Exercise compensates the metformin on T2DM-indcued muscle atrophy through autophagy prominent FLNC degradation

Exercise compensates the metformin on T2DM-indcued muscle atrophy through autophagy prominent FLNC degradation | 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 Research Article Exercise compensates the metformin on T2DM-indcued muscle atrophy through autophagy prominent FLNC degradation Jiao Lu, Mengqi Xiang, Anqi Zhao, Ye Xu, Wen Sun, Hongjian Xiao, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6749383/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract FLNC is localized in the Z-disc of skeletal muscle and plays an important role in maintaining mechanical stability. Its degradation and renewal are specifically mediated by autophagy. Previously, we revealed that exercise is superior to metformin in improving muscle atrophy by promoting autophagy and inhibiting the proteasome. Building on this foundation, we aim to explore whether exercise can compensate for the deficiency of metformin in enhancing autophagy-mediated FLNC degradation. In this study, db/db mice were used to establish a model of type 2 diabetes mellitus (T2DM). An exercise intervention was conducted using a treadmill protocol at speeds of 7–12 m/min for 30–40 minutes per day, five days a week. Metformin was administered daily via gavage at a dose of 300 mg/kg. A combined intervention was performed, in which metformin was administered first, followed by exercise. After eight weeks of intervention, the ultrastructure of skeletal muscle was evaluated using electron microscopy. The key proteins HSP70, BAG3, LC3, LAMP2, and Cathepsin D involved in BAG3-mediated FLNC degradation via selective autophagy were quantified using Western blotting and immunofluorescence co-localization. We discovered that abnormalities in autophagy within skeletal muscle lead to metabolic disorders of FLNC in the Z-disc, contributing to the breakdown of myofibrillar structure. Furthermore, compared to metformin, exercise enhanced FLNC degradation by promoting BAG3-mediated selective autophagy, which may compensate for the limitations of metformin in combination treatment. exercise metformin T2DM muscle atrophy FLNC autophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Type 2 diabetes mellitus (T2DM) is a globally prevalent chronic metabolic disease characterized by a prolonged course and multiple complications that can affect various organs, including the heart, liver, kidneys, eyes, skeletal muscles, and blood vessels (Zheng et al. 2018 ). In the later stages of T2DM, severe skeletal muscle atrophy emerges as one of the most significant complications that jeopardize overall health. This condition can occur even when blood sugar levels are well controlled (Purnamasari et al. 2022 ). Metformin is the first-line medication for the clinical management of T2DM; it effectively reduces blood sugar levels, regulates glucose and lipid metabolism, and improves insulin resistance, while also being characterized by safety, cost-effectiveness, and minimal side effects. However, multiple clinical follow-up studies have demonstrated that long-term use of metformin fails to ameliorate muscle atrophy associated with T2DM, particularly in the elderly, where this phenomenon is more pronounced (Kang et al. 2021 ). This suggests that the mechanisms underlying muscle atrophy may be unrelated to the benefits derived from metformin administration. Therefore, it is essential to elucidate the potential mechanisms behind the muscle atrophy side effects associated with metformin and to explore strategies to mitigate this adverse effect. Obstacles in protein synthesis and degradation are the primary mechanisms that induce skeletal muscle atrophy. When the rate of protein degradation exceeds that of protein synthesis, muscle fiber proteins are lost, leading to skeletal muscle atrophy (Eley and Tisdale 2007 ). Previous studies have shown that in T2DM-induced skeletal muscle atrophy, protein synthesis is reduced while hydrolysis is enhanced in the early stages due to disorders in the insulin signaling pathway (Xiang et al. 2023 ). As the disease progresses, there may be cross-talk between the proteasome system and the autophagy degradation system, characterized by the obstruction of cellular autophagy that promotes the degradation of the proteasome system. In skeletal muscle cells, selective autophagy specifically mediates the degradation and renewal of important structural proteins, such as filamin C (FLNC) in the Z-disc (Ruparelia et al. 2016 ). This structural protein plays a crucial role in mechanical stability, force transmission, and mechanotransduction, which are essential for maintaining myofibril stability. Abnormalities in its degradation can lead to the disintegration of the entire myofibril. This may be a significant factor that upregulates muscle atrophy proteins, MuRF1 and Atrogin-1, through the protease system's hydrolysis, ultimately resulting in skeletal muscle loss (Liu et al. 2017 ). Our previous study demonstrated that in skeletal muscle, metformin can activate the AMP activated protein kinase (AMPK) pathway to increase the generation of autophagosomes; however, it does not promote their degradation, leading to the accumulation of autophagosomes and a failure to improve muscle atrophy (Xiang et al. 2023 ; Lu et al. 2021 ). Therefore, the administration of metformin may result in abnormal degradation mediated by the lysosomal pathway in skeletal muscle cells, warranting further investigation into this mechanism. Exercise is medicine. Like metformin, it is recommended by the International Diabetes Federation as a first-line treatment plan for T2DM. Clinical studies have found that there are many "intersections" between exercise and metformin in the treatment of T2DM, such as reducing blood glucose levels and increasing insulin sensitivity. Furthermore, them both have the clear common targets in the mechanisms, which active the AMPK signaling pathway to regulate downstream effects, like glucose and lipid metabolism, autophagy, inflammatory response, etc (Wang et al. 2019 ; Vainshtein and Hood 2016 ). But they share different pathways on these effects: metformin, which brings out effects by inhibiting the metabolism, such as suppressing the production of glucose and ATP to lower blood sugar and improve insulin sensitivity; in contrast, exercise, which achieves this goal by increasing the turnover of energy substances and consumption to make body acquired adaptation. Therefore, the effects on combination treatment might vary for different organs or systems, with additive or antagonistic effects, and may also be complementary. Previous studies have revealed that the combination treatment brought out additive effects on glycemic control and T2DM-induced metabolic dysfunction-associated steatotic liver disease (MASLD), while demonstrated mediocre effects on the improvement of early cardiac fibrosis (Zhang et al. 2022 ; Liu et al. 2023 ). However, we discovered antagonistic effects on treatment of skeletal muscle atrophy induced by T2DM. As administration of metformin not reverse muscle atrophy, but promoted the expression of atrophy proteins, which was probably related to the inhibition of autophagic degradation (Xiang et al. 2023 ). Consequently, in this work, we aimed to clarify that abnormality of autophagy in skeletal muscle leading to myofibrillar breakdown may be a potential mechanism for T2DM-incduced muscle atrophy, and to explore whether exercise could compensate for the deficiency of metformin in improving autophagy disorder and induce a protective effect in reducing muscle atrophy. 2. Materials and Methods 2.1 Experimental animals In this study, 10 male BKS mice and 40 male BKS-db/db mice (7 weeks of age) were purchased from Jiangsu JiCui Yaokang Technology Co., Ltd. (Jiangsu, China) and allowed to acclimate for one week. The mice were housed at a temperature of (22 ± 2) °C and humidity levels of 50%-60%, with a 12:12 light/dark cycle. Following the acclimation period, the BKS mice were designated as the non-diabetic control group (Con, n = 10), while the BKS-db/db mice were randomly divided into four groups: the quiet feeding group (db/db, n = 10), the exercise intervention group (db/db + Ex, n = 10), the metformin administration group (db/db + Met, n = 10), and the exercise combined with metformin administration group (db/db + Ex + Met, n = 10). All experiments were performed in line with relevant guidelines and regulations including the EU Directive 2010/63/EU, and were approved by the Animal Ethics and Welfare Committee of Nanjing Sport Institute (Approval No. 2019-010). 2.2 Experimental protocol The db/db + Met and db/db + Ex + Met groups received metformin intragastrically at a dosage of 300 mg/kg daily for 8 weeks, while the other groups were administered ddH2O. Mice in the db/db + Ex and db/db + Ex + Met groups ran on a treadmill set at a 0°incline at speeds ranging from 7 to 12 m/min for 30 to 40 minutes per day over the course of 8 weeks (Xiang et al. 2023 ). 2.3 Immunofluorescent staining The skeletal muscles of three mice were fixed in 4% paraformaldehyde, dehydrated, cleared, waxed, and embedded, then sectioned into 4 µm slices. The slices were dewaxed to water, repaired with sodium citrate at high temperature, and blocked with goat serum (Solarbio, SL038, 1:9). They were then incubated overnight at 4°C with primary antibodies: rabbit anti-FLNC (NOVUS, NBP189300, 1:100), rabbit anti-Cathepsin D (Abcam, ab75852, 1:200), mouse anti-BAG3 (Proteintech, 68076-1-Ig, 1:100), and mouse anti-SQSTM1/p62 (Santa Cruz, sc-48402, 1:50). The sections were washed with phosphate-buffered saline (PBS) and incubated with secondary antibodies in a 37°C oven for one hour: goat anti-rabbit Alexa Fluor 488 (Cell Signaling, 4412S, 1:500), goat anti-rabbit Alexa Fluor 594 (Cell Signaling, 8889S, 1:500), and goat anti-mouse Alexa Fluor 594 (Cell Signaling, 8890S, 1:500). Immunofluorescence single-stained sections were incubated with DAPI and then mounted with antifade solution. Other sections were incubated with additional primary antibodies, specifically rabbit anti-LC3 (Proteintech, 14600-1-AP, 1:100) and rabbit anti-FLNC (NOVUS, NBP189300, 1:100), followed by incubation with a secondary antibody, goat anti-rabbit Alexa Fluor 488 (Abcam, ab150077, 1:500), and then DAPI. The sections were observed under a Zeiss microscope, and images were captured at 400× magnification. Five non-continuous fields were selected per sample, totaling 15 fields from three samples, which were analyzed using Image J. The positive areas of FLNC, BAG 3 and Cathepsin D were calculated as a percentage of positively stained area divided by the total muscle fiber tissue area. The Co-localization coefficients of BAG 3 + FLNC, and BAG 3 + LC 3 were calculated as BAG 3 and FLNC colocalized area / FLNC area, BAG 3 and LC 3 colocalized area / BAG 3 area respectively. The Co-localization coefficients of p62 + LC 3 was calculated by counting the colocalized points as previous studies did. 2.4 Transmission electron microscopy (TEM) A small piece of skeletal muscle tissue, approximately 1 mm³, was soaked in 4% paraformaldehyde, followed by fixation, dehydration, clearing, and embedding. The resulting block was carefully sectioned into ultrathin slices with a thickness of about 70 nm using an ultramicrotome (Leica). The sections were then stained with 3% uranyl acetate and lead citrate before imaging under a transmission electron microscope (TEM) (JEM-1400, Japan) at Beijing Jiaotong University. 2.5 Western blot We detected skeletal muscle tissue protein expression by western blot. Whole muscle protein was lysed in RIPA lysate (Beyotime, P0013B, China) supplemented with protease inhibitor (Beyotime, P1005, China) and phosphatase inhibitor (Beyotime, P1045, China), triturated and centrifuged to collect supernatant. Subsequently, protein levels were determined by BCA protein assay (Epizyme, Shanghai, China). Then samples were separated by 10% and 12.5% sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) gel, gel electrophoresis (90 V, 1.5 h), and polyvinylidene fluoride (PVDF) membranes (Millipore, Shanghai, China) were used for the transfer (110 V, 1 h). The membranes were blocked with 5% nonfat dry milk + Tris buffered saline plus Tween 20 (TBST) for 1.5 h, followed by overnight incubation at 4°C with appropriate primary antibodies: incubated with the primary antibodies: BAG3 (Proteintech, 68076-1-Ig, 1:20000), FLNC (NOVUS, NBP189300, 1:1000), HSP70 (SANTA, sc-66048, 1:1000), LAMP-2 (abcam, ab-125068, 1:2000), Cathepsin D (abcam, ab75852, 1:5000), GAPDH (Proteintech, 10494-1-AP, 1:20000). On the second day, the membranes were washed three times with TBST, incubated with the secondary antibodies: goat anti-mouse antibody (Cell Signaling, #7076, 1:2000), goat anti-rabbit antibody (Bioworld, BS13278, 1:10000) for 1.5 h at room temperature. The PVDF membranes wrer added the enhanced chemiluminescence (ECL) reagent, imitated using a Bio-Rad multicolor gel imaging machine, and then analyzed using Image Lab image processing software (Bio-Rad Laboratories, Hercules, CA, USA), GAPDH was used as a loading control to correct the values, and the differences were displayed with the relative value, which was normalized to the control. 2.6 Statistical analysis All data were processed using Excel and GraphPad Prism statistical software. The experimental data are expressed as mean ± standard error of the mean (X ± SEM). One-way analysis of variance (ANOVA) was employed to analyze the experimental results. Statistical significance was set at P < 0.05. 3. Results 3.1 Different effects of the three interventions on structure of skeletal muscle fibril As electron microscopy revealed that the skeletal muscle myofibrillar structure in the control (Con) group was intact, with neatly arranged Z-disks and a clear boundary between the I-band and A-band (Fig. 1 ). In contrast, the skeletal muscle myofibrillar structure of db/db mice exhibited damage, characterized by blurred Z-disks, an unclear boundary between the I-band and A-band, and high electron density lumps. After exercise intervention, myofibrillar structure were improved including the arrangement of Z-disks, I-band and A-band improved and became clear, and the high electron density lumps disappeared; interestingly, the Z-disk became widened. The improvement of myofibrillar structure was discounted with metformin intervention, as the high electron density lumps could still be found. Compared to exercise or metformin intervention, the combined intervention showed the best gain on myofibrillar structure, which was almost identical to the control group. Considering the significant changes of Z-disk in the electron microscopy results, we performed WB and immunofluorescence detection FLNC, a key protein located in Z-disk which could cross-link actin filaments and interact with numerous binding partners. Immunofluorescence showed that the green fluorescence of FLCN forms a band like pattern, arranged in parallel and perpendicular to the myofibrils in control group. Compared to control group, the arrangement of FLNC green fluorescence in db/db group was disordered and partially missing (Fig. 2 , A); importantly, there were many clumps of green fluorescence appeared in muscle fibers, which was consistent with the high electron density lumps in electron microscopy. Therefore, the FLNC immunopositive area were significantly increased. After exercise intervention, the clumps of green fluorescence were disappeared and the FLNC immunopositive area were significantly reduced, but some areas still had FLNC deficiency. While in the metformin intervention group, there were still some clumps of green fluorescence and the FLNC immunopositive area showed downward trend, but no significant difference. The combined intervention demonstrated the same trend with exercise intervention that no clumps of green fluorescence were observed and FLNC immunopositive area were significantly reduced. The results of WB also verified these founds that compared with WT group, the expression of FLNC was significantly increased in db/db group (Fig. 2 , B,C). Compared with db/db group, the expression of FLNC in db/db + Ex, and db/db + Ex + Met groups were significantly decreased, while in db/db + Met showed declined trend, but no significant difference. 3.2 All three interventions could facilitate the phagocytosis of FLNC protein by autophagosomes via the assistances of chaperones It is well proved that the degradation of FLNC depends on a specific autophagy pathway called chaperone-assisted selective autophagy (CASA) in skeletal muscle cells. Abnormal expression of CASA can impair FLNC degradation, leading to muscle atrophy due to myofibril collapse (Ruparelia et al. 2016 ; Rosati et al. 2011 ). CASA comprises three proteins: HSP70, HSPB8, and BAG3, which are essential for maintaining protein homeostasis in muscle and neuronal cells. Therefore, we primarily assessed the expression of BAG3 and HSP70 proteins. Compared to the control (Con) group, the expression of BAG3 in the skeletal muscle of db/db mice was significantly increased (Fig. 3 , B), while HSP70 expression showed no significant change (Fig. 3 , A). Following exercise and/or metformin intervention, BAG3 expression in skeletal muscle significantly decreased (Fig. 3 , B), whereas HSP70 expression remained unchanged (Fig. 3 , A). To evaluate the role of chaperone proteins on FLNC, we performed immunofluorescence staining for BAG3 and FLNC. FLNC stained the striations of skeletal muscle green, while BAG3 exhibited red fluorescent distributed near the striations, resulting in yellow fluorescent regions where the two proteins combined (Fig. 2 , C). Compared to the Con group, the immunofluorescence areas of BAG3 and FLNC were significantly reduced in db/db mice. While after exercise or combination intervention, these areas significantly increased in db/db mice. Metformin might make efforts on elevation of binding effects of BAG3 on FLNC, but the results did not show significant (Fig. 2 , B, C ). To assess the degradation of BAG3 through selective autophagy way, we performed immunofluorescence staining for BAG3 and LC3 (Fig. 4 , A). BAG3 displayed bright red spots, while LC3 exhibited green puncta; the areas of colocalization appeared as yellow fluorescence. Compared to the control (Con) group, the BAG3 + LC3 colocalization area were reduced in db/db mice. Following exercise and combination intervention, there were significant increase in colocalization area of BAG3 + LC3; however, no significant difference was observed between the metformin group (Fig. 4 , A, B). 3.3 Exercise demonstrated superiority on activation of lysosome to promote the autophagosomes degradation LC3 is located in the autophagosome membrane, while p62/SQSTM1 serves as a crucial adaptor for identifying and delivering specific organelles and protein aggregates to autophagosomes for degradation (Cha-Molstad et al. 2017 ). In this work, to assessed the degradation process of CASA, immunofluorescent labeling of p62 (red) and LC3 (green) was performed that showed in Fig. 4 , A. Their colocalization areas appearing as yellow fluorescence, which indicating the formation of autophagosomes. In this study, there was no difference on the number of fluorescence colocalization dots for p62 and LC3 between control (Con) group and db/db group. As expected, the p62 and LC3 colocalization dots were significantly increased after exercise and combination intervention, but demonstrated weak growth with metformin intervention (Fig. 4 A, B). Lysosomal associated membrane protein 2 (LAMP-2) is located on the lysosomal membrane, and it was elevated when autophagy activated (Hubert et al. 2016 ). We assessed the expression of LAMP-2 in db/db mice using Western blot analysis and found that its expression in db/db mice was significantly reduced compared to the control (Con) group. In contrast, the expression of LAMP-2 in the db/db + Ex and db/db + Ex + Met groups was significantly increased compared to the db/db group (Fig. 5 A). Cathepsin D is an important proteolytic enzyme that not only degrades proteins within lysosomes but also secretes them outside the cell (Di et al. 2021 ). Although there was no significant difference in the expression of Cathepsin D among the five groups (Fig. 5 C), immunofluorescence staining revealed that the positive area of Cathepsin D in the db/db + Ex and db/db + Ex + Met groups was significantly increased compared to db/db mice (Fig. 5 D, E). 4. Discussion In normal physiological processes, skeletal muscle cells require higher rates of protein degradation and anabolism (timely repair and renewal) to maintain their structure and function, particularly in response to wear caused by contraction and friction between thick and thin myofilaments (Bullard and Pastore 2019 ). However, an imbalance between protein synthesis and degradation is a significant contributor to muscle mass loss (Sartori et al. 2021 ). In type 2 diabetes mellitus (T2DM)-induced atrophy, skeletal muscle is characterized by elevated levels of muscle atrophy proteins, MuRF1 and Atrogin-1, which function as ubiquitin ligases and promote the proteolytic system that degrades structural proteins such as actin and myosin (Liu et al. 2017 ). Our results support this observation, as parts of the myofibrils in db/db mice exhibited significant structural damage, characterized by streaming and blurring in the light and dark bands of sarcomeres, as well as disorganization and clumpy electron density in the Z-disc. The alterations in the Z-disc are particularly concerning, as it plays a crucial role in mechanical stability, force transmission, and mechanotransduction. Disruption of proteins in the Z-disc can lead to the disintegration of the entire myofibril, resulting in increased protein degradation via the proteasome system (Mao and Nakamura 2020 ; Ma et al. 2011 ). Notably, the degradation and renewal of filamin C (FLNC), a key structural protein in the Z-disc, are specifically regulated through the autophagy-lysosomal pathway. Impairment of this pathway can lead to severe myofibril damage and muscle atrophy (Fujita et al. 2012 ). Given that previous studies have shown that autophagy is inhibited in the skeletal muscle of db/db mice (Xiang et al. 2023 ), we speculate that autophagy impairment may exacerbate myofibril disintegration, thereby promoting proteasome-mediated degradation. This creates a vicious cycle that further exacerbates muscular atrophy in T2DM. Further investigation has confirmed that the disordered arrangement and aggregation of filamin C (FLNC) may be responsible for the alteration of the Z-disc. These results indicate that dysfunction in FLNC degradation leads to the disintegration of the Z-disc, which is associated with myofibrillar myopathies induced by type 2 diabetes mellitus (T2DM). The degradation and turnover of FLNC are conserved processes that involve specific chaperones, such as HSP70 and BAG3, which utilize a unique degradation pathway through autophagy rather than the proteasome system. Under normal conditions, a structural form of HSP70, known as HSC70, plays a crucial role in the assembly and metabolism of FLNC. Subsequently, with the mediation of BAG3, FLNC can be degraded through selective autophagy, a process referred to as chaperone-assisted selective autophagy (CASA), and may also be degraded by lysosomal pathways. These processes are essential for skeletal muscle to undergo critical structural metabolism and maintain the stability of myofibrils. Our previous study found that as T2DM progresses, autophagy is suppressed due to the upregulation of the mTOR pathway. Furthermore, in this study, we found that the lysosomal degradation system is also compromised, likely affected by the overactive proteasome system. Consequently, damaged FLNC fails to be effectively degraded by CASA and aggregates, which affects the stability of the Z-disc and causes myofibril disruption in the muscle fibers of db/db mice. This may underlie the mechanism that leads to the upregulation of atrophy-related proteins and the loss of skeletal muscle mass. Therefore, improving skeletal muscle mass in T2DM may depend on the restoration of autophagy. In clinical settings, metformin has been effective in glycemic control and improving insulin resistance, but it has shown limited efficacy in alleviating muscle atrophy in late-stage type 2 diabetes mellitus (T2DM) (Kang et al. 2021 ; Sanchez-Rangel and Inzucchi 2017 ). Our previous research has also revealed that while metformin can effectively enhance the skeletal muscle insulin signaling pathway, it does not lead to an increase in muscle mass. The underlying reason for this may be that once autophagy impairment exacerbates the disintegration of myofibrils, it promotes proteasome-mediated degradation. Therefore, attempting to reverse muscle atrophy solely by improving the insulin signaling pathway is insufficient. Additionally, in this study, we observed the aggregation of filamin C (FLNC) and its chaperones within the myofibrils of the metformin group, indicating that metformin administration could increase the formation of autophagosomes in muscle fibers but failed to sufficiently activate lysosomes to degrade these aggregates. This phenomenon contradicts the classical understanding of metformin, which is thought to effectively activate autophagic flux through the AMPK pathway, primarily mediated by the inhibition of complex I in the mitochondrial oxidative respiratory chain, subsequently decreasing ATP synthesis (Wang et al. 2019 ). However, some studies have also suggested that metformin may inhibit autophagic flux in skeletal muscle, myocardium, and tumor cells, potentially due to lysosomal inhibition caused by metformin (Malin and Stewart 2020 ; L. et al. 2019). The inadequate intracellular ATP levels induced by metformin may obstruct V-ATPase, which functions as a proton pump transporting H + ions into lysosomes, resulting in abnormal lysosomal acidification (Sugawara and Ogawa 2022 ). Furthermore, recent research indicates that in the process of activating AMPK via the lysosomal pathway, metformin also inhibits V-ATPase activity, obstructing lysosomal maturation and acidification by directly binding to PEN2 (Ma et al. 2022 ). In this experiment, the inhibition of lysosomal activity in the metformin group further confirmed this mechanism. In conclusion, we discovered that metformin administration inhibited lysosomal hydrolysis activity and blocked the fusion of autophagosomes, leading to the accumulation of autophagosomes and impaired FLNC, which exacerbated the breakdown and hydrolysis of myofibrils. Similar phenomena have been reported in other studies, which found that metformin promoted the expression of atrophy-related proteins, MuRF1 and MAFbx32, and enhanced the activity of the ubiquitin-proteasome system (Kley et al. 2021 ). Skeletal muscles require a substantial amount of ATP for physical work. According to the use and disuse theory, exercise is more effective than disuse in mitigating the occurrence and development of muscle atrophy, as evidenced by both clinical and basic research. Although both metformin and exercise upregulate AMPK, their regulatory mechanisms differ significantly. Metformin activates AMPK primarily through the inhibition of ATP synthesis, while exercise promotes this effect by enhancing ATP consumption and energy turnover in skeletal muscle (Spaulding and Yan 2022 ; Tamargo-Gomez and Marino 2018 ). Additionally, exercise exerts long-term effects on skeletal muscle, enhancing mitochondrial respiration. This not only protects muscle cells from degradation due to insufficient lysosomal acidification resulting from low ATP levels (Heo et al. 2017 ), but also facilitates the production of reactive oxygen species (ROS), which further promotes autophagy (Gharehbagh et al. 2021 ). Our research verified this mechanism; impaired autophagic degradation via the lysosomal pathway was restored by aerobic exercise. Although the expression of HSP 70 was not significantly increased after exercise intervention in this work, which we thought due to the mice were stressed before being sacrificed. Aerobic exercise acted as optimal stimulation could increase the expression of HSP70 in muscle cells (Ohsawa and Kawano 2021 ; Lu et al. 2025 ), and it did strengthen the assembly of damaged FLNC with help of BAG 3. Consequently, the aggregation of FLNC significantly decreased, and the morphological structure of myofibrils improved. Interestingly, we observed a notable widening and deepening of electron density at the Z-disc, indicating muscle damage or atrophy following exercise intervention. However, considering the upregulation of HSP70 mediated by CASA in the exercise group, which facilitates the degradation and renewal of damaged FLNC, it is likely that the Z-disc was in a state of post-exercise repair rather than a pathological state of atrophy. While exercise can induce ultrastructural damage in skeletal muscle, such as a widened and disordered Z-disc, it also serves as a beneficial stimulus that effectively induces stress responses in skeletal muscle to initiate remodeling process (Orfanos et al. 2016 ). This mechanism might also be relate to the role of CASA during exercise-induced myofibrillar injury and repair (Pasiakos et al. 2014 ). Interestingly, in our previous work, the combination of exercise and metformin demonstrated an unexpected antagonistic effect on the regulation of AMPK levels in skeletal muscle due to their contradictory AMPK activation mechanisms. Fortunately, despite the antagonism, the enhancement of exercise on mitochondrial respiration allowed autophagic flux to be reversed through the ROS pathway induced by exercise (Kim et al. 2018 ; Triolo et al. 2022 ). Furthermore, in this experiment, the effects of exercise-induced degradation of FLNC through CASA were unaffected by metformin administration. Additionally, exercise compensated for the negative effects of metformin on lysosomes, leading to improved degradation and renewal of FLNC in skeletal muscle. Consequently, T2DM-induced myofibril disintegration decreased, and the state of muscle atrophy was ameliorated. Although no cumulative effects on skeletal muscle mass and muscle atrophy were observed in the combination group, surprisingly, the widening of the Z-disc in myofibrils caused by exercise was significantly improved by metformin administration. This phenomenon suggests that metformin may play a potential role in promoting the renewal of structural proteins, or that the combined intervention may have a synergistic effect on myofibril renewal. The specific mechanism still needs further investigation. 5. Conclusion We propose for the first time that during the course of T2DM, abnormalities in autophagy in skeletal muscle lead to metabolic disorders of FLNC in the Z-disc, promoting the breakdown of the myofibrillar structure and the overactivity of the protease system, which ultimately contributes to muscle atrophy. The inhibition of lysosomal activity caused by metformin creates obstacles for CASA-mediated degradation of FLNC, resulting in a failure to improve muscle atrophy. In contrast to metformin, exercise not only promotes the CASA pathway but also increases lysosomal activity, significantly enhancing FLNC degradation and alleviating muscle atrophy, which may compensate for the shortcomings of metformin in combination treatment. Declarations Declaration of Generative AI and AI‑assisted technologies in the writing process During the preparation of this work the authors did not use any AI-assisted technology. Author contributions J.L.: Conceptualization, Formal analysis, Investigation, Visualization, Writing – original draft, Writing-review&editing, Funding acquisition; M.X.: Formal analysis, Investigation, Visualization, Writing-review&editing; A.Z.: Investigation, Writing-review&editing; Y.X.: Investigation, Writing-review&editing; W.S.: Investigation, Writing-review&editing; H.X.: Investigation, Writing-review&editing; L.Z.: Investigation, Writing-review&editing; J.W.: Formal analysis, Investigation, Resources, Writing-review&editing; Q.T.: Conceptualization, Resources, Writing-review&editing; Y.Z.: Conceptualization, Investigation, Resources, Writing-review&editing,Funding acquisition. Funding source Major Programs for Basic Science (Natural Science) Research in Higher Education Institutions in Jiangsu Province (24KJA310002); Youth Project of National Natural Science Foundation of China (32000839)； Scientific research cultivation project of Nanjing Sport Institute (XSTD202408). Availability of data and materials The data sets used or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate This study was approved by the Animal Ethics and Welfare Committee of Nanjing Sport Institute (Nanjing,China) (Approval No. 2019-010). Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose. The authors have no competing interests to declare that are relevant to the content of this article. All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. The authors have no financial or proprietary interests in any material discussed in this article. 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HUBERT V, PESCHEL A, LANGER B, GROEGER M, REES A, KAIN R (2016) LAMP-2 is Required for Incorporating Syntaxin-17 into Autophagosomes and for Their Fusion with Lysosomes, Zenodo (CERN European Organization for Nuclear Research) KANG MJ, JUNG MOONJWLEEJOKIMJH, KIM EJ, WU SJOHJY, LEE SW, P. R., PARK, S. H., KIM HS (2021) Metformin Induces Muscle Atrophy by Transcriptional Regulation of Myostatin Via HDAC6 and FoxO3a. J Cachexia Sarcopenia Muscle, Vol. 13605–13620 KIM Y, TRIOLO M, ERLICH, A. T., HOOD DA (2018) Regulation of Autophagic and Mitophagic Flux During Chronic Contractile Activity-Induced Muscle Adaptations. Pflügers Archiv - Eur J Physiol, Vol. 471431–471440 KLEY RA, SCHRANK LEBERY, ZHUGE B, ORFANOS H, KOSTAN Z, ONIPE J, EGGERS ASELLUNGDGUETTSCHESAK, JACOBSEN B, KRESS F, MARCUS W, DJINOVIC-CARUGO K, van der VEN K, P. F. M., FUERST, D. O., VORGERD M (2021) FLNC-Associated Myofibrillar Myopathy: New Clinical, Functional, and Proteomic Data., NEUROLOGY-GENETICS , Vol. 7 L., C. 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Cell Death Dis, Vol. 2e141 RUPARELIA AA, OORSCHOT V, RAMM G, BRYSON-RICHARDSON RJ (2016) FLNC Myofibrillar Myopathy Results from Impaired Autophagy and Protein Insufficiency. Hum Mol Genet, Vol. 252131–252142 SANCHEZ-RANGEL E, INZUCCHI SE (2017) Metformin: clinical use in type 2 diabetes, Diabetologia , Vol. 601586–601593 SARTORI R, ROMANELLO V, SANDRI M (2021) Mechanisms of muscle atrophy and hypertrophy: implications in health and disease. Nat Commun, 12330 SPAULDING HR, YAN Z (2022) AMPK and the Adaptation to Exercise. Annu Rev Physiol, Vol. 84209–84227 SUGAWARA K, OGAWA W (2022) New Mechanism of Metformin Action Mediated by Lysosomal Presenilin Enhancer 2. J Diabetes Invest, Vol. 1412–1414 TAMARGO-GOMEZ I, MARINO G (2018) Ampk: Regulation Of Metabolic Dynamics In The Context Of Autophagy. Int J Mol Sci, 19 TRIOLO M, SLAVIN M, MORADI N, HOOD DA (2022) Time-dependent Changes in Autophagy, Mitophagy and Lysosomes in Skeletal Muscle During Denervation‐induced Disuse. J Physiol, Vol. 6001683–6001701 VAINSHTEIN A, HOOD DA (2016) The regulation of autophagy during exercise in skeletal muscle. J Appl Physiol, Vol. 120664–120673 WANG Y, AN H, LIU T, QIN C, SESAKI H, GUO S, RADOVICK S, HUSSAIN M, WONDISFORD MAHESHWARIA, O'ROURKE FE, B., HE L (2019) Metformin Improves Mitochondrial Respiratory Activity Through Activation of AMPK, Cell Reports , Vol. 291511-1523.e5 XIANG M, YUAN X, ZHANG N, ZHANG L, LIU Y, LIU J, GAO Y, XU Y, ZHANG SUNWTANGQ, Y., LU J (2023) Effects of Exercise, Metformin, and Combination Treatments on Type 2 Diabetic Mellitus-Induced Muscle Atrophy in Db/db Mice: Crosstalk Between Autophagy and the Proteasome. J Physiol Biochem, Vol. 80235–80247 ZHANG Y, LIU Y, LIU X, YUAN X, XIANG M, ZHANG LIUJ, ZHU L, LU S, TANG J, Q., CHENG S (2022) Exercise and Metformin Intervention Prevents Lipotoxicity-Induced Hepatocyte Apoptosis by Alleviating Oxidative and ER Stress and Activating the AMPK/Nrf2/HO-1 Signaling Pathway in Db/db Mice. Oxidative Med Cell Longev, Vol. 20221–20213 ZHENG Y, LEY SH, HU FB (2018) Global aetiology and epidemiology of type 2 diabetes mellitus and its complications. Nat Reviews Endocrinol 14(2):88–98 Additional Declarations No competing interests reported. Supplementary Files Highlights.docx Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6749383","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":464648054,"identity":"8a08fb63-97f9-4c6e-a236-9b25cbbf23e7","order_by":0,"name":"Jiao Lu","email":"","orcid":"","institution":"Nanjing Sports Institute","correspondingAuthor":false,"prefix":"","firstName":"Jiao","middleName":"","lastName":"Lu","suffix":""},{"id":464648055,"identity":"5e83d6c4-4db3-4ea3-a757-60e1f48ea8f0","order_by":1,"name":"Mengqi Xiang","email":"","orcid":"","institution":"Nanjing Sports Institute","correspondingAuthor":false,"prefix":"","firstName":"Mengqi","middleName":"","lastName":"Xiang","suffix":""},{"id":464648056,"identity":"28b87ffb-16a2-4d45-a036-90b219b48765","order_by":2,"name":"Anqi Zhao","email":"","orcid":"","institution":"Nanjing Sports Institute","correspondingAuthor":false,"prefix":"","firstName":"Anqi","middleName":"","lastName":"Zhao","suffix":""},{"id":464648057,"identity":"f952f698-6be5-4996-bea5-c88c5886f5fc","order_by":3,"name":"Ye Xu","email":"","orcid":"","institution":"Nanjing Sports Institute","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Xu","suffix":""},{"id":464648058,"identity":"e9bc988f-1476-408d-b668-29441fb65e23","order_by":4,"name":"Wen Sun","email":"","orcid":"","institution":"Nanjing Sports 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Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jingjie","middleName":"","lastName":"Wang","suffix":""},{"id":464648062,"identity":"f0496066-cf82-4228-884d-fb8ff93d1cf1","order_by":8,"name":"Qiang Tang","email":"","orcid":"","institution":"Nanjing Sports Institute","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Tang","suffix":""},{"id":464648063,"identity":"0076d0ff-5cd6-4521-acf2-d7573d732561","order_by":9,"name":"Yuan Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYDACCRiDvYGBIYGBgbGBeC08B0jWIpEApghrkZ/d/Ozh17bD8uaSbw9/eMBgI7vhAPOzB/i0MM45Zm4s23bYcOfsvDSgRWnGGw6wmRvg08IskWAmLdl2mHHD7RwzoF8OJ244wMMmgU8Lm0T6N5AW+w03zxh/SGD4T1gLj0SOmeTHNqDhN3gMgA47QFiLhEROmTTDufTkDWdyzCQSDJKNZx5mM8OrRX5G+jbJH2XWthuOnzH++KPCTrbvePMzvFpAgJmXDcYEBRUzIfVAwPjjDxGqRsEoGAWjYOQCAM+PSXKF7/KAAAAAAElFTkSuQmCC","orcid":"","institution":"Nanjing Sports Institute","correspondingAuthor":true,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-05-26 09:39:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6749383/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6749383/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83865010,"identity":"cbb5b0d7-0685-418e-a199-805c33af7536","added_by":"auto","created_at":"2025-06-03 21:28:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":261862,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron microscopy of mouse skeletal muscle fibers\u003c/p\u003e\n\u003cp\u003eThe black arrows indicate high electron density lumps.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6749383/v1/e1184266e36c0ff29b0ce1be.jpg"},{"id":83865014,"identity":"1492986c-3f66-4638-8790-43a4c71c155d","added_by":"auto","created_at":"2025-06-03 21:28:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":460780,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of FLNC in skeletal muscle\u003c/p\u003e\n\u003cp\u003e(A) Skeletal muscle sections were stained with fluorophore-labeled antibodies against FLNC (Alexa Fluor 488, green). White scale bars = 20μm. (B) Quantification of the percentage of FLNC area, (n=3 per group for fluorescence image analyses), each point represents an individual image. (C) Representative immunoblots of FLNC, GAPDH, and relative band densitometries of FLNC in skeletal muscle, (n = 5~7 per group). Data are mean ± SEM. #P \u0026lt; 0.05 vs. Con group, *P \u0026lt; 0.05 vs. db/db group.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6749383/v1/38b49800631f2b97582babe5.jpg"},{"id":83865015,"identity":"2cd3cb1e-849b-4eb1-8cf8-fc20e52070f5","added_by":"auto","created_at":"2025-06-03 21:28:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":587295,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of conjugation on chaperonin with FLNC\u003c/p\u003e\n\u003cp\u003e(A, B) Representative immunoblots of HSP70, BAG 3, GAPDH, vinculin, and relative band densitometries, (n = 5~7 per group). (C) Skeletal muscle sections were stained with fluorophore-labeled antibodies against BAG 3 (Alexa Fluor 594, red) and FLNC (Alexa Fluor 488, green). The white arrow indicates the areas of BAG3 + FLNC co-location, white scale bars = 20μm. (D, E) Quantitative percentage of BAG3 area and BAG3 area co-localize with FLNC, (n=3 per group for fluorescence image analyses), each point represents an individual image. Data are mean ± SEM. #P \u0026lt; 0.05 vs. Con group, *P \u0026lt; 0.05 vs. db/db group.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6749383/v1/8f3897edde130d504da92384.jpg"},{"id":83865016,"identity":"5fdf284e-4ae1-4640-8b92-76680571ed6d","added_by":"auto","created_at":"2025-06-03 21:28:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":743401,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence co-localization of BAG3 + LC3 and p62 + LC3\u003c/p\u003e\n\u003cp\u003e(A) Skeletal muscle sections were stained with fluorophore-labeled antibodies against BAG 3 (Alexa Fluor 594, red), LC3 (Alexa Fluor 488, green) and p62 (Alexa Fluor 594, red) and LC3 (Alexa Fluor 488, green). The white arrow indicates the areas of BAG3 + LC3 co-location or p62 + LC3 co-location, white scale bars = 20μm. (B) Quantitative percentage of BAG3 area co-localize with LC3, (n=3 per group for fluorescence image analyses), each point represents an individual image. (C) Point statistics of co-localization of p62 and LC3 by immunofluorescence, (n=3 per group for fluorescence image analyses), each point represents an individual image. Data are mean ± SEM. #P \u0026lt; 0.05 vs. Con group, *P \u0026lt; 0.05 vs. db/db group.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6749383/v1/d9a3df7a6b45d6ec7c29ed52.jpg"},{"id":83865094,"identity":"8898156d-5227-4ac5-b177-732dfecdcce0","added_by":"auto","created_at":"2025-06-03 21:36:44","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":276168,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of lysosome marker LAMP-2 and Cathepsin D in skeletal muscle\u003c/p\u003e\n\u003cp\u003e(A) Representative immunoblots of LAMP-2, GAPDH, Cathepsin D interdiate, Cathepsin D mature. (B,C) Relative band densitometries of LAMP-2 and Cathepsin D in skeletal muscle, (n = 5~7 per group). (D) Skeletal muscle sections were stained with fluorophore-labeled antibodies against Cathepsin D (Alexa Fluor 594, red). White scale bars = 20μm. (E) Quantitative percentage of Cathepsin D area, (n=3 per group for fluorescence image analyses), each point represents an individual image. Data are mean ± SEM. #P \u0026lt; 0.05 vs. Con group, *P \u0026lt; 0.05 vs. db/db group.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6749383/v1/0d9be7d79673da94ae422e32.jpg"},{"id":85870046,"identity":"df21d86d-e7c8-4c3e-8e26-144f9690156f","added_by":"auto","created_at":"2025-07-02 14:01:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3054321,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6749383/v1/435327bc-49a0-4c35-a846-5b2a9e9d5edf.pdf"},{"id":83865363,"identity":"7e47c85a-6055-48a4-a824-b635db28bb6b","added_by":"auto","created_at":"2025-06-03 21:44:44","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10675,"visible":true,"origin":"","legend":"","description":"","filename":"Highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-6749383/v1/87da012949ead536a28dd55d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exercise compensates the metformin on T2DM-indcued muscle atrophy through autophagy prominent FLNC degradation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eType 2 diabetes mellitus (T2DM) is a globally prevalent chronic metabolic disease characterized by a prolonged course and multiple complications that can affect various organs, including the heart, liver, kidneys, eyes, skeletal muscles, and blood vessels (Zheng et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the later stages of T2DM, severe skeletal muscle atrophy emerges as one of the most significant complications that jeopardize overall health. This condition can occur even when blood sugar levels are well controlled (Purnamasari et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Metformin is the first-line medication for the clinical management of T2DM; it effectively reduces blood sugar levels, regulates glucose and lipid metabolism, and improves insulin resistance, while also being characterized by safety, cost-effectiveness, and minimal side effects. However, multiple clinical follow-up studies have demonstrated that long-term use of metformin fails to ameliorate muscle atrophy associated with T2DM, particularly in the elderly, where this phenomenon is more pronounced (Kang et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This suggests that the mechanisms underlying muscle atrophy may be unrelated to the benefits derived from metformin administration. Therefore, it is essential to elucidate the potential mechanisms behind the muscle atrophy side effects associated with metformin and to explore strategies to mitigate this adverse effect.\u003c/p\u003e \u003cp\u003eObstacles in protein synthesis and degradation are the primary mechanisms that induce skeletal muscle atrophy. When the rate of protein degradation exceeds that of protein synthesis, muscle fiber proteins are lost, leading to skeletal muscle atrophy (Eley and Tisdale \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Previous studies have shown that in T2DM-induced skeletal muscle atrophy, protein synthesis is reduced while hydrolysis is enhanced in the early stages due to disorders in the insulin signaling pathway (Xiang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As the disease progresses, there may be cross-talk between the proteasome system and the autophagy degradation system, characterized by the obstruction of cellular autophagy that promotes the degradation of the proteasome system. In skeletal muscle cells, selective autophagy specifically mediates the degradation and renewal of important structural proteins, such as filamin C (FLNC) in the Z-disc (Ruparelia et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This structural protein plays a crucial role in mechanical stability, force transmission, and mechanotransduction, which are essential for maintaining myofibril stability. Abnormalities in its degradation can lead to the disintegration of the entire myofibril. This may be a significant factor that upregulates muscle atrophy proteins, MuRF1 and Atrogin-1, through the protease system's hydrolysis, ultimately resulting in skeletal muscle loss (Liu et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our previous study demonstrated that in skeletal muscle, metformin can activate the AMP activated protein kinase (AMPK) pathway to increase the generation of autophagosomes; however, it does not promote their degradation, leading to the accumulation of autophagosomes and a failure to improve muscle atrophy (Xiang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, the administration of metformin may result in abnormal degradation mediated by the lysosomal pathway in skeletal muscle cells, warranting further investigation into this mechanism.\u003c/p\u003e \u003cp\u003eExercise is medicine. Like metformin, it is recommended by the International Diabetes Federation as a first-line treatment plan for T2DM. Clinical studies have found that there are many \"intersections\" between exercise and metformin in the treatment of T2DM, such as reducing blood glucose levels and increasing insulin sensitivity. Furthermore, them both have the clear common targets in the mechanisms, which active the AMPK signaling pathway to regulate downstream effects, like glucose and lipid metabolism, autophagy, inflammatory response, etc (Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vainshtein and Hood \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). But they share different pathways on these effects: metformin, which brings out effects by inhibiting the metabolism, such as suppressing the production of glucose and ATP to lower blood sugar and improve insulin sensitivity; in contrast, exercise, which achieves this goal by increasing the turnover of energy substances and consumption to make body acquired adaptation. Therefore, the effects on combination treatment might vary for different organs or systems, with additive or antagonistic effects, and may also be complementary. Previous studies have revealed that the combination treatment brought out additive effects on glycemic control and T2DM-induced metabolic dysfunction-associated steatotic liver disease (MASLD), while demonstrated mediocre effects on the improvement of early cardiac fibrosis (Zhang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, we discovered antagonistic effects on treatment of skeletal muscle atrophy induced by T2DM. As administration of metformin not reverse muscle atrophy, but promoted the expression of atrophy proteins, which was probably related to the inhibition of autophagic degradation (Xiang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Consequently, in this work, we aimed to clarify that abnormality of autophagy in skeletal muscle leading to myofibrillar breakdown may be a potential mechanism for T2DM-incduced muscle atrophy, and to explore whether exercise could compensate for the deficiency of metformin in improving autophagy disorder and induce a protective effect in reducing muscle atrophy.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental animals\u003c/h2\u003e \u003cp\u003eIn this study, 10 male BKS mice and 40 male BKS-db/db mice (7 weeks of age) were purchased from Jiangsu JiCui Yaokang Technology Co., Ltd. (Jiangsu, China) and allowed to acclimate for one week. The mice were housed at a temperature of (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2) \u0026deg;C and humidity levels of 50%-60%, with a 12:12 light/dark cycle. Following the acclimation period, the BKS mice were designated as the non-diabetic control group (Con, n\u0026thinsp;=\u0026thinsp;10), while the BKS-db/db mice were randomly divided into four groups: the quiet feeding group (db/db, n\u0026thinsp;=\u0026thinsp;10), the exercise intervention group (db/db\u0026thinsp;+\u0026thinsp;Ex, n\u0026thinsp;=\u0026thinsp;10), the metformin administration group (db/db\u0026thinsp;+\u0026thinsp;Met, n\u0026thinsp;=\u0026thinsp;10), and the exercise combined with metformin administration group (db/db\u0026thinsp;+\u0026thinsp;Ex\u0026thinsp;+\u0026thinsp;Met, n\u0026thinsp;=\u0026thinsp;10). All experiments were performed in line with relevant guidelines and regulations including the EU Directive 2010/63/EU, and were approved by the Animal Ethics and Welfare Committee of Nanjing Sport Institute (Approval No. 2019-010).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental protocol\u003c/h2\u003e \u003cp\u003eThe db/db\u0026thinsp;+\u0026thinsp;Met and db/db\u0026thinsp;+\u0026thinsp;Ex\u0026thinsp;+\u0026thinsp;Met groups received metformin intragastrically at a dosage of 300 mg/kg daily for 8 weeks, while the other groups were administered ddH2O. Mice in the db/db\u0026thinsp;+\u0026thinsp;Ex and db/db\u0026thinsp;+\u0026thinsp;Ex\u0026thinsp;+\u0026thinsp;Met groups ran on a treadmill set at a 0\u0026deg;incline at speeds ranging from 7 to 12 m/min for 30 to 40 minutes per day over the course of 8 weeks (Xiang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Immunofluorescent staining\u003c/h2\u003e \u003cp\u003eThe skeletal muscles of three mice were fixed in 4% paraformaldehyde, dehydrated, cleared, waxed, and embedded, then sectioned into 4 \u0026micro;m slices. The slices were dewaxed to water, repaired with sodium citrate at high temperature, and blocked with goat serum (Solarbio, SL038, 1:9). They were then incubated overnight at 4\u0026deg;C with primary antibodies: rabbit anti-FLNC (NOVUS, NBP189300, 1:100), rabbit anti-Cathepsin D (Abcam, ab75852, 1:200), mouse anti-BAG3 (Proteintech, 68076-1-Ig, 1:100), and mouse anti-SQSTM1/p62 (Santa Cruz, sc-48402, 1:50). The sections were washed with phosphate-buffered saline (PBS) and incubated with secondary antibodies in a 37\u0026deg;C oven for one hour: goat anti-rabbit Alexa Fluor 488 (Cell Signaling, 4412S, 1:500), goat anti-rabbit Alexa Fluor 594 (Cell Signaling, 8889S, 1:500), and goat anti-mouse Alexa Fluor 594 (Cell Signaling, 8890S, 1:500). Immunofluorescence single-stained sections were incubated with DAPI and then mounted with antifade solution. Other sections were incubated with additional primary antibodies, specifically rabbit anti-LC3 (Proteintech, 14600-1-AP, 1:100) and rabbit anti-FLNC (NOVUS, NBP189300, 1:100), followed by incubation with a secondary antibody, goat anti-rabbit Alexa Fluor 488 (Abcam, ab150077, 1:500), and then DAPI. The sections were observed under a Zeiss microscope, and images were captured at 400\u0026times; magnification. Five non-continuous fields were selected per sample, totaling 15 fields from three samples, which were analyzed using Image J. The positive areas of FLNC, BAG 3 and Cathepsin D were calculated as a percentage of positively stained area divided by the total muscle fiber tissue area. The Co-localization coefficients of BAG 3\u0026thinsp;+\u0026thinsp;FLNC, and BAG 3\u0026thinsp;+\u0026thinsp;LC 3 were calculated as BAG 3 and FLNC colocalized area / FLNC area, BAG 3 and LC 3 colocalized area / BAG 3 area respectively. The Co-localization coefficients of p62\u0026thinsp;+\u0026thinsp;LC 3 was calculated by counting the colocalized points as previous studies did.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Transmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eA small piece of skeletal muscle tissue, approximately 1 mm\u0026sup3;, was soaked in 4% paraformaldehyde, followed by fixation, dehydration, clearing, and embedding. The resulting block was carefully sectioned into ultrathin slices with a thickness of about 70 nm using an ultramicrotome (Leica). The sections were then stained with 3% uranyl acetate and lead citrate before imaging under a transmission electron microscope (TEM) (JEM-1400, Japan) at Beijing Jiaotong University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Western blot\u003c/h2\u003e \u003cp\u003eWe detected skeletal muscle tissue protein expression by western blot. Whole muscle protein was lysed in RIPA lysate (Beyotime, P0013B, China) supplemented with protease inhibitor (Beyotime, P1005, China) and phosphatase inhibitor (Beyotime, P1045, China), triturated and centrifuged to collect supernatant. Subsequently, protein levels were determined by BCA protein assay (Epizyme, Shanghai, China). Then samples were separated by 10% and 12.5% sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) gel, gel electrophoresis (90 V, 1.5 h), and polyvinylidene fluoride (PVDF) membranes (Millipore, Shanghai, China) were used for the transfer (110 V, 1 h). The membranes were blocked with 5% nonfat dry milk\u0026thinsp;+\u0026thinsp;Tris buffered saline plus Tween 20 (TBST) for 1.5 h, followed by overnight incubation at 4\u0026deg;C with appropriate primary antibodies: incubated with the primary antibodies: BAG3 (Proteintech, 68076-1-Ig, 1:20000), FLNC (NOVUS, NBP189300, 1:1000), HSP70 (SANTA, sc-66048, 1:1000), LAMP-2 (abcam, ab-125068, 1:2000), Cathepsin D (abcam, ab75852, 1:5000), GAPDH (Proteintech, 10494-1-AP, 1:20000). On the second day, the membranes were washed three times with TBST, incubated with the secondary antibodies: goat anti-mouse antibody (Cell Signaling, #7076, 1:2000), goat anti-rabbit antibody (Bioworld, BS13278, 1:10000) for 1.5 h at room temperature. The PVDF membranes wrer added the enhanced chemiluminescence (ECL) reagent, imitated using a Bio-Rad multicolor gel imaging machine, and then analyzed using Image Lab image processing software (Bio-Rad Laboratories, Hercules, CA, USA), GAPDH was used as a loading control to correct the values, and the differences were displayed with the relative value, which was normalized to the control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data were processed using Excel and GraphPad Prism statistical software. The experimental data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (X\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM). One-way analysis of variance (ANOVA) was employed to analyze the experimental results. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Different effects of the three interventions on structure of skeletal muscle fibril\u003c/h2\u003e\n \u003cp\u003eAs electron microscopy revealed that the skeletal muscle myofibrillar structure in the control (Con) group was intact, with neatly arranged Z-disks and a clear boundary between the I-band and A-band (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, the skeletal muscle myofibrillar structure of db/db mice exhibited damage, characterized by blurred Z-disks, an unclear boundary between the I-band and A-band, and high electron density lumps. After exercise intervention, myofibrillar structure were improved including the arrangement of Z-disks, I-band and A-band improved and became clear, and the high electron density lumps disappeared; interestingly, the Z-disk became widened. The improvement of myofibrillar structure was discounted with metformin intervention, as the high electron density lumps could still be found. Compared to exercise or metformin intervention, the combined intervention showed the best gain on myofibrillar structure, which was almost identical to the control group.\u003c/p\u003e\n \u003cp\u003eConsidering the significant changes of Z-disk in the electron microscopy results, we performed WB and immunofluorescence detection FLNC, a key protein located in Z-disk which could cross-link actin filaments and interact with numerous binding partners. Immunofluorescence showed that the green fluorescence of FLCN forms a band like pattern, arranged in parallel and perpendicular to the myofibrils in control group. Compared to control group, the arrangement of FLNC green fluorescence in db/db group was disordered and partially missing (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, A); importantly, there were many clumps of green fluorescence appeared in muscle fibers, which was consistent with the high electron density lumps in electron microscopy. Therefore, the FLNC immunopositive area were significantly increased. After exercise intervention, the clumps of green fluorescence were disappeared and the FLNC immunopositive area were significantly reduced, but some areas still had FLNC deficiency. While in the metformin intervention group, there were still some clumps of green fluorescence and the FLNC immunopositive area showed downward trend, but no significant difference. The combined intervention demonstrated the same trend with exercise intervention that no clumps of green fluorescence were observed and FLNC immunopositive area were significantly reduced. The results of WB also verified these founds that compared with WT group, the expression of FLNC was significantly increased in db/db group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, B,C). Compared with db/db group, the expression of FLNC in db/db\u0026thinsp;+\u0026thinsp;Ex, and db/db\u0026thinsp;+\u0026thinsp;Ex\u0026thinsp;+\u0026thinsp;Met groups were significantly decreased, while in db/db\u0026thinsp;+\u0026thinsp;Met showed declined trend, but no significant difference.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.2 All three interventions could facilitate the phagocytosis of FLNC protein by autophagosomes via the assistances of chaperones\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eIt is well proved that the degradation of FLNC depends on a specific autophagy pathway called chaperone-assisted selective autophagy (CASA) in skeletal muscle cells. Abnormal expression of CASA can impair FLNC degradation, leading to muscle atrophy due to myofibril collapse (Ruparelia et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Rosati et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). CASA comprises three proteins: HSP70, HSPB8, and BAG3, which are essential for maintaining protein homeostasis in muscle and neuronal cells. Therefore, we primarily assessed the expression of BAG3 and HSP70 proteins. Compared to the control (Con) group, the expression of BAG3 in the skeletal muscle of db/db mice was significantly increased (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, B), while HSP70 expression showed no significant change (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, A). Following exercise and/or metformin intervention, BAG3 expression in skeletal muscle significantly decreased (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, B), whereas HSP70 expression remained unchanged (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, A). To evaluate the role of chaperone proteins on FLNC, we performed immunofluorescence staining for BAG3 and FLNC. FLNC stained the striations of skeletal muscle green, while BAG3 exhibited red fluorescent distributed near the striations, resulting in yellow fluorescent regions where the two proteins combined (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, C). Compared to the Con group, the immunofluorescence areas of BAG3 and FLNC were significantly reduced in db/db mice. While after exercise or combination intervention, these areas significantly increased in db/db mice. Metformin might make efforts on elevation of binding effects of BAG3 on FLNC, but the results did not show significant (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, B, C ).\u003c/p\u003e\n \u003cp\u003eTo assess the degradation of BAG3 through selective autophagy way, we performed immunofluorescence staining for BAG3 and LC3 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, A). BAG3 displayed bright red spots, while LC3 exhibited green puncta; the areas of colocalization appeared as yellow fluorescence. Compared to the control (Con) group, the BAG3\u0026thinsp;+\u0026thinsp;LC3 colocalization area were reduced in db/db mice. Following exercise and combination intervention, there were significant increase in colocalization area of BAG3\u0026thinsp;+\u0026thinsp;LC3; however, no significant difference was observed between the metformin group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, A, B).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Exercise demonstrated superiority on activation of lysosome to promote the autophagosomes degradation\u003c/h2\u003e\n \u003cp\u003eLC3 is located in the autophagosome membrane, while p62/SQSTM1 serves as a crucial adaptor for identifying and delivering specific organelles and protein aggregates to autophagosomes for degradation (Cha-Molstad et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this work, to assessed the degradation process of CASA, immunofluorescent labeling of p62 (red) and LC3 (green) was performed that showed in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, A. Their colocalization areas appearing as yellow fluorescence, which indicating the formation of autophagosomes. In this study, there was no difference on the number of fluorescence colocalization dots for p62 and LC3 between control (Con) group and db/db group. As expected, the p62 and LC3 colocalization dots were significantly increased after exercise and combination intervention, but demonstrated weak growth with metformin intervention (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, B).\u003c/p\u003e\n \u003cp\u003eLysosomal associated membrane protein 2 (LAMP-2) is located on the lysosomal membrane, and it was elevated when autophagy activated (Hubert et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). We assessed the expression of LAMP-2 in db/db mice using Western blot analysis and found that its expression in db/db mice was significantly reduced compared to the control (Con) group. In contrast, the expression of LAMP-2 in the db/db\u0026thinsp;+\u0026thinsp;Ex and db/db\u0026thinsp;+\u0026thinsp;Ex\u0026thinsp;+\u0026thinsp;Met groups was significantly increased compared to the db/db group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Cathepsin D is an important proteolytic enzyme that not only degrades proteins within lysosomes but also secretes them outside the cell (Di et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although there was no significant difference in the expression of Cathepsin D among the five groups (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC), immunofluorescence staining revealed that the positive area of Cathepsin D in the db/db\u0026thinsp;+\u0026thinsp;Ex and db/db\u0026thinsp;+\u0026thinsp;Ex\u0026thinsp;+\u0026thinsp;Met groups was significantly increased compared to db/db mice (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD, E).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn normal physiological processes, skeletal muscle cells require higher rates of protein degradation and anabolism (timely repair and renewal) to maintain their structure and function, particularly in response to wear caused by contraction and friction between thick and thin myofilaments (Bullard and Pastore \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, an imbalance between protein synthesis and degradation is a significant contributor to muscle mass loss (Sartori et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In type 2 diabetes mellitus (T2DM)-induced atrophy, skeletal muscle is characterized by elevated levels of muscle atrophy proteins, MuRF1 and Atrogin-1, which function as ubiquitin ligases and promote the proteolytic system that degrades structural proteins such as actin and myosin (Liu et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our results support this observation, as parts of the myofibrils in db/db mice exhibited significant structural damage, characterized by streaming and blurring in the light and dark bands of sarcomeres, as well as disorganization and clumpy electron density in the Z-disc. The alterations in the Z-disc are particularly concerning, as it plays a crucial role in mechanical stability, force transmission, and mechanotransduction. Disruption of proteins in the Z-disc can lead to the disintegration of the entire myofibril, resulting in increased protein degradation via the proteasome system (Mao and Nakamura \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Notably, the degradation and renewal of filamin C (FLNC), a key structural protein in the Z-disc, are specifically regulated through the autophagy-lysosomal pathway. Impairment of this pathway can lead to severe myofibril damage and muscle atrophy (Fujita et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Given that previous studies have shown that autophagy is inhibited in the skeletal muscle of db/db mice (Xiang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we speculate that autophagy impairment may exacerbate myofibril disintegration, thereby promoting proteasome-mediated degradation. This creates a vicious cycle that further exacerbates muscular atrophy in T2DM.\u003c/p\u003e \u003cp\u003eFurther investigation has confirmed that the disordered arrangement and aggregation of filamin C (FLNC) may be responsible for the alteration of the Z-disc. These results indicate that dysfunction in FLNC degradation leads to the disintegration of the Z-disc, which is associated with myofibrillar myopathies induced by type 2 diabetes mellitus (T2DM). The degradation and turnover of FLNC are conserved processes that involve specific chaperones, such as HSP70 and BAG3, which utilize a unique degradation pathway through autophagy rather than the proteasome system. Under normal conditions, a structural form of HSP70, known as HSC70, plays a crucial role in the assembly and metabolism of FLNC. Subsequently, with the mediation of BAG3, FLNC can be degraded through selective autophagy, a process referred to as chaperone-assisted selective autophagy (CASA), and may also be degraded by lysosomal pathways. These processes are essential for skeletal muscle to undergo critical structural metabolism and maintain the stability of myofibrils. Our previous study found that as T2DM progresses, autophagy is suppressed due to the upregulation of the mTOR pathway. Furthermore, in this study, we found that the lysosomal degradation system is also compromised, likely affected by the overactive proteasome system. Consequently, damaged FLNC fails to be effectively degraded by CASA and aggregates, which affects the stability of the Z-disc and causes myofibril disruption in the muscle fibers of db/db mice. This may underlie the mechanism that leads to the upregulation of atrophy-related proteins and the loss of skeletal muscle mass. Therefore, improving skeletal muscle mass in T2DM may depend on the restoration of autophagy.\u003c/p\u003e \u003cp\u003eIn clinical settings, metformin has been effective in glycemic control and improving insulin resistance, but it has shown limited efficacy in alleviating muscle atrophy in late-stage type 2 diabetes mellitus (T2DM) (Kang et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sanchez-Rangel and Inzucchi \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our previous research has also revealed that while metformin can effectively enhance the skeletal muscle insulin signaling pathway, it does not lead to an increase in muscle mass. The underlying reason for this may be that once autophagy impairment exacerbates the disintegration of myofibrils, it promotes proteasome-mediated degradation. Therefore, attempting to reverse muscle atrophy solely by improving the insulin signaling pathway is insufficient. Additionally, in this study, we observed the aggregation of filamin C (FLNC) and its chaperones within the myofibrils of the metformin group, indicating that metformin administration could increase the formation of autophagosomes in muscle fibers but failed to sufficiently activate lysosomes to degrade these aggregates. This phenomenon contradicts the classical understanding of metformin, which is thought to effectively activate autophagic flux through the AMPK pathway, primarily mediated by the inhibition of complex I in the mitochondrial oxidative respiratory chain, subsequently decreasing ATP synthesis (Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, some studies have also suggested that metformin may inhibit autophagic flux in skeletal muscle, myocardium, and tumor cells, potentially due to lysosomal inhibition caused by metformin (Malin and Stewart \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; L. \u003cem\u003eet al.\u003c/em\u003e 2019). The inadequate intracellular ATP levels induced by metformin may obstruct V-ATPase, which functions as a proton pump transporting H\u003csup\u003e+\u003c/sup\u003e ions into lysosomes, resulting in abnormal lysosomal acidification (Sugawara and Ogawa \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, recent research indicates that in the process of activating AMPK via the lysosomal pathway, metformin also inhibits V-ATPase activity, obstructing lysosomal maturation and acidification by directly binding to PEN2 (Ma et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this experiment, the inhibition of lysosomal activity in the metformin group further confirmed this mechanism. In conclusion, we discovered that metformin administration inhibited lysosomal hydrolysis activity and blocked the fusion of autophagosomes, leading to the accumulation of autophagosomes and impaired FLNC, which exacerbated the breakdown and hydrolysis of myofibrils. Similar phenomena have been reported in other studies, which found that metformin promoted the expression of atrophy-related proteins, MuRF1 and MAFbx32, and enhanced the activity of the ubiquitin-proteasome system (Kley et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSkeletal muscles require a substantial amount of ATP for physical work. According to the use and disuse theory, exercise is more effective than disuse in mitigating the occurrence and development of muscle atrophy, as evidenced by both clinical and basic research. Although both metformin and exercise upregulate AMPK, their regulatory mechanisms differ significantly. Metformin activates AMPK primarily through the inhibition of ATP synthesis, while exercise promotes this effect by enhancing ATP consumption and energy turnover in skeletal muscle (Spaulding and Yan \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tamargo-Gomez and Marino \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, exercise exerts long-term effects on skeletal muscle, enhancing mitochondrial respiration. This not only protects muscle cells from degradation due to insufficient lysosomal acidification resulting from low ATP levels (Heo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), but also facilitates the production of reactive oxygen species (ROS), which further promotes autophagy (Gharehbagh et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Our research verified this mechanism; impaired autophagic degradation via the lysosomal pathway was restored by aerobic exercise. Although the expression of HSP 70 was not significantly increased after exercise intervention in this work, which we thought due to the mice were stressed before being sacrificed. Aerobic exercise acted as optimal stimulation could increase the expression of HSP70 in muscle cells (Ohsawa and Kawano \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and it did strengthen the assembly of damaged FLNC with help of BAG 3. Consequently, the aggregation of FLNC significantly decreased, and the morphological structure of myofibrils improved. Interestingly, we observed a notable widening and deepening of electron density at the Z-disc, indicating muscle damage or atrophy following exercise intervention. However, considering the upregulation of HSP70 mediated by CASA in the exercise group, which facilitates the degradation and renewal of damaged FLNC, it is likely that the Z-disc was in a state of post-exercise repair rather than a pathological state of atrophy. While exercise can induce ultrastructural damage in skeletal muscle, such as a widened and disordered Z-disc, it also serves as a beneficial stimulus that effectively induces stress responses in skeletal muscle to initiate remodeling process (Orfanos et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This mechanism might also be relate to the role of CASA during exercise-induced myofibrillar injury and repair (Pasiakos et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, in our previous work, the combination of exercise and metformin demonstrated an unexpected antagonistic effect on the regulation of AMPK levels in skeletal muscle due to their contradictory AMPK activation mechanisms. Fortunately, despite the antagonism, the enhancement of exercise on mitochondrial respiration allowed autophagic flux to be reversed through the ROS pathway induced by exercise (Kim et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Triolo et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, in this experiment, the effects of exercise-induced degradation of FLNC through CASA were unaffected by metformin administration. Additionally, exercise compensated for the negative effects of metformin on lysosomes, leading to improved degradation and renewal of FLNC in skeletal muscle. Consequently, T2DM-induced myofibril disintegration decreased, and the state of muscle atrophy was ameliorated. Although no cumulative effects on skeletal muscle mass and muscle atrophy were observed in the combination group, surprisingly, the widening of the Z-disc in myofibrils caused by exercise was significantly improved by metformin administration. This phenomenon suggests that metformin may play a potential role in promoting the renewal of structural proteins, or that the combined intervention may have a synergistic effect on myofibril renewal. The specific mechanism still needs further investigation.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eWe propose for the first time that during the course of T2DM, abnormalities in autophagy in skeletal muscle lead to metabolic disorders of FLNC in the Z-disc, promoting the breakdown of the myofibrillar structure and the overactivity of the protease system, which ultimately contributes to muscle atrophy. The inhibition of lysosomal activity caused by metformin creates obstacles for CASA-mediated degradation of FLNC, resulting in a failure to improve muscle atrophy. In contrast to metformin, exercise not only promotes the CASA pathway but also increases lysosomal activity, significantly enhancing FLNC degradation and alleviating muscle atrophy, which may compensate for the shortcomings of metformin in combination treatment.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of Generative AI and AI‑assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work the authors did not use any AI-assisted technology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.L.: Conceptualization, Formal analysis, Investigation, Visualization, Writing – original draft, Writing-review\u0026amp;editing, Funding acquisition; M.X.: Formal analysis, Investigation, Visualization, Writing-review\u0026amp;editing; A.Z.: Investigation, Writing-review\u0026amp;editing; Y.X.: Investigation, Writing-review\u0026amp;editing; W.S.: Investigation, Writing-review\u0026amp;editing; H.X.: Investigation, Writing-review\u0026amp;editing; L.Z.: Investigation, Writing-review\u0026amp;editing; J.W.: Formal analysis, Investigation, Resources, Writing-review\u0026amp;editing; Q.T.: Conceptualization, Resources, Writing-review\u0026amp;editing; Y.Z.: Conceptualization, Investigation, Resources, Writing-review\u0026amp;editing,Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding source\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMajor Programs for Basic Science (Natural Science) Research in Higher Education Institutions in Jiangsu Province (24KJA310002);\u003c/p\u003e\n\u003cp\u003eYouth Project of National Natural Science Foundation of China (32000839)；\u003c/p\u003e\n\u003cp\u003eScientific research cultivation project of Nanjing Sport Institute (XSTD202408).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets used or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Animal Ethics and Welfare Committee of Nanjing Sport Institute (Nanjing,China) (Approval No. 2019-010).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003eAll authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e\n\u003cp\u003eThe authors have no financial or proprietary interests in any material discussed in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBULLARD B, PASTORE A (2019) Through Thick and Thin: Dual Regulation of Insect Flight Muscle and Cardiac Muscle Compared, \u003cem\u003eJournal of muscle research and cell motility\u003c/em\u003e, Vol. 4099\u0026thinsp;\u0026ndash;\u0026thinsp;110\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCHA-MOLSTAD H, FENG YUJE, LEE Z, KIM SH, YANG JG, HAN P, YOO BSUNGKW, SHIM YDHWANGJMCGUIRET, SONG SM, GANIPISETTI HD, WANG S, LEE NJANGJM, MOOK-JUNG MJKIMSJLEEKHHONGJTCIECHANOVERA, XIE IKIMKP (2017) X., KWON, Y. 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Nat Reviews Endocrinol 14(2):88\u0026ndash;98\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"exercise, metformin, T2DM, muscle atrophy, FLNC, autophagy","lastPublishedDoi":"10.21203/rs.3.rs-6749383/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6749383/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFLNC is localized in the Z-disc of skeletal muscle and plays an important role in maintaining mechanical stability. Its degradation and renewal are specifically mediated by autophagy. Previously, we revealed that exercise is superior to metformin in improving muscle atrophy by promoting autophagy and inhibiting the proteasome. Building on this foundation, we aim to explore whether exercise can compensate for the deficiency of metformin in enhancing autophagy-mediated FLNC degradation. In this study, db/db mice were used to establish a model of type 2 diabetes mellitus (T2DM). An exercise intervention was conducted using a treadmill protocol at speeds of 7\u0026ndash;12 m/min for 30\u0026ndash;40 minutes per day, five days a week. Metformin was administered daily via gavage at a dose of 300 mg/kg. A combined intervention was performed, in which metformin was administered first, followed by exercise. After eight weeks of intervention, the ultrastructure of skeletal muscle was evaluated using electron microscopy. The key proteins HSP70, BAG3, LC3, LAMP2, and Cathepsin D involved in BAG3-mediated FLNC degradation via selective autophagy were quantified using Western blotting and immunofluorescence co-localization. We discovered that abnormalities in autophagy within skeletal muscle lead to metabolic disorders of FLNC in the Z-disc, contributing to the breakdown of myofibrillar structure. Furthermore, compared to metformin, exercise enhanced FLNC degradation by promoting BAG3-mediated selective autophagy, which may compensate for the limitations of metformin in combination treatment.\u003c/p\u003e","manuscriptTitle":"Exercise compensates the metformin on T2DM-indcued muscle atrophy through autophagy prominent FLNC degradation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 21:28:39","doi":"10.21203/rs.3.rs-6749383/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4194c638-64d0-445d-a0fa-eb6d3ddc56e3","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-02T13:53:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-03 21:28:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6749383","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6749383","identity":"rs-6749383","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}