of miR-130 in skeletal muscle exists[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. We found that the miR-130 level in skeletal muscle was upregulated in obese rats than in rats fed with normal chow, which was significantly abrogated by DR, ET or DR\u0026thinsp;+\u0026thinsp;ET, with DR\u0026thinsp;+\u0026thinsp;ET exhibiting the most pronounced inhibitory ability. Similarly, high-fat diet suppressed levels of PPARγ, a target of miR-130 in adipocytes, obese rats were beneficially reversed by DR, ET or DR\u0026thinsp;+\u0026thinsp;ET, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). To further elucidate the role of miR-130 in the function of skeletal muscle cells, L6 and C2C12 skeletal muscle cells were transfected with miR-130 mimics. The miR-130 levels were significantly promoted by transfection of the mimics (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and dramatically reduced proliferation rate in both L6 and C2C12 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Besides, increased activity of creatine kinase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) and expression of muscle-specific protein, i.e. myogenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), are main characteristics of muscle differentiation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePPARγ expressed in skeletal muscle cells can protect against high-fat diet induced insulin resistance and muscle dysfunction [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In this study, we found that transfection of miR-130 mimics significantly inhibited luciferase activity of pRL-PPARγ-3\u0026rsquo;-UTR, but failed to alter the activity of the mutated version in both L6 and C2C12 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The inhibitory effect of miR-130 on PPARγ expression was also indicated by the reduction of PPARγ mRNA expression in L6 and C2C12 cells transfected with miR-130 mimics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We then sought to investigate the role of PPARγ in proliferation and differentiation of skeletal muscle cells. Specifically, the proliferation rates of L6 and C2C12 cells were dramatically increased in cells with forced expression of PPARγ. Moreover, the improving effect of PPARγ on myogenic differentiation was evidenced by elevated creatine kinases activity and upregulated myogenin expression in L6 and C2C12 cells transfected with plasmid expressing PPARγ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). These findings collectively suggest that miR-130 inhibited PPARγ expression, potentially through binding to PPARγ 3\u0026rsquo;-UTR in L6 and C2C12 cells, mechanistically influencing skeletal muscle protein synthesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study systematically investigated the protective effects and underlying mechanisms of dietary restriction (DR) and aerobic exercise training (ET) against high-fat diet (HFD)-induced obesity and associated skeletal muscle impairment through in vivo and in vitro models. Our results demonstrate that both DR and ET, as independent interventions, significantly improved the cross-sectional area and protein content of the soleus muscle, activated the IGF-1/Akt/mTOR signaling pathway, and suppressed miR-130 expression in skeletal muscle, thereby alleviating muscle atrophy and functional decline. Notably, although DR and ET each produced significant benefits, their combination (DR\u0026thinsp;+\u0026thinsp;ET) did not yield synergistic or additive effects, suggesting possible shared signaling pathways or cellular mechanisms leading to effect saturation.\u003c/p\u003e\u003cp\u003eAt the mechanistic level, this study provides the first evidence in skeletal muscle cells that miR-130 inhibits PPARγ expression by directly targeting its 3\u0026prime;-UTR, thereby impairing muscle cell proliferation and differentiation. Overexpression of miR-130 significantly suppressed proliferation and differentiation in L6 and C2C12 cells, accompanied by reduced expression of the myogenic marker myogenin and decreased creatine kinase activity. These effects were reversed by restoring PPARγ expression.\u003c/p\u003e\u003cp\u003eIn conclusion, DR and ET play important roles in ameliorating obesity-induced skeletal muscle injury by modulating the IGF-1/Akt/mTOR/miR-130/PPARγ axis. These findings provide new theoretical insights and potential therapeutic targets for managing obesity-related sarcopenia. Future studies should further elucidate the mechanisms underlying the lack of synergistic effects between DR and ET and explore optimized strategies for clinical application.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eThere are no conflicts of interest to declare.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis work was supported by the Fundamental Research Funds for the Central Universities of Shaanxi Normal University (2019TS085 and TD2020043Y).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e**Qiyi Zhang:** Conceptualization, Data curation, Writing - Original Draft, Writing - Review \u0026amp;amp; Editing, Software, Formal analysis, Visualization., **Tianqi Liu:** Investigation, Validation, Visualization, Writing - Original Draft., **Xiaoxiong He** : Conceptualization, Supervision, Funding acquisition, Writing - Original Draft, Writing - Review \u0026amp;amp; Editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work was supported by the Fundamental Research Funds for the Central Universities of Shaanxi Nor-mal University (2019TS085 and TD2020043Y).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang, Y., Zhao, L., Gao, L., Pan, A. \u0026amp; Xue, H. Health policy and public health implications of obesity in China. \u003cem\u003eLancet Diabetes Endocrinol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 446\u0026ndash;461 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBristow, C., Meurer, C., Simmonds, J. \u0026amp; Snell, T. Anti-obesity public health messages and risk factors for disordered eating: a systematic review. \u003cem\u003eHealth Promot. Int.\u003c/em\u003e \u003cb\u003e35\u003c/b\u003e, 1551\u0026ndash;1569 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRichter, E. A., Hargreaves, M. \u0026amp; Exercise GLUT4, and Skeletal Muscle Glucose Uptake. \u003cem\u003ePhysiol. Rev.\u003c/em\u003e \u003cb\u003e93\u003c/b\u003e, 993\u0026ndash;1017 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWu, H. \u0026amp; Ballantyne, C. M. Skeletal muscle inflammation and insulin resistance in obesity. \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e \u003cb\u003e127\u003c/b\u003e, 43\u0026ndash;54 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCokorinos, E. C. et al. Activation of Skeletal Muscle AMPK Promotes Glucose Disposal and Glucose Lowering in Non-human Primates and Mice. \u003cem\u003eCell Metabol.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 1147\u0026ndash;1159e10 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAkhmedov, D. \u0026amp; Berdeaux, R. The effects of obesity on skeletal muscle regeneration. \u003cem\u003eFront. Physiol.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 371\u0026ndash;371 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBeals, J. W., Burd, N. A., Moore, D. R. \u0026amp; van Vliet Obesity Alters the Muscle Protein Synthetic Response to Nutrition and Exercise. \u003cem\u003eFront. Nutr.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 87 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCosta, R. R. S. et al. High fat diet induces central obesity, insulin resistance and microvascular dysfunction in hamsters. \u003cem\u003eMicrovasc. Res.\u003c/em\u003e \u003cb\u003e82\u003c/b\u003e, 416\u0026ndash;422 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eColleluori, G. et al. Aerobic Plus Resistance Exercise in Obese Older Adults Improves Muscle Protein Synthesis and Preserves Myocellular Quality Despite Weight Loss. \u003cem\u003eCell Metabol.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 261\u0026ndash;273e6 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhmad, S. S., Ahmad, K., Lee, E. J., Lee, Y. H. \u0026amp; Choi, I. Implications of Insulin-Like Growth Factor-1 in Skeletal Muscle and Various Diseases. \u003cem\u003eCells\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 1773 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChristoffolete, M. A. et al. Muscle IGF-1-induced skeletal muscle hypertrophy evokes higher insulin sensitivity and carbohydrate use as preferential energy substrate. \u003cem\u003eBiomed Res Int\u003c/em\u003e 282984\u0026ndash;282984 (2015). (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoshida, T. \u0026amp; Delafontaine, P. Mechanisms of IGF-1-Mediated Regulation of Skeletal Muscle Hypertrophy and Atrophy. \u003cem\u003eCells\u003c/em\u003e 9, (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRommel, C. et al. Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. \u003cem\u003eNat. Cell Biol.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e, 1009\u0026ndash;1013 (2001).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOchi, E., Ishii, N. \u0026amp; Nakazato, K. Time Course Change of IGF1/Akt/mTOR/p70s6k Pathway Activation in Rat Gastrocnemius Muscle During Repeated Bouts of Eccentric Exercise. \u003cem\u003eJ. Sports Sci. Med.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 170\u0026ndash;175 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSullivan, B. P. et al. Skeletal muscle IGF-1 is lower at rest and after resistance exercise in humans with obesity. \u003cem\u003eEur. J. Appl. Physiol.\u003c/em\u003e \u003cb\u003e120\u003c/b\u003e, 2835\u0026ndash;2846 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHamarneh, S. R. et al. Relationship Between Serum IGF-1 and Skeletal Muscle IGF-1 mRNA Expression to Phosphocreatine Recovery After Exercise in Obese Men With Reduced GH. \u003cem\u003eJ. Clin. Endocrinol. Metabolism\u003c/em\u003e. \u003cb\u003e100\u003c/b\u003e, 617\u0026ndash;625 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeiuliis, J. A. MicroRNAs as regulators of metabolic disease: pathophysiologic significance and emerging role as biomarkers and therapeutics. \u003cem\u003eInt. J. Obes.\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e, 88\u0026ndash;101 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eG\u0026uuml;ller, I. \u0026amp; Russell, A. P. MicroRNAs in skeletal muscle: their role and regulation in development, disease and function. \u003cem\u003eJ. Physiol.\u003c/em\u003e \u003cb\u003e588\u003c/b\u003e, 4075\u0026ndash;4087 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHorak, M., Novak, J. \u0026amp; Bienertova-Vasku, J. Muscle-specific microRNAs in skeletal muscle development. \u003cem\u003eDev. Biol.\u003c/em\u003e \u003cb\u003e410\u003c/b\u003e, 1\u0026ndash;13 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChu, X. et al. miR-130 aggravates acute myocardial infarction-induced myocardial injury by targeting PPAR-γ. \u003cem\u003eJ. Cell. Biochem.\u003c/em\u003e \u003cb\u003e119\u003c/b\u003e, 7235\u0026ndash;7244 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee Eun et al. miR-130 Suppresses Adipogenesis by Inhibiting Peroxisome Proliferator-Activated Receptor γ Expression. \u003cem\u003eMol. Cell. Biol.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 626\u0026ndash;638 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYuan, Y., Peng, W., Liu, Y. \u0026amp; Xu, Z. Circulating miR-130 and its target PPAR-γ may be potential biomarkers in patients of coronary artery disease with type 2 diabetes mellitus. \u003cem\u003eMol. Genet. Genomic Med.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, e909\u0026ndash;e909 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAl-Rawaf, H. A. Circulating microRNAs and adipokines as markers of metabolic syndrome in adolescents with obesity. \u003cem\u003eClin. Nutr.\u003c/em\u003e \u003cb\u003e38\u003c/b\u003e, 2231\u0026ndash;2238 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, Y. et al. Circulating miR-130b mediates metabolic crosstalk between fat and muscle in overweight/obesity. \u003cem\u003eDiabetologia\u003c/em\u003e \u003cb\u003e56\u003c/b\u003e, 2275\u0026ndash;2285 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHall, K. D. et al. Calorie for Calorie, Dietary Fat Restriction Results in More Body Fat Loss than Carbohydrate Restriction in People with Obesity. \u003cem\u003eCell Metabol.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, 427\u0026ndash;436 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchoenfeld, B. J. Does Exercise-Induced Muscle Damage Play a Role in Skeletal Muscle Hypertrophy? \u003cem\u003eThe J. Strength. \u0026amp; Conditioning Research\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePedersen, B. K. \u0026amp; Febbraio, M. A. Muscles, exercise and obesity: skeletal muscle as a secretory organ. \u003cem\u003eNat. Reviews Endocrinol.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 457\u0026ndash;465 (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen, H. T., Chung, Y. C., Chen, Y. J., Ho, S. Y. \u0026amp; Wu, H. J. Effects of Different Types of Exercise on Body Composition, Muscle Strength, and IGF-1 in the Elderly with Sarcopenic Obesity. \u003cem\u003eJ. Am. Geriatr. Soc.\u003c/em\u003e \u003cb\u003e65\u003c/b\u003e, 827\u0026ndash;832 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eL\u0026oacute;pez-Lluch, G. \u0026amp; Navas, P. Calorie restriction as an intervention in ageing. \u003cem\u003eJ. Physiol.\u003c/em\u003e \u003cb\u003e594\u003c/b\u003e, 2043\u0026ndash;2060 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSharples, A. P. et al. Longevity and skeletal muscle mass: the role of IGF signalling, the sirtuins, dietary restriction and protein intake. \u003cem\u003eAging Cell.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 511\u0026ndash;523 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMassart, J. et al. Endurance exercise training-responsive miR-19b-3p improves skeletal muscle glucose metabolism. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 5948\u0026ndash;5948 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKeller, P. et al. A transcriptional map of the impact of endurance exercise training on skeletal muscle phenotype. \u003cem\u003eJ. Appl. Physiol. (1985)\u003c/em\u003e. \u003cb\u003e110\u003c/b\u003e, 46\u0026ndash;59 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNielsen, S. et al. Muscle specific microRNAs are regulated by endurance exercise in human skeletal muscle. \u003cem\u003eJ. Physiol.\u003c/em\u003e \u003cb\u003e588\u003c/b\u003e, 4029\u0026ndash;4037 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu, T. et al. Intermittent fasting, exercise, and dietary modification induce unique transcriptomic signatures of multiple tissues governing metabolic homeostasis during weight loss and rebound weight gain. \u003cem\u003eJ. Nutr. Biochem.\u003c/em\u003e \u003cb\u003e130\u003c/b\u003e, 109649 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDuan, Y. C. et al. Swimming Exercise Ameliorates Hypertension-Induced Kidney Dysfunction via Alleviating Renal Interstitial Fibrosis and Apoptosis. \u003cem\u003eKidney Blood Press. Res.\u003c/em\u003e \u003cb\u003e46\u003c/b\u003e, 219\u0026ndash;228 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhmad, K., Choi, I. \u0026amp; Lee, Y. H. Implications of Skeletal Muscle Extracellular Matrix Remodeling in Metabolic Disorders: Diabetes Perspective. \u003cem\u003eInternational J. Mol. Sciences\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArgil\u0026eacute;s, J. M., Busquets, S., Alvarez, B. \u0026amp; L\u0026oacute;pez-Soriano, F. J. Mechanism for the increased skeletal muscle protein degradation in the obese zucker rat. \u003cem\u003eJ. Nutr. Biochem.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 244\u0026ndash;248 (1999).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBassel-Duby, R. \u0026amp; Olson, E. N. Signaling Pathways in Skeletal Muscle Remodeling. \u003cem\u003eAnnu. Rev. Biochem.\u003c/em\u003e \u003cb\u003e75\u003c/b\u003e, 19\u0026ndash;37 (2006).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSpeakman, J. R. \u0026amp; Mitchell, S. E. Caloric restriction. \u003cem\u003eMol. Aspects Med.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e, 159\u0026ndash;221 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBartel, D. P. \u0026amp; MicroRNAs Genomics, Biogenesis, Mechanism, and Function. \u003cem\u003eCell\u003c/em\u003e \u003cb\u003e116\u003c/b\u003e, 281\u0026ndash;297 (2004).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen, D., Hu, S., Wu, Z., Liu, J. \u0026amp; Li, S. The Role of MiR-132 in Regulating Neural Stem Cell Proliferation, Differentiation and Neuronal Maturation. \u003cem\u003eCell. Physiol. Biochem.\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e, 2319\u0026ndash;2330 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePan, X., He, Y., Chen, Z., Yan, G. \u0026amp; Ma, G. Circulating miR-130 is a potential bio signature for early prognosis of acute myocardial infarction. \u003cem\u003eJournal of Thoracic Disease; Vol 12, No 12 (December\u003c/em\u003e : Journal of Thoracic Disease (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jtd.amegroups.com/article/view/47461\u003c/span\u003e\u003cspan address=\"https://jtd.amegroups.com/article/view/47461\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHernandez-Quiles, M., Broekema, M. F. \u0026amp; Kalkhoven, E. PPARgamma in metabolism, immunity, and cancer: unified and diverse mechanisms of action. \u003cem\u003eFront. Endocrinol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 624112 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang, Y. et al. Specific muscle targeted delivery of miR-130a loaded lipid nanoparticles: a novel approach to inhibit lipid accumulation in skeletal muscle and obesity. \u003cem\u003eJ. Nanobiotechnol.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 159 (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAmin, R. H., Mathews, S. T., Camp, H. S., Ding, L. \u0026amp; Leff, T. Selective activation of PPARγ in skeletal muscle induces endogenous production of adiponectin and protects mice from diet-induced insulin resistance. \u003cem\u003eAm. J. Physiology-Endocrinology Metabolism\u003c/em\u003e. \u003cb\u003e298\u003c/b\u003e, E28\u0026ndash;E37 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGrygiel-G\u0026oacute;rniak, B. Peroxisome proliferator-activated receptors and their ligands: nutritional and clinical implications - a review. \u003cem\u003eNutr. J.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 17 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu, P., Zhai, Y. \u0026amp; Wang, J. The Role of PPAR and Its Cross-Talk with CAR and LXR in Obesity and Atherosclerosis. \u003cem\u003eInternational J. Mol. Sciences\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Obesity, Dietary restriction, Aerobic exercise, Skeletal muscle","lastPublishedDoi":"10.21203/rs.3.rs-8116495/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8116495/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the effects of dietary restriction (DR) combined with aerobic exercise training (ET) on high-fat diet (HFD)-induced obesity and associated skeletal muscle impairment in male Sprague-Dawley rats, as well as the underlying mechanisms. An 8-week DR\u0026thinsp;+\u0026thinsp;ET intervention effectively increased the cross-sectional area and protein content of the soleus muscle. Both DR and ET alone promoted the activation of the IGF-1/Akt/mTOR signaling pathway and reduced the protein expression of peroxisome proliferator-activated receptor γ (PPARγ), a target of miR-130 in adipocytes, in HFD-induced obese rats. However, no additional benefit was observed with combined DR\u0026thinsp;+\u0026thinsp;ET treatment. Consistent with these findings, miR-130 expression was upregulated in the skeletal muscle of obese rats compared with those fed a normal diet, and this increase was significantly attenuated by DR, ET, or DR\u0026thinsp;+\u0026thinsp;ET, with DR\u0026thinsp;+\u0026thinsp;ET showing the strongest inhibitory effect. To further explore the role of miR-130 in skeletal muscle cells, L6 and C2C12 cells were transfected with miR-130 mimics. Overexpression of miR-130 markedly suppressed proliferation and differentiation in these cells, accompanied by reduced creatine kinase activity and decreased myogenin expression\u0026mdash;both key markers of myogenic differentiation. Moreover, miR-130 overexpression inhibited the luciferase activity of a reporter vector containing the PPARγ-3\u0026prime;-UTR, and this inhibition was abolished by mutation of the PPARγ-3\u0026prime;-UTR, indicating a direct regulatory mechanism affecting protein synthesis in skeletal muscle. In summary, DR and ET each alleviated obesity-related skeletal muscle impairment by activating IGF-1/Akt/mTOR signaling and suppressing miR-130 expression, but their combination did not produce synergistic effects.\u003c/p\u003e","manuscriptTitle":"Dietary restriction and aerobic exercise alleviate obesity-related skeletal muscle impairment via the miR-130/PPARγ axis and IGF-1/Akt/mTOR signaling activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-03 08:16:59","doi":"10.21203/rs.3.rs-8116495/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-30T16:43:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-23T11:43:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-12T18:11:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"181964645573211203145703167455476159007","date":"2025-12-06T14:56:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"116173376903587980069225099774446723295","date":"2025-12-05T16:03:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213450247097782025296962107077550160299","date":"2025-12-01T15:48:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-01T14:53:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-01T14:34:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-25T09:13:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-22T14:40:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-22T14:37:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"27def198-ee58-4c08-92dd-4f7b6ea8862f","owner":[],"postedDate":"December 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58952605,"name":"Biological sciences/Cell biology"},{"id":58952606,"name":"Health sciences/Diseases"},{"id":58952607,"name":"Health sciences/Endocrinology"},{"id":58952608,"name":"Biological sciences/Molecular biology"},{"id":58952609,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-04-07T16:00:58+00:00","versionOfRecord":{"articleIdentity":"rs-8116495","link":"https://doi.org/10.1038/s41598-026-46630-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-01 15:57:39","publishedOnDateReadable":"April 1st, 2026"},"versionCreatedAt":"2025-12-03 08:16:59","video":"","vorDoi":"10.1038/s41598-026-46630-7","vorDoiUrl":"https://doi.org/10.1038/s41598-026-46630-7","workflowStages":[]},"version":"v1","identity":"rs-8116495","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8116495","identity":"rs-8116495","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}