Study The Effect Of Axin1 On Contraction Regulated Skeletal Muscle Glucose Metabolism And Muscle Development And Regeneration

Objective:Skeletal muscle accounts for about 40%-60% of human body weight and plays an important role in maintaining material metabolism and body movement.Skeletal muscle is not only the body’s largest energy metabolism organ,but also an endocrine organ(secreting growth factors such as myostatin,insulin-like growth factor,etc).Skeletal muscle diseases are common in metabolic diseases(obesity and diabetes),genetic diseases(progressive skeletal muscular dystrophy,such as DMD)and aging-related diseases(sarcopenia).Axin1 is a structure scaffold protein that participates in a variety of signaling pathways.Recently,it has been reported that Axin1 participates in insulin signal to promote glucose uptake in adipose tissue.Exercise/muscle contraction and insulin are the physiological effects of promoting glucose uptake in skeletal muscle.Unlike insulin,the molecular mechanism of exercise/muscle contraction promoted glucose uptake in skeletal muscle is not clear.Therefore,illustration the underlying mechanism is helpful for prevention and treatmeant of diabetes.In addition,the development of skeletal muscle involves cell proliferation and differentiation.When skeletal muscle is injured,muscle satellite cells are activated to start regeneration.Under obesity and diabetes,skeletal muscle mass decreases,exercise and metabolic dysfunction.However,the mechanism of how obesity and diabetes affecting skeletal muscle development and regeneration after injury is not fully understood yet.These need to clarify.Methods:Part 1: C2C12 murine skeletal muscle cell lines and a variety of mouse models were used to explore the role of Axin1 in contraction-stimulated skeletal muscle glucose uptake.C57BL/6 mice exercised in treadmill.Electrical plus stimulation(EPS)made C2C12 myotube contraction.Up-regulate or down-regulate AMPK activity with activators,inhibitors,siRNA and adenovirus,Axin1 or Tiam1 levels,respectively.Pull down experiment detected Rac1 activity.ELISA measured GLUT4 translocation.2-NBDG detected glucose uptake.Western blot detected protein level and phosphorylation.Part 2: Explore the role of Axin1 in the development and regeneration of skeletal muscle in obese and diabetic mice.The expression of Axin1 in C2C12 skeletal muscle cells was regulated with plasmids and siRNA.Western blot,qRT-PCR,immunofluorescence and flow cytometry were used to detect the effect of Axin1 on the proliferation and differentiation of skeletal muscle cells.The role of Axin1 on cell proliferation and differentiation was detected in insulin resistance muscel cells induced by high insulin.Mouse skeletal muscle injury was induced by cardiotoxin(CTX)in vivo.qRT-PCR and Western blot were used to detect the expression of Axin1 and skeletal muscle regeneration related genes and proteins,respectively.Skeletal muscle regeneration was detected by HE and immunofluorescence staining.In vivo experiments,the mice were induced obesity by high-fat diet feeding.The effect of Axin1 on improving skeletal muscle regeneration in obese mice was detected after knocking down Axin1 by siRNA.Results:The first part results showed: 1.Muscle contraction consumed energy,upregulated AMP/ATP ratio,glycogen utilization and lactic acid production.2.Exercise/muscle contraction upregulated Axin1 protein level and enhanced its interaction with AMPK to activate AMPK,as well as promoting glucose uptake.3.AMPK activates Rac1 via Tiam1 and participates in contraction to promote glucose uptake in skeletal muscle.The second part results showed: 1.Knockdown of Axin1 expression inhibited skeletal muscle cell proliferation and promoted cell differentiation.Overexpression of Axin1 promoted cell proliferation and inhibited cell differentiation.The expression of Axin1 and cell proliferation increases,while differentiation decreased in insulin resistant muscle cells.2.Axin1 expression is down-regulated and β-catenin expression is increased during skeletal muscle regeneration,which activates the Wnt signaling pathway in CTX-induced muscle damage.3.Axin1 protein level increased,differentiation and regeneration impaired in skeletal muscle of obese and diabetic mice.Knocking down of Axin1 improved skeletal muscle regeneration in obesity mice.Conclusion:Axin1 participates in a variety of signaling pathways and plays an important role in skeletal muscle.Exercise/muscle contraction promotes skeletal muscle glucose uptake through AMPK/Axin1-Tiam1-Rac1 signaling pathway.Axin1 is involved in the development and regeneration of skeletal muscle.Down-regulating Axin1 inhibits skeletal muscle cell proliferation and promotes differentiation and promote skeletal muscle regeneration in obese mice.