| Background and ObjectiveObesity is a chronic metabolic disease characterized by excessive accumulation and abnormal distribution of fat.Obesity is not only an independent condition,but also one of the most important risk factors for type 2 diabetes,cardiovascular disease,hypertension,stroke and a variety of cancers.With the change of life-styles,the incidence of obesity is increasing year by year in China and around the world,and it has become one of the major diseases that seriously threaten human health.In addition to participating in body movements,skeletal muscle is the largest metabolic organ accounting for 40%of the body’s weight.Skeletal muscle maintains the metabolic homeostasis of the body mainly through the use of glucose and fatty acids,preventing the occurrence and development of obesity and metabolic diseases such as type 2 diabetes.Skeletal muscle will be injured when being exposed to pathological factors such as high glucose and high fat,leading to impaired regulation on metabolism.Once this happens,an important compensation for the body is to initiate skeletal muscle regeneration.Skeletal muscle regeneration refers to the activation of satellite cells located on the surface of muscle fibers by inflammatory factors produced by damaged muscle cells.Satellite cells are a kind of stem cells that are cabable of undergoing proliferation,differentiation,fusion and growth,and finally form new skeletal muscle fibers to repair the damaged ones.It can be seen that the impaired skeletal muscle regeneration will seriously affect the skeletal muscle motor function and metabolic regulation function,and further aggravate the metabolic disorder that has already occurred in the individuals.The cellular repressor of E1A-stimulated genes（CREG）is a secreted small molecule glycoprotein that is widely expressed in various tissues and cells.The study found that CREG knockout mice died early during embryogenesis,indicating that the CREG gene is essential for embryonic growth and development.In addition,CREG can promote the differentiation of embryonic stem cells into a variety of cells,including mesodermal-derived endothelial cells and cardiomyocytes.These results suggest that CREG is a key gene in regulating cell differentiation.In addition,our laboratory recently found in animal models that obesity inhibited the expression of CREG in fat and liver tissues,while CREG transgene alleviated obesity,fatty liver and insulin resistance induced by high-fat diet,suggesting that CREG also plays essential roles in metabolic regulation.Based on the above findings,we proposed a hypothesis that CREG may play an important role in obesity induced skeletal muscle regeneration dysfunction by regulating skeletal muscle cell differentiation.Therefore,by using the obese mouse as in vivo model and myoblast C2C12 cells as in vitro model,this study aims to determine the role of CREG on skeletal muscle regeneration and skeletal muscle cell differentiation,and to further clarify its molecular mechanism preliminaryly.Methods and ResultsPart I.Role study1.Establishment of obese mouse model and detection of CREG expression in skeletal muscleMethods:Wild-type C57BL/6 mice were fed with high-fat diet for 12 weeks to establish an obese mouse model.The body weight,fasting blood glucose,blood lipid,insulin,glucose tolerance and insulin tolerance were measured to evaluated whether the obese model was successful established.The expression of CREG in skeletal muscle of obese mice was detected by real-time PCR,western blot and immunohistochemical（IHC）staining.Results:Compared with the control mice receiving normal diet,the mice fed the high-fat diet for 12 weeks showed significant obesity,elevated fasting blood glucose and blood lipid levels,increased serum insulin,and impaired glucose tolerance and insulin tolerance,indicating that the obesity model was established successfully.There was no difference in the transcriptional level of CREG in skeletal muscle of obese mice compared with the control group,but the protein level was significantly lower than that of the control group.IHC staining showed that the expression of CREG in skeletal muscle of obese mice was decreased.2.Establishment of skeletal muscle regeneration modelMethods:A model of skeletal muscle regeneration was established by injecting cardiotoxin（CTX）into skeletal muscle to simulate acute injury of skeletal muscle.The expression of myogenic regulatory factors Myf5,MyoD,Myogenin and mature skeletal muscle marker MyHC were detected by western blot and real-time PCR.Results:Compared with the control group injected with normal saline,the expression of myogenic regulatory factors Myf5,MyoD and Myogenin increased significantly at the protein and mRNA levels after injection of CTX,while the expression of mature skeletal muscle marker MyHC decreased,indicating that CTX successfully induced skeletal muscle regeneration.3.Detection of CREG expression during skeletal muscle regeneration in obese miceMethods:CTX was injected into skeletal muscle of normal-fed mice or high-fat fed obese mice to induce skeletal muscle regeneration.Injection of the normal saline was used as a control to CTX.Western blot,real-time PCR and IHC staining were used to detect CREG expression.Results:Compared with the skeletal muscle tissue of the control group,the expression of CREG in the skeletal muscle tissue of the normal and high-fat fed obese mice was significantly increased,indicating an association of increased CREG expression with skeletal muscle regeneration.Moreover,compared with normal-fed mice,CREG expression was decreased in regenerative skeletal muscle tissue of high-fat-fed obese mice.IHC staining further confirmed that skeletal muscle regeneration was attenuated and CREG expression was decreased in high-fat-fed obese mice compared with normal-fed mice.Since the newly formed regenerative skeletal muscle is mainly composed of incomplete differentiated satellite cells after injury,the above results suggest that skeletal muscle regeneration dysfunction in obese mice may be associated with decreased expression of CREG in satellite cells.4.Identification of CREG+/-mouse model and detection on changes in skeletal muscle regenerationMethods:Western blot and quantitative PCR were used to genotype the breeding CREG+/-mouse model.CTX was injected into skeletal muscle of WT mice and CREG+/-mice,and skeletal muscle tissues were collected 3 days and 15 days after CTX injection respectively,and skeletal muscle fiber status was detected by H&E staining.RESULTS:Compared with WT mice,CREG expression in skeletal muscle of CREG+/-mouse was significantly reduced at the protein and mRNA levels,indicating that the CREG+/-mouse model was successfully established.H&E staining on day 3 and day15 of skeletal muscle CTX injury showed that CREG+/-mouse skeletal muscle regenerated muscle fiber diameter was significantly reduced compared with WT mice,while undamaged muscle fibers showed no significant difference,indicating CREG+/-mouse skeletal muscle regeneration is weakened.5.Establishment of in vitro myoblast cell line C2C12 differentiation model and detection of CREG expressionMethods:C2C12 cells were induced to differentiate using 2%horse serum.Western blot and real-time PCR were used to detect the expression changes of CREG duiring myogenic differentiation of C2C12 cells.Results:With the C2C12 differentiation,the expression of CREG at the protein and mRNA levels continued to increase,suggesting that CREG may be involved in the differentiation of myoblasts into skeletal muscle cells.6.Determination of the role of CREG in the differentiation of C2C12 into skeletal muscle cellsMethods:C2C12 cells with low expression and overexpression of CREG were realized by RNA interference and adenovirus infection,respectively,which were futther induced to differentiate into skeletal muscle cells.Western blot,real-time PCR,immunofluorescence staining and creatine kinase（CK）activity measured by ELISA were used to evaluate the differentiation of C2C12,and BrdU kit was employed to access the proliferation of C2C12.Results:Compared with the control group,the expression of MyHC in the CREG low expression group was significantly reduced at the level of protein and RNA,the number of MyHC-positive skeletal muscle cells was also decreased,and the activity of CK was declined.In contrast,the expression of MyHC in CREG over-expressing group was significantly increased,the number of MyHC-positive differentiated skeletal muscle cells and CK activity increased.BrdU proliferation assay showed that there was no significant change in C2C12 proliferation between CREG low expression group and CREG overexpression group compared with the control group.The above results suggest that CREG promotes the differentiation of C2C12 cells without affecting their proliferation.7.Establish of an in vitro cytological model mimicking obesity,and determination of the expression change and effects of CREGMethods:To simulate the obese state in vivo,C2C12 cells were treated with different concentrations（0.1mM,0.2mM,0.3mM and 0.4mM）of PA to establish an in vitro model.Western blot and real-time PCR were used to detect the expression of CREG and MyHC in C2C12 after PA stimulation.An optimal concentration of PA was employed for the following in vitro study.CREG overexpressing C2C12 cells were further treaded with the optimal PA concentration.Western blot,real-time PCR,and immunofluorescence staining were performed for detection of MyHC expression and CK activity assay was also done by ELISA.Results:After stimulation of C2C12 cells with different concentrations of PA,the expression of CREG at the protein level decreased in a dose-dependent manner,while the mRNA level did not change significantly.MyHC showed a dose-dependent decrease in both protein and transcriptional levels.The 0.4 mM was chosen as the optimal concentration of PA.The C2C12 cells with normal expression and overexpression of CREG were treated with 0.4 mM PA.Compared with the normal expression group,the expression of MyHC in the CREG overexpression group was significantly increased at the protein and transcription levels,the number of cells positive for MyHC staining was significantly increased,and the CK activity was significantly enhanced.These results indicate that PA inhibits C2C12 cell differentiation by decreasing CREG expression.8.Identification of the Tg-CREG mouse model to detect skeletal muscle regeneration in high-fat feeding conditionsMethods:Western blot and quantitative PCR were used to genotype the breeding Tg-CREG mouse model.CTX was injected into skeletal muscle of normal-diet WT mice,high-fat-diet WT mice,and high-fat-diet Tg-CREG mice,and skeletal muscle tissues were collected before CTX injection,3 days and 15 days after injection.H&E staining was used to detect skeletal muscle fiber status.RESULTS:Compared with WT mice,the expression of CREG in skeletal muscle of Tg-CREG mice was significantly increased at the protein and mRNA levels,indicating that the Tg-CREG mouse model was successfully established.H&E staining on day 3 and day 15 of skeletal muscle CTX injury showed that skeletal muscle regenerated muscle fiber diameter was significantly reduced in high-fat-diet WT mice compared with normal-diet WT mice,while the diameter of regenerated muscle fibers of high-fat-diet Tg-CREG mice was significantly larger than that of high fat diet WT mice,indicating that CREG transgene improved the damage of skeletal muscle regeneration caused by obesity.The above in vivo animal and in vitro cell experiments show that CREG plays a key role in obesity-induced skeletal muscle regeneration dysfunction.Part II.Mechanism study1.Screening for CREG interacting proteinsMethods:His-tagged recombinant human CREG protein was added to C2C12 cells,and protein precipitation complex was obtained by immunoprecipitation technique using anti-His antibody.The components of protein precipitation were detected by mass spectrometry,and the target proteins that may interact with CREG protein and participate in skeletal muscle differentiation were screened by literature review.The expression of CREG and target protein was further detected by immunofluorescence staining in C2C12cells.Results:The precipitated protein complex was successfully obtained from C2C12cells by immunoprecipitation.By mass spectrometry,a total of 35 proteins that may interact with CREG were obtained.Combined with the literature review,CBL was identified as a target protein.Immunofluorescence staining showed that both CREG and CBL were expressed in the perinuclear nucleus region and colocalized.2.Clarification of the relationship between CREG and CBLMETHODS:CBL expression was detected by western blot and quantitative PCR in C2C12 with low expression of CREG and overexpression of CREG.RESULTS:Compared with the control group,CBL in the low expression group of CREG was significantly increased at the protein level,while CBL in the high expression group of CREG was significantly decreased at the protein level.Quantitative PCR results showed that there was no difference between CREG low expression group and CREG high expression group and control group.This result suggests that CREG negatively regulates CBL expression at protein level.3.Determination of the effect of CBL on C2C12 differentiationMETHODS:C2C12 cells with low expression and high expression of CBL were established by RNA interference and adenovirus infection.The expression of MyHC was detected by western blot and immunofluorescence staining.CK activity was detected by ELISA kit.RESULTS:Compared with the control group,the expression of MyHC in the low expression CBL group was significantly increased at the protein level,the differentiation of MyHC-positive skeletal muscle cells was increased,and the CK activity of the differentiated cells was enhanced,while the expression of MyHC in the high expression CBL group was significantly decreased at the protein level and MyHC was positive.The differentiation of skeletal muscle cells is reduced,and the CK activity of differentiated cells is weakened.These results indicate that CBL has a negative regulatory effect on skeletal muscle cell differentiation.4.Illuminating whether CREG regulates C2C12 differentiation through CBLMETHODS:CREG and CBL were simultaneously expressed and overexpressed by RNA interference and adenovirus infection in C2C12 cells.The expression of MyHC was detected by western blot and immunofluorescence staining,and CK activity was detected by ELISA.RESULTS:Compared with the low expression group of CREG,the expression of MyHC in the low expression group of CREG and CBL was significantly increased at the protein level,the differentiation of MyHC-positive skeletal muscle cells was increased,and the CK activity of differentiated cells was enhanced.Compared with the CREG overexpression group,the expression of MyHC in the overexpressed group of CREG and CBL was significantly decreased at the protein level,the differentiation of MyHC-positive skeletal muscle cells was decreased,and the CK activity of differentiated cells was decreased.5.Defining the regulation of CREG on CBL in skeletal muscleMethods:The expression of CBL in skeletal muscle of WT mice,CREG+/-mice and Tg-CREG mice was detected by western blot.RESULTS:The expression of CBL in skeletal muscle of CREG+/-mice was significantly higher than that of WT mice and the expression of CBL in skeletal muscle of Tg-CREG mice was significantly lower than that of WT mice.This part of the cytology experiments showed that CREG promoted C2C12differentiation by inhibiting CBL;animal experiments showed that CREG negatively regulated the expression of CBL in skeletal muscle.The resulsts in this part demonstrates that CREG promotes C2C12 differentiation by inhibiting CBL.Conclusions1.CREG was up-regulated during skeletal muscle regeneration in mice,and skeletal muscle regeneration was impaired in obese mice accompanied by decreased CREG expression,suggesting a positive correlation between CREG expression and skeletal muscle regeneration.2.CREG+/-mice have reduced skeletal muscle regeneration.3.Knockdown of CREG inhibits skeletal muscle cell differentiation,and overexpression of CREG promotes skeletal muscle cell differentiation,indicating that CREG is a key regulator of skeletal muscle cell differentiation.4.At the cellular level,PA inhibits CREG expression and skeletal muscle cell differentiation.Overexpression of CREG rescues skeletal muscle differentiation dysfunction induced by PA.At the body level,CREG transgenes can improve the damage of skeletal muscle regeneration caused by high fat,suggesting that obesity may cause skeletal muscle regeneration dysfunction by inhibiting CREG.5.At the cellular level,CBL expression increased and skeletal muscle cell differentiation decreased after knocking down CREG.Simultaneous knockdown of CBL could reduce the differentiation of skeletal muscle cells.After overexpression of CREG,CBL expression decreased,skeletal muscle cell differentiation increased,and overexpression of CBL inhibited Increased differentiation of skeletal muscle cells.This suggests that CBL is negatively regulated by CREG and mediates the regulation of CREG on skeletal muscle cell differentiation.At the body level,CREG negatively regulates the expression of CBL in skeletal muscle.This topic preliminarily explored the role and mechanism of CREG in skeletal muscle regeneration induced by obesity.It suggests that CREG may be a key target for the prevention and treatment of skeletal muscle regeneration caused by obesity. |
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