Differential Protein Metabolism and Regeneration in Gastrocnemius Muscles in High-fat Diet Fed Mice and Pre-hibernation Daurian Ground Squirrels: A Comparison between Pathological and Healthy Obesity

© 2021 Academia Sinica, Taiwan Open Access Differential Protein Metabolism and Regeneration in Gastrocnemius Muscles in High-fat Diet Fed Mice and Pre-hibernation Daurian Ground Squirrels: A Comparison between Pathological and Healthy Obesity Xia Yan1,2,§, Qiaohua Niu2,§, Xuli Gao1,2, Shenyang Shen2, Nan He2, Huiping Wang1,2, Rongrong Fang2, Yunfang Gao1,2,*, and Hui Chang1,2,* 1Shaanxi Key Laboratory for Animal Conservation, Northwest University, Xi’an, 710069, P.R. China. *Correspondence: E-mail: [email protected] (Chang); [email protected] (Y. Gao). Tel: +8615389060926. Fax: 029-88303572. E-mail: [email protected] (Yan); [email protected] (X. Gao); [email protected] (Wang) 2Key Laboratory of Resource Biology and Biotechnology in Western China (College of Life Sciences, Northwest University), Ministry of Education, Xi’an, 710069, P.R. China. E-mail: [email protected] (Niu); [email protected] (Shen); [email protected] (Fang) §XY and QN contributed equally to this work. Received 27 May 2020 / Accepted 31 December 2020 / Published 8 March 2021 Communicated by Pung-Pung Hwang We focused on pathological obesity induced by excessive fat intake (nutritional obesity) in non-hibernator and healthy obesity due to pre-hibernation (PRE) fat storage in hibernator to study the effects of different types of obesity on skeletal muscle protein metabolism and cell regeneration. Kunming mice were fed with high-fat diet for 3 months to construct a pathological obesity model. Daurian ground squirrels fattened naturally before hibernation were used as a healthy obesity model. Body weight, adipose tissue wet weight, gastrocnemius muscle wet weight, muscle fiber cross-sectional area (CSA) and fiber type distribution were measured. The protein expression levels related to protein degradation (MuRF-1, atrogin-1, calpain1, calpain2, calpastatin, desmin, troponin T, Beclin-1, LC3-II), protein synthesis (P-Akt, P-mTORC1, P-S6K1, P-4E-BP1) and cell regeneration (MyoD, myogenin, myostatin) were detected by Western blot. As a result, the body weight and adipose tissue wet weight were both significantly increased in high fat obese (OB) mice and pre-hibernation fat (PRE) ground squirrels. The muscle wet weight, ratio of muscle wet weight to body weight, and muscle fiber CSA were significantly decreased, while the percentage of MHC I fiber isoform was significantly increased in gastrocnemius muscle of OB mice compared with the control (CON) group. The protein expression levels of P-Akt, P-mTORC1, P-4E-BP1 and myogenin were significantly decreased, while those of calpain1, calpain2, MuRF-1 and myostatin were significantly increased in the OB mice. In the ground squirrels, the muscle wet weight, muscle fiber CSA and percentage of MHC I fiber isoform all showed no change in the gastrocnemius muscle in the PRE group compared with the summer active (SA) group. The protein expression levels of P-Akt, P-mTORC1, P-S6K1 and MyoD were significantly increased, while those of Beclin-1 and LC3-II were significantly decreased in the PRE ground squirrels. This study demonstrated that the decrease in protein expression levels in the Akt/ mTOR pathway (P-Akt, P-mTORC1 and P-4E-BP1) and cell regeneration (myogenin) and the increase in protein expression levels of the calpain pathway (calpain1 and calpain2) and ubiquitin-proteasome pathway (MuRF-1) were involved in the mechanism of muscle atrophy in gastrocnemius muscle of the pathologically obese Kunming mice induced by high-fat diet. In contrast, the increased protein expression Citation: Yan X, Niu Q, Gao X, Shen S, He N, Wang H, Fang R, Gao Y, Chang H. 2021. Differential protein metabolism and regeneration in gastrocnemius muscles in high-fat diet fed mice and pre-hibernation Daurian ground squirrels: a comparison between pathological and healthy obesity. Zool Stud 60:6. doi:10.6620/ZS.2021.60-06. Zoological Studies 60: 6 (2021) doi:10.6620/ZS.2021.60-06 1 © 2021 Academia Sinica, Taiwan levels of the Akt/mTOR pathway (P-Akt, P-mTORC1 and P-S6K1) and cell regeneration (MyoD), and the decreased protein expression levels of the autophagy lysosomal pathway (Beclin-1 and LC3-II) were involved in the mechanism of anti-atrophy in gastrocnemius muscle of the healthy obese ground squirrels fattened before hibernation. Key words: High-fat diet, Pre-hibernation fattening, Protein metabolism, Cell regeneration, Skeletal muscle atrophy. BACKGROUND Skeletal muscle accounts for more than 40% of a mammal’s weight, and is not only the main organ for providing physical strength and exercise but also the main place for material and energy consumption (Chang et al. 2016; Li and Ji 2018). Nutritional obesity causes skeletal muscle atrophy; it occurs in a variety of muscles, including the gastrocnemius muscle, soleus muscle and tibialis anterior muscle (Adhikary et al. 2019; Akhmedov and Berdeaux 2013; Lee et al. 2018; Tong et al. 2019), and is mainly characterized by a decrease in muscle mass, reduction in cross-sectional area of muscle fibers, and changes in the types of fiber and myosin isoforms (Dumitru et al. 2018; Tong et al. 2019). Meanwhile, the expressions of atrogin-1 and MuRF-1, as atrogenes, are up-regulated in muscle atrophy induced by obesity (Adhikary et al. 2019; Bodine and Baehr 2014). Interestingly, lipid-storing hibernating species acquire and store large amounts of fat during the prehibernation fattening stage, and then go into hibernation. After the fat storage and before hibernation, the weight of the hibernator is often increased greatly (Xing et al. 2012). However, at least in hibernators like Japanese black bears (Ursus thibetanus japonicus), the obesity in pre-hibernation is not accompanied by a series of health problems, such as type 2 diabetes and hyperglycemia, which are common in non-hibernators with the same percentage of fat (Kamine et al. 2012). Thus, the obesity in hibernators caused by the fattening before hibernation is called “healthy obesity” (Rigano et al. 2017). Our previous research showed that there was no change in the muscle wet weight and cross-sectional area in gastrocnemius muscle, plantaris muscle or extensor digitorum longus muscle between the pre-hibernation (PRE, squirrels that finished natural fattening in lateautumn) and the summer active (SA, squirrels that were in active state in June) Daurian ground squirrels (Spermophilus dauricus) (Zhang et al. 2019; Ma et al. 2019). Therefore, the gastrocnemius muscle, the largest muscle on the calf of the ground squirrel, does not undergo muscle atrophy during fat storage and before hibernation. Skeletal muscle atrophy is caused by an imbalance in protein synthesis and degradation (Schiaffino et al. 2013). Protein synthesis is mainly regulated by the insulin-like growth factor-1 (IGF-1)/protein kinase B (PKB, also called Akt) signaling pathway, which affects the transcription and translation processes in protein synthesis by activating the mammalian target of rapamycin (mTOR) (Andres-Mateos et al. 2013). In this pathway, ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) are the most critical downstream effectors of mammalian target of rapamycin in complex 1 (mTORC1) (Gao et al. 2018). The reduced activations of Akt, p70S6K and mTOR result in reduced protein synthesis and reduced muscle growth in the plantaris muscle of male obese C57BL/6 mice (Mus musculus) induced by a 14-week high-fat and low-carbohydrate diet, the gastrocnemius muscle of male obese Wistar rats (Rattus norvegicus) induced by a 16-week highfat and high-carbohydrate Western-type diet, and the gastrocnemius muscle of male obese C57BL/6 mice induced by a 10-week high-fat diet (Sishi et al. 2011; Sitnick et al. 2009; Tong et al. 2019). However, it has been reported that there is no difference in P-mTOR or P-Akt levels in the muscles of grizzly bears (Ursus arctos horribilis) after finishing fattening before hibernation (in October) compared with the SA period (in May) (Rigano et al. 2017). In view of the differential expression of proteins and the possible changes in skeletal muscle, it is necessary to further compare the expression of the Akt-mTOR signal in the two rodent obesity models. Protein degradation in skeletal muscle involves the calpain pathway, the ubiquitin-proteasome pathway, the autophagy lysosomal pathway and others (Jackman and Kandarian 2004). Calpain is the initiator of skeletal muscle protein degradation, which triggers the activation of the ubiquitin-proteasome pathway (Jackman and Kandarian 2004). Calpain1 can be activated by micromolar Ca2+, and calpain2 requires millimolar Ca2+ to be activated; these are the two most widely studied isozyme molecules (Goll et al. 2003). page 2 of 18Zoological Studies 60:06 (2021) © 2021 Academia Sinica, Taiwan Calpastatin is a specific, endogenous inhibitor of calpain (Goll et al. 2003). It has been reported that there are higher calpain1 and calpain2 mRNA levels in skeletal muscles in 5-month-old male obese (fa/fa) Zucker rats than in lean rats when they are in a sedentary state (Hsieh et al. 2008). However, our previous study reported that the protein expression levels of calpain1, calpain2 and calpastatin show no difference between the PRE and SA group in the lateral gastrocnemius muscle, plantaris muscle and extensor digitorum longus muscle in Daurian ground squirrels (Ma et al. 2019). The ubiquitin-proteasome pathway includes three catalytic proteins: ubiquitin-activating enzyme E1, ubiquitin-binding enzyme E2s and ubiquitinprotein ligase E3s (Hershko and Ciechanover 1998). Atrogin-1 and muscle ring finger-1 (MuRF-1) are two of the most common E3s (Glass 2005). MuRF-1 level was significantly increased by 97.4%, while atrogin-1 showed no change in the gastrocnemius muscle of male obese Wistar rats induced by 16 weeks of high-fat and high-carbohydrate Western-type diet feeding (Sishi et al. 2011). However, in Daurian ground squirrels, the expression levels of MuRF-1 and atrogin-1 in the lateral gastrocnemius muscle, plantaris muscle, extensor digitorum longus muscle were not changed between the pre-hibernation group and the SA group (Ma et al. 2019). The other pathway involved in protein degradation is the autophagy lysosomal pathway (Sandri 2008). Autophagy is a highly conserved pathway responsible for the hydrolysis of long-lived proteins and organelles in cells (Sala et al. 2014), which maintains homeostasis by degrading the cell’s own components and is usually activated in conditions of nutrient deprivation or starvation (Mizushima et al. 2004; Mordier et al. 2000). Beclin-1 is one of the important components of the Class III Phosphotidylinositol-3kinase (PI3KC3) complex and plays a necessary role in the process of autophagosome nucleation (Mei et al. 2016). Microtubule-associated protein 1A/1B-light chain 3 (LC3) is another important molecule near the downstream of the autophagic pathway and is involved in the formation of autophagosomes, which is an indicator of autophagy lysosomal pathway activation (Klionsky et al. 2012). Previous studies showed that the expression of LC3 did not change between the obesity and control groups in the gastrocnemius muscle of C57BL/6 male mice induced by a 10-week highfat diet (Herrenbruck and Bollinger 2019). Similarly, the expression of LC3, LC3-II/I and Beclin-1 was also not significantly different between the obesity and control group in the soleus muscle and plantaris muscle of female Sprague Dawley rats induced by a 16week high-fat diet (Campbell et al. 2015). However, the protein expression level of Beclin-1 in the soleus muscle was significantly increased, while the protein expression levels of LC3-II and the ratio of LC3-II/I were significantly decreased in pre-hibernation group compared with SA group in Daurian ground squirrels (Chang et al. 2020). Excessive protein degradation causes muscle mass loss and promotes muscle atrophy. Therefore, our study selected the largest skeletal muscle of the hind calf-the gastrocnemius muscle to study the role of the calpain pathway, the ubiquitin-proteasome pathway and the autophagy lysosomal pathway in muscle atrophy in the pathological obesity model and the healthy obesity model. Maintenance of skeletal muscle mass is also associated with cell regeneration (Sartorelli and Fulco 2004). During the regeneration of skeletal muscle cells, the expression of myogenicity differentiation factor (MyoD) and myogenin play an important role in the proliferation and differentiation of satellite cells (Akhmedov and Berdeaux 2013). However, myostatin, a negative regulator of cell regeneration, can reduce myocyte proliferation and differentiation (Lee 2004) and inhibit the expression of MyoD and myogenin (Akhmedov and Berdeaux 2013). It has been reported that the obese mice induced by a high-fat diet display attenuated muscle regeneration after injury more than control mice (D’Souza et al. 2015). In addition, the expression of myostatin in skeletal muscle was increased in high-fat diet and ob/ob mice as well as in extremely obese women (Allen et al. 2008; Hittel et al. 2009). Therefore, it is necessary to clarify whether the regeneration is different in skeletal muscle between the pathological obesity model and the healthy obesity model. Why do pre-hibernation ground squirrels prevent skeletal muscle atrophy caused by fattening before hibernation? Based on the previous research on protein metabolism, we hypothesize that the skeletal muscle can be maintained through regulating protein synthesis, degradation and cell regeneration in the healthy obesity ground squirrels caused by pre-hibernation fattening, which is different from the muscle atrophy in pathologically obese mice induced by high-fat diet. In this study, the Daurian ground squirrels after natural fattening were used as a healthy animal model of obesity, whereas the Kunming mice fed with highfat diet for 3 months were used as a pathological model of nutritional obesity. We examined the body weight, the adipose tissue wet weight, the muscle wet weight, the cross-sectional area, the muscle fiber type and the protein expressions of MuRF-1, atrogin-1, calpain1, calpain2, calpastatin, troponin T, desmin, Beclin-1, LC3-II, P-Akt, P-mTORC1, P-S6K1, P-4EBP1, MyoD, myogenin and myostatin, intending to compare and clarify the similarities and differences page 3 of 18Zoological Studies 60:06 (2021) © 2021 Academia Sinica, Taiwan of protein expression levels of key signal molecules related to protein metabolism and cell regeneration in gastrocnemius muscle between pathological obesity and healthy obesity, which will provide experimental evidence for screening key targets for anti-atrophy induced by obesity in fattening before hibernation. MATERIALS AND METHODS Animal model of pathological obesity All animal experiments were approved by the Experimental Animal Protection Committee of the Ministry of Health of the People’s Republic of China. Four-week-old male Kunming mice, weighing 23 to 25 grams, were purchased from Chengdu Dashuo Experimental Animal Company. The mice were placed in plastic cages with sawdust. There were 2–3 mice per cage, and food and water were provided ad libitum. They were kept in a standard animal room at 18–25℃ where the light and darkness corresponded to the local sunrise and sunset. The sawdust in the cage was replaced every two days to keep it clean. The mice were randomly divided into two groups after being fed a normal diet for one week. (1) Contol group (CON group, n = 6): fed with normal diet; (2) obesity group (OB group, n = 6): fed with high-fat diet, as the pathological obesity model. The normal feed and high-fat feed were provided by Chengdu Dashuo Experimental Animal Company. Individuals were then stored at room temperature and protected from light. The composition of normal diet and high fat diet is shown in table 1. After feeding for 3 months, both groups of mice were sacrificed and sampled. Animal model of healthy obesity As described by our laboratory previously (Chang et al. 2018), the Daurian ground squirrels were captured from the Weinan plain in the Shaanxi province of China. After returning to the laboratory, all ground squirrels were put in plastic cages with sawdust, with one ground squirrel per cage; food and water were provided ad libitum. The ground squirrels were maintained in an animal room at 18–25℃ where the light and darkness corresponded to the local sunrise and sunset. The sawdust in the cage was replaced every two days to keep it clean. There were two groups of ground squirrels in the experiment (n = 6, three females and three males in each group): (1) Summer active group (SA group): active squirrels in June with a body temperature (Tb) of 36–38°C; captured and sacrificed in end of June. (2) Pre-hibernation group (PRE group): squirrels that had finished fattening in nature; captured and sacrificed in late-autumn (end of September, 30–40 d before hibernation). Sample collection After the body weight was recorded, the mice were anesthetized with 2 mg/kg sodium pentobarbital intraperitoneally, while the ground squirrels were anesthetized with 90 mg/kg sodium pentobarbital intraperitoneally. Then the gastrocnemius muscles from both legs were separated and weighed immediately. Subsequently, the muscle samples were frozen in liquid nitrogen and stored at -80℃ for follow-up experiments. The adipose tissues—including mesenteric adipose, perirenal adipose and back scapula subcutaneous adipose—were also separated and weighed. The animals were sacrificed by an overdose injection of sodium pentobarbital after sampling was completed. Immunofluorescent analysis The muscle fiber cross-sectional area (CSA) and fiber type composition were measured by Table 1. Composition of the experimental diets (A) Composition of normal diet (B) Composition of high fat diet Components Content Components Content Corn 25.4 g Patterned animal base 50 g Wheat 30.6 g Soy flour 5 g Soybean 13 g Fish meal 5 g Fish meal 6 g Milk powder 10 g Rice bran 6 g Peanut 6 g Wheat bran 10 g Egg yolk powder 5 g Soybean meal 5 g Lard 12 g Other 4 g Salt 2 g Total 100g Sucrose 5 g page 4 of 18Zoological Studies 60:06 (2021) © 2021 Academia Sinica, Taiwan immunofluorescent analysis, as described by our laboratory previously (Chang et al. 2018). Briefly, the mid-belly of gastrocnemius muscle was cut into 10-μm thick frozen muscle cross-sections at -20℃ with a cryostat (Leica, Wetzlar, CM1850, Germany). Then the slides with sections were fixed in 4% paraformaldehyde for 30 min, and subsequently permeabilized in 0.1% Triton X-100 (dissolve in PBS) for 30 min. The slides were then blocked with 1% bovine serum albumin (BSA) in PBS for 60 min at room temperature, and immediately incubated at 4℃ overnight with the antilaminin rabbit polyclonal antibody (1:200, Boster, BA1761-1) to visualize the myofiber interstitial tissue and the anti-skeletal slow myosin mouse monoclonal antibody (1:200, Boster, BM1533) to visualize the type I MHC in both muscles. The slides were rinsed twice in PBS and incubated with Alexa Fluor 647-labeled IgG secondary antibody (1:200; Thermo Fisher Scientific, #A21245) and FITC-labeled IgG secondary antibody (1:200; Sigma, F1010) for 60 min. Image-Pro Plus 6.0 software was used to measure the CSA of at least 10 different visual fields of each sample. Western blots As described previously by our lab (Chang et al. 2018). The total protein, extracted from the frozen gastrocnemius muscles of mice and ground squirrels by homogenization, was put into a sample buffer (pH 6.8, 100 mM Tris, 4% SDS, 5% glycerol, 5% 2-β-mercaptoethanol, and bromophenol blue). Then, the muscle protein extracts were separated by SDS-PAGE (10% Laemmli gels with an acrylamide/bisacrylamide ratio of 29:1 for P-Akt, P-mTORC1, P-S6K1, P-4EBP1, calpain1, calpain2, calpastatin, troponin T, desmin, atrogin-1, MuRF-1, Beclin-1, LC3, MyoD, myogenin and myostatin). After electrophoresis, the total protein bands were visualized by putting the gel on the UV transilluminator and irradiating for 2 min; the Syngene G:BOX system (Syngene, Frederick, MD) was then used to take photographs of the gel. The protein gel was transferred electrically to PVDF membranes (0.45 μm) using a Bio-Rad semi-dry transfer apparatus. Then, the membranes were blocked with 5% skim milk dissolved in TBST (10 mM Tris–HCl, 150 mM NaCl, 0.05% Tween-20. pH 7.6) for 2 h at room temperature and incubated with the primary antibodies of P-Akt (Ser473) (1:1,000, abcam, 81283), P-mTORC1 (Ser2448) (1:1,000, sigma, 4504476), P-S6K1 (Thr389) (1:1,000, Cell Signaling Technology (CST), 9205S), P-4E-BP1 (Thr37/46) (1:1,000, CST, 2855S), calpain1 (1:1,000, CST, 2556S), calpain2 (1:1,000, CST, 2539S), calpastatin (1:1,000, CST, 4146S), troponin T (1:1,000, Sigma, T6277), desmin (1:1,000, CST, 4024S), atrogin-1 (1:1,000, Proteintech, 12866-1-AP), MuRF-1 (1:1000, Abcam, ab172479), Beclin-1 (1:1000, CST, 3738), LC3 (1:1000, abcam, ab48394), MyoD (1:1,000, proteintech, 18943-1-AP), myogenin (1:1,000, abcam, 124800) and myostatin (1:1,000, proteintech, 19142-1-AP) in TBST containing 0.1% bovine serum albumin (BSA) at 4℃ overnight. Then the membrane was washed with TBST four times (10 minutes/time) and incubated with HRPconjugated anti-mouse secondary antibody (1:10000, Thermo Fisher Scientific, A28177) or HRP-conjugated anti-rabbit secondary antibody (1:5000, Thermo Fisher Scientific, A27036) for 2 h at room temperature. Then the PVDF membrane was washed three times (10 min/ time). The fluorescent bands were visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific, NCI5079). The NIH Image J software was used to carry out quantification analysis. The density of the immunoblot band in each individual lane was standardized using the summed densities from a group of total protein bands in the same lane. Statistical analyses An independent-samples t-test was used to determine the significant differences between OB and CON mice, SA and PRE ground squirrels. All western blot data were corrected for false discovery rate (FDR). All data were analyzed using SPSS 24 and expressed as means ± SEM. p < 0.05 was considered to be statistically significant. RESULTS Body weight The composition of normal diet and high fat diet is shown in table 1. The fat calorie percentage of high fat diet is 61%. As shown in table 2, after being fed a high-fat diet or normal diet for 3 months, the OB mice gained 10.6% higher body weight than the CON group (CON: 46.3 ± 0.5 g vs. OB: 51.2 ± 1.1 g, p < 0.05). In ground squirrels, the body weight of the PRE group was significantly increased by 67.9% (SA: 200.3 ± 2.0 g vs. PRE: 336.3 ± 15.6 g, p < 0.05) compared with the SA group at experiment time. Adipose tissue wet weight, muscle wet weight and the ratio of muscle wet weight to body weight As shown in table 3, compared with the CON mice, the mesenteric adipose wet weight in the OB group was significantly increased by 1.1-fold from page 5 of 18Zoological Studies 60:06 (2021) © 2021 Academia Sinica, Taiwan 0.89 g to 1.89 g (p < 0.05), while the perirenal adipose wet weight was significantly increased by 1.1-fold from 0.30 g to 0.63 g (p < 0.05). Compared with the SA ground squirrels, the mesenteric adipose wet weight in the PRE group was significantly increased by 24.4-fold from 0.74 g to 18.78 g (p < 0.05), the perirenal adipose wet weight was significantly increased by 8.8-fold from 0.32 g to 3.13 g (p < 0.05), and the subcutaneous adipose wet weight was significantly increased by 34.6fold from 0.35 g to 12.47 g (p < 0.05). As shown in figure 1A, the wet weight of the gastrocnemius muscle in the OB mice was significantly reduced by 8.8% from 361.75 mg to 329.80 mg compared with the CON group (p < 0.05). However, there was no change in the gastrocnemius muscle wet weight between the PRE and SA ground squirrels. As shown in figure 1B, the ratio of muscle wet weight/body weight was significantly decreased by 17.4% in the OB mice compared with the CON group (p < 0.05), while the ratio was significantly decreased by 40.3% in the PRE ground squirrels compared with the SA group (p < 0.05). Fiber type distribution and fiber CSA The fiber type distribution and CSA of the Table 3. Adipose tissue wet weight CON OB SA PRE Mesenteric adipose wet weight (g) 0.89 ± 0.12 1.89 ± 0.21 * 0.74 ± 0.20 18.78 ± 2.74 # Perirenal adipose wet weight (g) 0.30 ± 0.03 0.63 ± 0.08 * 0.32 ± 0.13 3.13 ± 0.37 # Subcutaneous adipose wet weight (g) - - 0.35 ± 0.17 12.47 ± 1.15 # CON: control mice, OB: mice after 3 months of ingesting high fat diet, SA: summer active Daurian ground squirrels, PRE: pre-hibernation, squirrels that finished natural fattening, sacrificed in late-autumn (end of September, 30–40 d before hibernation). Values are mean ± SEM, n = 6. *p < 0.05 compared with CON group. #p < 0.05 compared with SA group. Table 2. Body weight for all groups CON OB SA PRE Body weight before high fat-fed (g) 24.1 ± 0.3 24.5 ± 0.2 - - Body weight at experiment time (g) 46.3 ± 0.5 51.2 ± 1.1 * 200.3 ± 2.0 336.3 ± 15.6 # CON: control mice, OB: mice after 3 months of ingesting high fat diet, SA: summer active Daurian ground squirrels, PRE: pre-hibernation, squirrels that finished natural fattening, sacrificed in late-autumn (end of September, 30–40 d before hibernation). Values are mean ± SEM, n = 6. *p < 0.05 compared with CON group. #p < 0.05 compared with SA group. Fig. 1. Skeletal muscle wet weight and the ratio of muscle wet weight/body weight. (A) The changes in muscle wet weight in mice and Daurian ground squirrels. (B) The changes in the ratio of muscle wet weight/body weight in mice and Daurian ground squirrels. CON: control mice, OB: mice after 3 months of ingesting high fat diet, SA: summer active Daurian ground squirrels, PRE: pre-hibernation, squirrels that finished natural fattening, sacrificed in late-autumn (end of September, 30-40 d before hibernation). Values are mean ± SEM, n = 6. *p < 0.05 compared with CON group. #p < 0.05 compared with SA group. page 6 of 18 Zoological Studies 60:06 (2021) © 2021 Academia Sinica, Taiwan gastrocnemius muscle fibers were measured by immunofluorescent staining. As shown in figure 2B, the percentage of MHC I fiber isoform in the gastrocnemius muscles was significantly increased (58.1%, p < 0.05) in the OB mice compared with the CON group, whereas it had no significant difference between the PRE and SA ground squirrels. Compared with the CON mice, the CSA of the gastrocnemius muscle was significantly reduced (-14.7%, p < 0.05) in the OB group, whereas it was not significantly different between the PRE and SA ground squirrels (Fig. 2C). Relative protein levels of MuRF-1 and atrogin-1 We analyzed the expression levels of MuRF-1 and atrogin-1, which are molecular markers of muscle atrophy and the E3s in the ubiquitin proteasome pathway (Bodine et al. 2001). Compared with the CON mice, the expression of MuRF-1 was significantly increased in the OB group (18.1%, p < 0.05), while it was not significantly different between the PRE and SA ground squirrels (Fig. 3B). The expression level of atrogin-1 showed no change in the CON or OB mice. In addition, there was no significant difference in the PRE or SA ground squirrels (Fig. 3C). Relative protein levels of calpain1, calpain2, calpastatin, desmin and troponin T We determined the molecules in the calpain pathway that initiate protein degradation, including calpain1 and calpain2, which act on degradation, their inhibitor calpastatin, and their substrates desmin and troponin T (Yang et al. 2014). The expression levels of calpain1 and calpain2 were significantly increased in the gastrocnemius muscle in the OB mice (60.1% and 22.4%, respectively, p < 0.05) compared with the CON group, while there was no significant difference between the PRE and SA ground squirrels (Fig. 4B and 4C). The expression level of calpastatin showed no significant difference between the OB and CON mice, or between the PRE and SA ground squirrels (Fig. 4D). As the substrates of calpain, both desmin and troponin T showed a significant decrease in the OB mice compared with the CON group (-33.6% and -29.8%, respectively, Fig. 2. MHC I isoform distribution and single fiber cross-sectional area (CSA). (A) Representative immunofluorescent images of MHC I (green) fibers in gastrocnemius muscles in each group. Red represents laminin stain of myofiber interstitial tissue. Scale bar = 50 μm. (B) The changes in MHC I isoform distribution (%) in mice and Daurian ground squirrels. (C) The changes in muscle single fiber CSA in mice and Daurian ground squirrels. CON: control mice, OB: mice after 3 months of ingesting high fat diet, SA: summer active Daurian ground squirrels, PRE: pre-hibernation, squirrels that finished natural fattening, sacrificed in late-autumn (end of September, 30–40 d before hibernation). Values are mean ± SEM, n = 6. *p < 0.05 compared with CON group. page 7 of 18Zoological Studies 60:06 (2021) © 2021 Academia Sinica, Taiwan p < 0.05). However, both desmin and troponin T was not significantly different between the PRE and SA ground squirrels (Fig. 4E and 4F). Relative protein levels of Beclin-1 and LC3-II Beclin-1 and LC3-II are important molecules in the autophagy pathway of protein degradation (Chang et al. 2020). Compared with the CON mice, the expression levels of Beclin-1 and LC3-II had no significant differences in the OB group. However, both Beclin-1 and LC3-II were significantly decreased in the PRE ground squirrels compared with the SA group (-11.8% and -35.5%, respectively, p < 0.05, Fig. 5B and 5C). Relative protein levels of P-Akt, P-mTORC1, P-S6K1 and P-4E-BP1 The Akt//mTOR pathway is an important way to regulate protein synthesis, and S6K1 and 4EBP1 are the most important effectors downstream of mTORC1 (Schiaffino et al. 2013). Here, we measured the expression levels of phosphorylated Akt, mTORC1, S6K1 and 4E-BP1 to compare the differences in protein synthesis in obesity mice and ground squirrels. As shown in figure 6, the relative protein levels of P-Akt and P-mTORC1 were significantly decreased in the OB mice compared with the CON group (-33.8% and -25.0%, respectively, p < 0.05). In contrast, both P-Akt and P-mTORC1 were significantly increased in the PRE ground squirrels compared with the SA group (22.3% and 28.9%, respectively, p < 0.05, Fig. 6B and 6C). The relative protein level of P-S6K1 showed no change between the OB and CON group in mice (Fig. 6D), while it was significantly increased in the PRE ground squirrels compared with that in the SA group (30.5%, p < 0.05, Fig. 6D). In addition, the protein expression level of P-4E-BP1 in the OB mice was significantly decreased compared with the CON group (-26.1%, p < 0.05), while it showed an increased trend but no significant difference in the PRE ground squirrels compared with that in the SA group (40.1%, p = 0.05, Fig. 6E). Fig. 3. The protein expression levels of MuRF-1 and atrogin-1. (A) Representative immunoblots of MuRF-1 and atrogin-1 in gastrocnemius muscles in each group. (B) The relative protein expression level of MuRF-1 in mice and Daurian ground squirrels. (C) The relative protein expression level of atrogin-1 in mice and Daurian ground squirrels. CON: control mice, OB: mice after 3 months of ingesting high fat diet, SA: summer active Daurian ground squirrels, PRE: pre-hibernation, squirrels that finished natural fattening, sacrificed in late-autumn (end of September, 30–40 d before hibernation). Values are mean ± SEM, n = 6. *p < 0.05 compared with CON group. page 8 of 18Zoological Studies 60:06 (2021) © 2021 Academia Sinica, Taiwan Fig. 4. The protein expression levels of calpain1, calpain2, calpastatin, desmin and troponin T. (A) Representative immunoblots of calpain1, calpain2, calpastatin, desmin and troponin T in gastrocnemius muscles in each group. (B) The relative protein expression level of calpain1 in mice and Daurian ground squirrels. (C) The relative protein expression level of calpain2 in mice and Daurian ground squirrels. (D) The relative protein expression level of calpastatin in mice and Daurian ground squirrels. (E) The relative protein expression level of desmin in mice and Daurian ground squirrels. (F) The relative protein expression level of troponin T in mice and Daurian ground squirrels. CON: control mice, OB: mice after 3 months of ingesting high fat diet, SA: summer active Daurian ground squirrels, PRE: pre-hibernation, squirrels that finished natural fattening, sacrificed in late-autumn (end of September, 30-40 d before hibernation). Values are mean ± SEM, n = 6. *p < 0.05 compared with CON group. page 9 of 18Zoological Studies 60:06 (2021) © 2021 Academia Sinica, Taiwan al. 2012). Desmin is not only a substrate of the calpain system, but also an intermediate filament protein expressed during muscle differentiation. A decrease in desmin is a signal of impaired muscle fiber maturation (Hawke et al. 2003). Our results showed that desmin expression was significantly decreased in the OB mice, but showed no change in the PRE ground squirrels, which suggested that the muscle fiber maturation is impaired in the OB mice, but not in the PRE ground squirrels. Thus, pathological obesity induced by a highfat diet caused a decline in the expressions of proteins related to cell regeneration, while healthy obesity caused by pre-hibernation fattening showed an increase in protein expressions related to cell regeneration, which might be involved in the mechanism of muscle atrophy and anti-muscle atrophy in obese mice and ground squirrels, respectively. CONCLUSIONS In conclusion, the gastrocnemius muscle showed atrophy in pathological obese mice induced by highfat diet, but it showed anti-atrophy in healthy obese ground squirrels caused by pre-hibernation fattening. The decrease in the protein expression levels of the Akt/mTOR pathway and cell regeneration, the increase in the protein expression levels of the calpain system and ubiquitin-proteasome pathway were involved in the mechanism of muscle atrophy in the gastrocnemius muscle of the OB mice. However, the increased protein expression levels of the Akt/mTOR pathway and cell regeneration, and the decreased protein expression levels of the autophagy lysosomal pathway, were involved in the mechanism of anti-atrophy in gastrocnemius muscle of the healthy obese ground squirrels fattened before hibernation. The present study not only clarified the differential protein metabolism and regeneration in gastrocnemius muscles in highfat diet fed mice and pre-hibernation Daurian ground squirrels, but also provided experimental evidence for screening key targets for anti-obesity muscular atrophy in fattening ground squirrels before hibernation. List of abbreviations OB, obesity group. CON, control group. PRE, pre-hibernation. SA, summer active. IGF-1, insulin like grow factor-1. Akt, protein kinase B. mTORC1, mammalian target of rapamycin in complex 1. S6K1, ribosomal protein S6 kinase 1. 4E-BP1, 4E binding protein 1. MuRF-1, muscle ring finger-1. LC3, Microtubule-associated protein 1A/1B-light chain 3. MyoD, myogenicity differentiation factor. CSA, cross-sectional area. TNF-α, Tumor necrosis factor α. NF-κB, Nuclear factor kappa-B. IKKb, IkB kinase b. p38 MAPK, p38 mitogen-activated protein kinase. Acknowledgments: This study was supported by funds from the National Nature Science Foundation of China (31640072), the Shaanxi Province Natural Science Basic Research Program (2020JM-428). We sincerely thank Yihan Wang and Yutong Wang for helping us revise the English writing. Authors’ contributions: Conceived and designed the experiments: HC and YG. Performed the experiments: XY, QN, XG, SS, NH and RF. Analyzed the data: HC, XY, QN and HW. Wrote the paper: QN and HC. All authors read and approved the final manuscript. Competing interests: XY, QN, XG, SS, NH, HW, RF, YG, and HC declare that they have no conflict of interests. Availability of data and materials: The datasets used or analysed during the current study are available from the corresponding author upon reasonable request. Consent for publication: All authors have read and approved the submission of this manuscript to Zoological Studies. Ethics approval consent to participate: All animal experiments were approved by the Experimental Animal Protection Committee of the Ministry of Health of the People’s Republic of China. REFERENCES Abrigo J, Rivera JC, Aravena J, Cabrera D, Simon F, Ezquer F et al. 2016. High fat diet-induced skeletal muscle wasting is decreased by mesenchymal stem cells administration: Implications on oxidative stress, ubiquitin proteasome pathway activation, and myonuclear apoptosis. Oxid Med Cell Longev. doi:10.1155/2016/9047821. Adhikary S, Kothari P, Choudhary D, Tripathi AK, Trivedi R. 2019. Glucocorticoid aggravates bone micro-architecture deterioration and skeletal muscle atrophy in mice fed on high-fat diet. Steroids 149:108416. doi:10.1016/j.steroids.2019.05.008. page 16 of 18Zoological Studies 60:06 (2021) © 2021 Academia Sinica, Taiwan Akhmedov D, Berdeaux R. 2013. The effects of obesity on skeletal muscle regeneration. Front Physiol 4:371. doi:10.3389/ fphys.2013.00371. Allen DL, Cleary AS, Speaker KJ, Lindsay SF, Uyenishi J, Reed JM et al. 2008. Myostatin, activin receptor iib, and follistatinlike-3 gene expression are altered in adipose tissue and skeletal muscle of obese mice. Am J Physiol-Endoc M 294:E918–E927. doi:10.1152/ajpendo.00798.2007. Andres-Mateos E, Brinkmeier H, Burks TN, Mejias R, Files DC, Steinberger M et al. 2013. Activation of serum/glucocorticoidinduced kinase 1 (SGK1) is important to maintain skeletal muscle homeostasis and prevent atrophy. EMBO Mol Med 5:80–91. doi:10.1002/emmm.201201443. Ballinger MA, Andrews MT. 2018. Nature’s fat-burning machine: Brown adipose tissue in a hibernating mammal. J Exp Biol 221(Pt Suppl 1):jeb162586. doi:10.1242/jeb.162586. Bodine SC, Baehr LM. 2014. Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1. Am J PhysiolEndoc M 307:E469–E484. doi:10.1152/ajpendo.00204.2014. Bodine SC, Latres E, Baumhueter S, Lai VKM, Nunez L, Clarke BA et al. 2001. Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294:1704–1708. doi:10.1126/ science.1065874. Bonaldo P, Sandri M. 2013. Cellular and molecular mechanisms of muscle atrophy. Dis Model Mech 6:25–39. doi:10.1242/ dmm.010389. Borst SE, Conover CF. 2005. High-fat diet induces increased tissue expression of TNF-α. Life Sci 77:2156–2165. doi:10.1016/ j.lfs.2005.03.021. Campbell TL, Mitchell AS, McMillan EM, Bloemberg D, Pavlov D, Messa I et al. 2015. High-fat feeding does not induce an autophagic or apoptotic phenotype in female rat skeletal muscle. Exp Biol Med (Maywood) 240:657–668. doi:10.1177/1535370214557223. Chang H, Jiang S-F, Dang K, Wang H-P, Xu S-H, Gao Y-F. 2016. iTRAQ-based proteomic analysis of myofibrillar contents and relevant synthesis and proteolytic proteins in soleus muscle of hibernating Daurian ground squirrels (Spermophilus dauricus). Proteome Sci 14:16. doi:10.1186/s12953-016-0105-x. Chang H, Lei T, Ma X, Zhang J, Wang H, Zhang X et al. 2018. Muscle-specific activation of calpain system in hindlimb unloading rats and hibernating daurian ground squirrels: A comparison between artificial and natural disuse. J Comp Physiol B 188:863–876. doi:10.1007/s00360-018-1176-z. Chang H, Peng X, Yan X, Zhang J, Xu S, Wang H et al. 2020. Autophagy and Akt-mTOR signaling display periodic oscillations during torpor-arousal cycles in oxidative skeletal muscle of Daurian ground squirrels (Spermophilus dauricus). J Comp Physiol B 190:113–123. doi:10.1007/s00360-019-012455. Chen B, Shan T. 2019. The role of satellite and other functional cell types in muscle repair and regeneration. J Muscle Res Cell M 40:1–8. doi:10.1007/s10974-019-09511-3. De Larichaudy J, Zufferli A, Serra F, Isidori AM, Naro F, Dessalle K et al. 2012. Tnf-αand tumor-induced skeletal muscle atrophy involves sphingolipid metabolism. Skelet Muscle 2:2. doi:10.1186/2044-5040-2-2. D’Souza DM, Trajcevski KE, Al-Sajee D, Wang DC, Thomas M, Anderson JE et al. 2015. Diet-induced obesity impairs muscle satellite cell activation and muscle repair through alterations in hepatocyte growth factor signaling. Physiol Rep 3:e12506. doi:10.14814/phy2.12506. Dina A, Inga R, Alexandra V, Eitan S, Shenhav C. 2018. Gsk3-β promotes calpain-1-mediated desmin filament depolymerization and myofibril loss in atrophy. J cell biol 217:3698–3714. doi:10.1083/jcb.201802018. Dumitru A, Radu BM, Radu M, Cretoiu SM. 2018. Muscle changes during atrophy. In: J. Xiao (ed.) Muscle atrophy, Adv Exp Med Biol No. 1088. pp. 73–92. doi:10.1007/978-981-13-1435-3_4. Egerman MA, Glass DJ. 2014. Signaling pathways controlling skeletal muscle mass. Crit Rev Biochem Mol 49:59–68. doi:10.3 109/10409238.2013.857291. Fu WW, Hu HX, Dang K, Chang H, Du B, Wu X et al. 2016. Remarkable preservation of Ca2+ homeostasis and inhibition of apoptosis contribute to anti-muscle atrophy effect in hibernating daurian ground squirrels. Sci Rep-UK 6:27020. doi:10.1038/ srep27020. Gao Y, Arfat Y, Wang H, Goswami N. 2018. Muscle atrophy induced by mechanical unloading: Mechanisms and potential countermeasures. Front Physiol 9:235. doi:10.3389/ fphys.2018.00235. Glass DJ. 2005. Skeletal muscle hypertrophy and atrophy signaling pathways. Int J Biochem Cell B 37:1974–1984. doi:10.1016/ j.biocel.2005.04.018. Goll DE, Thompson VF, Li H, Wei W, Cong J. 2003. The calpain system. Physiol Rev 83:731–801. doi:10.1152/ physrev.00029.2002. Hawke TJ, Meeson AP, Jiang N, Graham S, Hutcheson K, DiMaio JM et al. 2003. P21 is essential for normal myogenic progenitor cell function in regenerating skeletal muscle. Am J Physiol-Cell Ph 285:C1019–C1027. doi:10.1152/ajpcell.00055.2003. Herrenbruck AR, Bollinger LM. 2019. Role of skeletal muscle autophagy in high-fat-diet-induced obesity and exercise. Nutr Rev 78:56–64. doi:10.1093/nutrit/nuz044. Hershko A, Ciechanover A. 1998. The ubiquitin system. Annu rev biochem 67:425–479. doi:10.1146/annurev.biochem.67.1.425. Hittel DS, Berggren JR, Shearer J, Boyle K, Houmard JA. 2009. Increased secretion and expression of myostatin in skeletal muscle from extremely obese women. Diabetes 58:30–38. doi:10.2337/db08-0943. Hsieh YY, Chang CC, Hsu KH, Tsai FJ, Chen CP, Tsai HD. 2008. Effect of exercise training on calpain systems in lean and obese Zucker rats. Int J Biol Sci 4:300–308. doi:10.7150/ijbs.4.300. Huang JR, Zhu XP. 2016. The molecular mechanisms of calpains action on skeletal muscle atrophy. Physiol Res 65:547–560. doi:10.33549/physiolres.933087. Jackman RW, Kandarian SC. 2004. The molecular basis of skeletal muscle atrophy. Am J Physiol-Cell Ph 287:C834–C843. doi:10.1152/ajpcell.00579.2003. Kamine A, Shimozuru M, Shibata H, Tsubota T. 2012. Changes in blood glucose and insulin responses to intravenous glucose tolerance tests and blood biochemical values in adult female Japanese black bears (Ursus thibetanus japonicus). Jpn J Vet Res 60:5–13. doi:10.14943/jjvr.60.1.5. Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, AcevedoArozena A, Adeli K et al. 2012. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8:445–544. doi:10.4161/auto.19496. Lee S, Kim M-B, Kim C, Hwang J-K. 2018. Whole grain cereal attenuates obesity-induced muscle atrophy by activating the PI3K/Akt pathway in obese C57BL/6N mice. Food Sci Biotechnol 27:159–168. doi:10.1007/s10068-017-0277-x. Lee SJ. 2004. Regulation of muscle mass by myostatin. Annu Rev Cell Dev Biol 20:61–86. doi:10.1146/annurev. cellbio.20.012103.135836. Li Z, Ji GE. 2018. Ginseng and obesity. J Ginseng Res 42:1–8. doi:10.1016/j.jgr.2016.12.005. Lokireddy S, Wijesoma IW, Sze SK, McFarlane C, Kambadur page 17 of 18Zoological Studies 60:06 (2021) © 2021 Academia Sinica, Taiwan R, Sharma M. 2012. Identification of atrogin-1-targeted proteins during the myostatin-induced skeletal muscle wasting. Am J Physiol-Cell Ph 303:C512–C529. doi:10.1152/ ajpcell.00402.2011. Ma X, Chang H, Wang Z, Xu S, Peng X, Zhang J et al. 2019. Differential activation of the calpain system involved in individualized adaptation of different fast-twitch muscles in hibernating daurian ground squirrels. J Appl Physiol (Bethesda, Md. : 1985) 127:328–341. doi:10.6084/m9.figshare.8061893. Mei Y, Ramanathan A, Glover K, Stanley C, Sanishvili R, Chakravarthy S et al. 2016. Conformational flexibility enables the function of a BECN1 region essential for starvation-mediated autophagy. Biochemistry 55:1945–1958. doi:10.1021/acs. biochem.5b01264. Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y. 2004. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 15:1101–1111. doi:10.1091/mbc.e03-090704. Mordier S, Deval C, Bechet D, Tassa A, Ferrara M. 2000. Leucine limitation induces autophagy and activation of lysosomedependent proteolysis in C2C12 myotubes through a mammalian target of rapamycin-independent signaling pathway. J Biol Chem 275:29900–29906. doi:10.1074/jbc.M003633200. Ohno Y, Matsuba Y, Hashimoto N, Sugiura T, Ohira Y, Yoshioka T et al. 2016. Suppression of myostatin stimulates regenerative potential of injured antigravitational soleus muscle in mice under unloading condition. Int J Med Sci 13:680–685. doi:10.7150/ ijms.16267. Rigano KS, Gehring JL, Evans Hutzenbiler BD, Chen AV, Nelson OL, Vella CA et al. 2017. Life in the fat lane: Seasonal regulation of insulin sensitivity, food intake, and adipose biology in brown bears. J Comp Physiol B 187:649–676. doi:10.1007/s00360-0161050-9. Sala D, Ivanova S, Plana N, Ribas V, Duran J, Bach D et al. 2014. Autophagy-regulating TP53INP2 mediates muscle wasting and is repressed in diabetes. J Clin Invest 124:1914–1927. doi:10.1172/ JCI72327. Sandri M. 2008. Signaling in muscle atrophy and hypertrophy. Physiology 23:160–170. doi:10.1152/physiol.00041.2007. Sartorelli V, Fulco M. 2004. Molecular and cellular determinants of skeletal muscle atrophy and hypertrophy. Sci STKE 2004:re11. doi:10.1126/stke.2442004re11. Sartori R, Milan G, Patron M, Mammucari C, Blaauw B, Abraham R et al. 2009. Smad2 and 3 transcription factors control muscle mass in adulthood. Am J Physiol Cell Physiol 296:C1248–1257. doi:10.1152/ajpcell.00104.2009. Schiaffino S, Dyar KA, Ciciliot S, Blaauw B, Sandri M. 2013. Mechanisms regulating skeletal muscle growth and atrophy. Febs J 280:4294–4314. doi:10.1111/febs.12253. Schiaffino S, Mammucari C. 2011. Regulation of skeletal muscle growth by the IGF1-Akt/PKB pathway: Insights from genetic models. Skelet Muscle 1:4. doi:10.1186/2044-5040-1-4. Shortreed KE, Krause MP, Huang JH, Dhanani D, Moradi J, Ceddia RB et al. 2009. Muscle-specific adaptations, impaired oxidative capacity and maintenance of contractile function characterize diet-induced obese mouse skeletal muscle. PLoS ONE 4:e7293. doi:10.1371/journal.pone.0007293. Sishi B, Loos B, Ellis B, Smith W, du Toit EF, Engelbrecht A-M. 2011. Diet-induced obesity alters signalling pathways and induces atrophy and apoptosis in skeletal muscle in a prediabetic rat model. Exp Physiol 96:179–193. doi:10.1113/ expphysiol.2010.054189. Sitnick M, Bodine SC, Rutledge JC. 2009. Chronic high fat feeding attenuates load-induced hypertrophy in mice. J Physiol-London 587:5753–5765. doi:10.1113/jphysiol.2009.180174. Smith IJ, Lecker SH, Hasselgren PO. 2008. Calpain activity and muscle wasting in sepsis. Am J Physiol-Endoc M 295:E762– E771. doi:10.1152/ajpendo.90226.2008. Smith RE, Hock RJ. 1963. Brown fat: Thermogenic effector of arousal in hibernators. Science 140:199–200. doi:10.1126/ science.140.3563.199. Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO et al. 2004. The IGF-1/PI3K/Akt pathway prevents short article expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors. Mol Cell 14:395–403. doi:10.1016/S1097-2765(04)00211-4. Tokuyasu KT, Dutton AH, Singer SJ. 1983. Immunoelectron microscopic studies of desmin (skeletin) localization and intermediate filament organization in chicken skeletal muscle. J Cell Biol 96:1727–1735. doi:10.1083/jcb.96.6.1727. Tong T, Kim M, Park T. 2019. α-lonone attenuates high-fat dietinduced skeletal muscle wasting in mice via activation of camp signaling. Food Funct 10:1167–1178. doi:10.1039/c8fo01992d. Trendelenburg AU, Meyer A, Rohner D, Boyle J, Hatakeyama S, Glass DJ. 2009. Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and myotube size. Am J Physiol Cell Physiol 296:C1258–1270. doi:10.1152/ ajpcell.00105.2009. Xing X, Sun M-Y, Peng X, Song S-Y, Yang M. 2012. Expression of orexigenic and anorexigenic neuropeptides before and during hibernation in the daurian ground squirrel (Spermophilus dauricus). In: T. Ruf, C. Bieber, W. Arnold and E. Millesi (eds.) Living in a seasonal world: Thermoregulatory and metabolic adaptations. p 543–556. Springer Berlin Heidelberg, Berlin, Heidelberg. doi:10.1007/978-3-642-28678-0_47. Yang C-X, He Y, Gao Y-F, Wang H-P, Goswami N. 2014. Changes in calpains and calpastatin in the soleus muscle of Daurian ground squirrels during hibernation. Comp Biochem Phys A 176:26–31. doi:10.1016/j.cbpa.2014.05.022. Zhang D, Lee JH, Kwak SE, Shin HE, Zhang Y, Moon HY et al. 2019. Effect of a single bout of exercise on autophagy regulation in skeletal muscle of high-fat high-sucrose diet-fed mice. J Obes Metab Syndr 28:175–185. doi:10.7570/jomes.2019.28.3.175. Zhang J, Li X, Ismail F, Xu S, Wang Z, Peng X et al. 2020. Priority strategy of intracellular Ca2+ homeostasis in skeletal muscle fibers during the multiple stresses of hibernation. Cells 9:42. doi:10.3390/cells9010042. Zhang J, Wei Y, Qu T, Wang Z, Xu S, Peng X et al. 2019. Prosurvival roles mediated by the perk signaling pathway effectively prevent excessive endoplasmic reticulum stress-induced skeletal muscle loss during high-stress conditions of hibernation. J Cell Physiol 234:19728–19739. doi:10.1002/jcp.28572. page 18 of 18Zoological Studies 60:06 (2021)