| Purpose:In this study,a rat model of type 2 diabetes mellitus(T2DM)was established by highfat diet and streptozotocin injection(STZ).The effect of exercise on improving insulin resistance and hyperglycemia was studied,and the mechanism of exercise inhibiting excessive mitochondrial fission and improving insulin resistance was clarified from the perspective of Irisin regulating AMPK/Drp1 pathway changes.To provide theoretical basis for exercise prevention and treatment of T2DM.Method:After one week of adaptive feeding,the 8-week-old Wistar male rats were injected with high fat diet(4 weeks)+STZ,and the blood glucose levels detected on the third and seventh days after injection were 16.7mmol/L.The rats were randomly divided into 4 groups(8 in each group):(1)normal control group(Con);2)T2DM group(DM);(3)T2DM exercise intervention group(DMEx);(4)T2DM exercise intervention plus RGDyk antagonism Irisin group(DMExRg).The rats in exercise group ran on the treadmill for 8 weeks,5 times a week,45min for each time,and the treadmill speed was 18m/min.RGDyk was administered intravenously for 8 weeks,twice a week,at a dose of 2.5mg/kg and a concentration of lmg/ml,and the other groups received an equal dose of 10%DMSO solution.After the experiment,the rats were anesthetized and killed,and abdominal aorta blood samples and soleus muscle samples were collected.Serum lipid concentration(TG,TC,LDL-C and HDL-C),fasting blood glucose(FBG)and fasting insulin(FIN)levels of rats were detected by automatic biochemical analyzer and Elisa kit,and HOMA-IR was calculated.The expression of Drp1 in rat soleus muscle was detected by immunofluorescence.The gene expression levels of FNDC5,AMPK,Drp1,Fis1 and MFF and the protein expression levels of Irisin,p-AMPK,AMPK,Drpl,Fis1 and MFF in rat skeletal muscle tissue were detected by RT-qPCR and Western blot.Mean ± SD was used to describe the data.Univariate analysis of variance between groups was performed using GraphPad Prism statistical software,and the statistical difference between the two groups was analyzed by t test,p<0.05 is a significant difference.Results:1.Changes of rat body weight and soleus muscle weight:there was no significant difference in experimental precursor weight among all groups.After the experiment,the body weight of DM group rats(p<0.05)and soleus muscle weight(p<0.05)compared with Con group,body weight of rats in DMEx group was significantly decreased(p<0.05)and soleus muscle weight(p<0.05)were significantly higher than those in DM group,while the body weight and soleus muscle weight of rats in DMExRg group were not significantly different from those in DMEx group.2.Serum Irisin of DM group(p<0.05)and HDL-C(p<0.05)was significantly lower than that in Con group,while serum TC(p<0.05),TG(p<0.05)and LDL-C(p<0.05)level and FBG level(p<0.05),FIN level(p<0.05)and HOMA-IR(p<0.05)was significantly higher than that of Con group.Compared with DM group,serum Irisin in DMEx group(p<0.05)and HDL-C(p<0.05),while serum TC(p<0.05),TG(p<0.05)and LDL-C(p<0.05)level and FBG level(p<0.05),FIN level(p<0.05)and HOMA-IR(p<0.05)were significantly decreased.Compared with DMEx group,serum Irisin of rats in DMExRg group(p<0.05),TC(p<0.05)and TG(p<0.05)level and FIN level(p<0.05)and HOMA-IR(p<0.05)significantly increased serum HDL-C level(p<0.05),but there was no significant difference in serum LDL-C level and FBG level.3.Changes in immunofluorescence analysis of rat soleus muscle:The fluorescence intensity of Drp1,a key protein regulating mitochondrial fission,was significantly higher in the DM group than in the Con group(p<0.05),and 8-week exercise intervention significantly inhibited the increase trend of Drp1 fluorescence intensity in skeletal muscle of DM group(p<0.05),however,when Irisin receptor signal was blocked by RGDyk,the effect of exercise on Drp1 expression was also eliminated(p<0.05).4.Changes of gene and protein expression in rat soleus muscle:Mitochondrial fission factor Drpl(p<0.05),Fis1(p<0.05)and MFF(p<0.05)mRNA and protein expression were significantly higher than those in Con group,while FNDC5 mRNA(p<0.05)and Irisin(p<0.05)and p-AMPK(p<0.05),the protein expression of Con group was lower than that of Con group,and AMPK mRNA expression was not significantly changed from Con group.Compared with DM group,FNDC5 mRNA in DMEx group(p<0.05)and Irisin(p<0.05)and p-AMPK(p<0.05),while Drp1(p<0.05),Fis1(p<0.05)and MFF(p<0.05)mRNA and protein expression decreased significantly,while AMPK mRNA expression showed no significant difference.Compared with DMEx group,rats in DMExRg group had Drp1(p<0.05)and Fis1(p<0.05)mRNA expression and protein expression were significantly increased,while p-AMPK(p<0.05)The protein expression level was significantly decreased,but the expressions of FNDC5 mRNA,AMPK mRNA,Irisin protein,MFF gene and protein were not significantly changed.Conclusion:1.8-week moderate-intensity treadmill exercise intervention can effectively improve insulin resistance,hyperglycemia and lipid metabolism disorders in T2DM rats.2.Exercise significantly inhibited gene and protein expressions of mitochondrial fission factors Drp1,Fisl and MFF in skeletal muscle of diabetic rats,and improved mitochondrial over-fission.3.Exercise may regulate AMPK/Drp1 signaling pathway by promoting Irisin secretion,thereby reducing excessive fission of skeletal muscle mitochondria and improving insulin resistance in T2DM rats. |
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