Atg7 Leads To Aggravation From Obesity-triggered Muscular Atrophy By Repressing Akt Phosphorylation

Background:Sarcopenia is a common high incidence of old age,characterized by loss of muscle mass and strength with age.According to clinical case reports,individuals with muscle loss are often accompanied by an increase in fat mass,especially in the muscle and visceral compartments.The presence of absolute or relatively low muscle mass plus a body mass index greater than 30 or overall weight is called“sarcopenic obesity”.The other view is that obesity is not only a comorbid condition of sarcopenia,but may be an important cause of sarcopenia.Many obesity-related abnormalities are also involved in muscle growth regulation.Based on the above two perspectives,the causal relationship between obesity and sarcopenia and whether obesity can induce muscle atrophy need to be further clarified.Autophagy is an intracellular degradation process in which damaged organelles and long-life proteins are degraded and recycled for maintenance of normal intracellular homeostasis.However,autophagy overactivation can lead to many metabolic diseases and related complications.The level of autophagy in adipose tissue was positively correlated with the degree of obesity in obese patients and obese mice.In mice with targeted deletion of Atg7 in adipose tissue,fat mass was significantly reduced due to defective differentiation of white adipose cells,and the white adipose tissue of such mice showed characteristics of brown adipose tissue,contributing to enhanced insulin sensitivity.It has been shown that some key components of autophagy are upregulated during muscular atrophy and induction precedes muscular atrophy.Skeletal muscle-specific gene knockout Atg7 mice increased glucose clearance and energy expenditure.However,whether autophagy and key proteins of autophagy pathway play a regulatory role in obesity-induced muscle atrophy remains unknown.Akt is a co-regulator of both myoprotein synthesis and degradation pathways:In promoting myoprotein synthesis,over-expression of Akt in adult muscle fibers by electroporation completely blocks denervation induced muscle atrophy.The key role of Akt in regulating muscle size was further demonstrated by the production of transgenic mice overexpressing Akt in skeletal muscle,the mechanism by which Akt controls protein synthesis through m TOR,a key kinase downstream of insulin and nutrition-sensitive pathways required for cell growth.Akt inhibits myoglobin degradation by negatively regulating Fox O transcription factors and thereby blocking the increase of muscle tissue-specific ubiquitin ligases,atrogin-1 and TRIM63.Therefore,maintaining Akt activity is an important target for improving muscle atrophy.Objective:The purpose of this study was to identify obesity as the inducing factor of muscle atrophy and to further clarify the mechanism of obesity-induced muscle atrophy.Materials and Methods:1.Part I:In this part,adult mice were selected as research objects and given high-fat diet stimulation for different lengths of time.Glucose tolerance,glucose lipid serological indicators and body fat distribution were detected to evaluate obesity modeling.Grasping force and endurance of mice were measured by mouse scratching apparatus and mouse cycle fatigue tester to evaluate the behavioral ability of skeletal muscle of mice.To determine the potential effect of high fat diet on skeletal muscle atrophy,gastrimius muscle weight and index were measured,and morphological changes of skeletal muscle in mice were assessed by hematoxylin-eosin staining.2.Part II:Western blot was used to detect the Atg7/LC3/P62 autophagy pathway and Akt/Fox O3a/TRIM63/My HC myosin degradation pathway in obese mice fed with high fat for 20 weeks to clarify the activation of autophagy pathway.Further,adeno-associated virus gastrocnemius injection was used to knock down Atg7 expression.The experiment was divided into four groups:WT-ND group（sh NC adeno-associated virus injection was given normal diet）,Atg7^△SM-ND group（sh Atg7 adeno-associated virus injection was given normal diet）,WT-HF group（sh NC adeno-associated virus injection was given high-fat diet）,Atg7^△SM-HF group（sh Atg7 adenovirus injection was given high-fat diet）.Grasping force and endurance of mice were measured by mouse scratching instrument and mouse cycle fatigue tester to evaluate the behavioral ability of skeletal muscle in each group.The weight and index of gastrimius muscle were detected,and morphological changes of skeletal muscle in each group were assessed by hematoxylin-eosin staining.The above pathways of gastrimius muscle in each group were detected by Western blot to explore whether autophagy related protein Atg7 is involved in the regulation of muscle atrophy induced by obesity.3.Part III:In this part,palmitic acid was used to stimulate mouse skeletal muscle cells C2C12 to construct cell models in vitro,and Atg7 was overexpressed and knocked down on this basis.Western blot was used to detect the above pathways in each group of cells.Immunofluorescence detection of My HC protein expression confirmed the regulatory role of Atg7 in myocyte atrophy.Actinomycin and MG132 or CQ were used to treat palmitic acid-stimulated C2C12 cells,and the effects of Atg7 on My HC half-life and degradation were evaluated in combination with Atg7 intervention,and the main pathway of Atg7 regulating My HC degradation was identified.Western blot was used to detect the Akt/Fox O3a/TRIM63/My HC myosin degradation pathway of C2C12 cells stimulated by palmitic acid and combined with Atg7 knockdown,Akt knockdown or Akt phosphorylation inhibitor MK2206.The binding of Atg7 to Akt protein was detected by GST-pulldown,and the binding changes of Atg7 to Akt in C2C12 cells stimulated by palmitic acid were detected by immunoprecipitation,so as to clarify the action mode of Atg7 regulating Akt.Analyzing the mechanism of Atg7 regulating obesity-induced muscular atrophy by inhibiting Akt phosphorylation.Data（n>6）were expressed as mean±SEM.Data（n≤6）were expressed as mean±SD.T-test was used for comparison between Two groups.One-way or two-way ANOVA was used for comparison between three groups or more.P<0.05 means that the data results are statistically significant,P<0.01 represents significant statistical difference in data results.Unidirectional or bidirectional variance was used for analysis,and SPSS software（Windows version 13.0）was used for post-bonferroni test to determine individual differences between groups.The statistical graph was analyzed by Graph Pad Prism Version 5.01.Results:1.Part I:In this part of the study,a high-fat diet was used to construct an obesity model.From the third week,the body weight of high-fat diet group（HF group）was significantly higher than that of normal diet group（ND group）,and the serum triglyceride（P<0.05）and total cholesterol content（P<0.05）was significantly higher than that of normal diet group,and the trend was further expanded after 20 weeks of high fat diet.Fasting blood glucose of mice fed high fat diet for 20 weeks was significantly higher than that of normal diet group（P<0.05）,glucose tolerance curve and area under curve within 120 min were significantly increased compared with normal diet group（P<0.01）.The results of body fat distribution showed that the lean body weight of mice in the high-fat diet group was significantly lower than that in the normal diet group（P<0.05）.Gastrocnemius muscle weight（P<0.05）and gastrocnemius index（P<0.01）were significantly lower than those in normal diet group.The results of H&E staining showed that the cross section and number of muscle fibers of mice fed with high fat for 8 weeks decreased significantly,and there were obvious gaps in the arrangement of muscle fibers between mice fed with high fat for 8 weeks and mice fed with normal diet.The cross section and number of muscle fibers of mice fed with high fat for 20 weeks and mice fed with normal diet decreased significantly,and the arrangement of muscle fibers was loose and there was a large gap.The grasping and endurance test results showed that the grasping and running time of mice after 8 weeks of high-fat diet were significantly lower than that of normal diet group.2.Part II:In this part of the study,Western blot was first used to detect the Atg7/LC3/p62 autophagy pathway and Akt/Fox O3a/TRIM63/My HC myoprotein degradation pathway in the gastrocnemius muscle of obese mice fed with high fat for 20weeks（P<0.01）,My HC content decreased significantly（P<0.05）,p-Akt/Akt and p-Fox O3a/Fox O3a ratio were significantly decreased（P<0.05）,Atg7,LC3-II/I ratio and P62 were significantly increased（P<0.01）.The mice model of Atg7^△SM-HF increased lean body weight（P<0.01）,decreased fat distribution（P<0.01）,gastrocnemius muscle mass of mice（P<0.05）and mouse gastrocnemius index（P<0.01）increase.The results of H&E staining showed that the muscle fibers of gastnemius muscle of Atg7^△SM-HF mice were all arranged,and the cross-sectional area of muscle fibers（P<0.01）and muscle fiber number（P<0.01）compared with WT-HF group,the running time of mice（P<0.01）,running wheel distance（P<0.01）and grip（P<0.01）significantly increased compared with WT-HF group.Signal pathway detection results showed that THE TRIM63 content in Atg7^△SM-HF mice was significantly decreased compared with that in WT-HF group（P<0.01）,My HC content increased significantly（P<0.01）,p-Akt/Akt and p-Fox O3a/Fox O3a ratio were significantly increased（P<0.05）.3.Part III:In this part of the study,C2C12 cells were stimulated by 0.75 m M palmitic acid for 24 h and 48 h to detect the above signaling pathways.The results showed that under palmitic acid stimulation,TRIM63 content was significantly increased,My HC content was significantly decreased,p-Akt/Akt and p-Fox O3a/Fox O3a ratio were significantly decreased.Atg7,LC3-II/I ratio and p62 were significantly increased.Overexpression of Atg7 resulted in significantly decreased protein expression of p-Fox O3a and p-Akt,significantly increased expression of TRIM63,significantly decreased My HC,My HC immunofluorescence showed significantly shortened myocyte diameter（P<0.01）;Knocking down Atg7 resulted in increased expression of p-Fox O3a and p-Akt proteins,significantly decreased expression of TRIM63,and significantly increased My HC.Knocking down Atg7 partially improved the diameter shortening of myocytes under palmitic acid stimulation,and maintained the diameter of myocytes（P<0.01）.The My HC of the control group decreased significantly after treated with actinomyone for 1 h after 0-2 h treatment with protein synthase inhibitor actinomyone,and its half-life was speculated to be 0.5-1 h,while the half-life of the control group was greater than 2 h after knocking down Atg7.My HC protein was decreased by palmitic acid stimulation.My HC protein was re-accumulated in MG132 group after treatment with proteasome inhibitor MG132 and autophagolysosome inhibitor CQ respectively.However,CQ alone did not improve My HC protein degradation under palmitic acid stimulation,and overexpression of Atg7 significantly stimulated My HC expression decline.It accumulates after administration of the proteasome inhibitor MG132.Transfection of sh Akt plasmid revealed that knockdown of Akt resulted in the decrease of p-Fox O3a and the activation of TRIM63 stimulated by palmitic acid.When Atg7 was knocked down and Akt was knocked down,fox O3A-mediated TRIM63 pathway was reactivated,and the expression of My HC decreased.After Atg7 knockdown and MK2206administration,Fox O3a-mediated TRIM63 pathway was reactivated and My HC expression was decreased.GST-Pulldown results showed that Atg7 directly binds Akt in vitro,and immunoprecipitation results showed that the combination of Atg7 and Akt increased under palmitic acid stimulation,and the expression of p-Akt decreased.Conclusion:1.High-fat diet resulted in decreased muscle mass,muscle fiber number and muscle function in mice;2.Autophagy related protein Atg7 was activated in gastnemius tissue and palmitic acid-stimulated muscle cells under high fat diet or palmitic acid treatment;3.Intracellular knockdown of Atg7 in gastrocnemius muscle tissue and palmitic acid stimulated muscle cells can improve palmitic acid-induced muscle atrophy;4.Atg7 inhibition of Akt phosphorylation promotes palmitic acid-stimulated myoprotein degradation,possibly through Atg7 binding to Akt inhibition of its phosphorylation.