| With the improvement of people’s living standards,the level of pet ownership is also continuously improving.Overfeeding of high fat diet(HFD,the full text of the abbreviation can be found in the appendix,the same below)will make animals more prone to obesity,insulin resistance and diabetes,which will bring huge risks to animal health.Insulin resistance is a chronic syndrome characterized by decreased sensitivity of some tissues and cells to insulin,resulting in hyperglycemia and hyperinsulinemia.The occurrence of insulin resistance is a typical precursor of type II diabetes(T2DM).However,during the process of insulin resistance induced by high fat diet,how the body’s skeletal muscle and myocardium change in energy and mitochondrial dynamics has not yet been fully elucidated.Therefore,this study successfully induced insulin resistance in C57BL6 mice through 12 weeks of high fat diet.On the first day of the 6th,8th,10 th,and 12 th week of feeding high fat diet,the clinical manifestations,weight,body shape,and overall status of each group of mice were observed to analyze their insulin resistance.On this basis,the histopathological changes,and the expression of related genes at the protein level of skeletal muscle and myocardium in each group of mice were detected to analyze the changes in mitochondrial energy metabolism of skeletal muscle and myocardium in mice during the process of insulin resistance,hoping to provide some reference for further research in the future.In this experiment,C57BL6 mice were used as experimental animals and were divided into two groups,namely,the control group(Con group)and the high fat diet group(HFD group).On the first day of the 6th,8th,10 th,and 12 th weeks of the experiment,observe the overall state of the two groups of mice,measure their weight,and conduct behavioral analysis.Use the grip reflex test to detect their grip and traction abilities.Use the open field test to measure the total distance of movement and the number of spans of each group of mice;Then use blood glucose test paper and insulin kit to detect the fasting blood glucose and fasting insulin indicators of mice in each group.Use glucose tolerance test and calculate insulin resistance index to evaluate the insulin resistance of mice in each group;The content of serum myocardial enzymes in the two groups of mice was detected;Then take photos and record the general anatomy of skeletal muscle and myocardium in each group of mice;The contents of Mitochondrial respiratory chain complex I(COX-Ⅰ)、Mitochondrial respiratory chain complex Ⅴ(COX-Ⅴ)、 Adenosine triphosphate(ATP)、phosphofructokinase(PFK)and Lactate dehydrogenase(LDH)in skeletal muscle and myocardium of mice in each group were detected using a kit;HE staining,PAS staining,and Masson staining were performed on skeletal muscle and myocardial tissue to observe the histopathological changes of skeletal muscle and myocardium in each group of mice;The degree of mitochondrial damage and the changes of muscle fibers in skeletal and myocardial tissues were observed using transmission electron microscopy;Protein immunoblotting and immunofluorescence methods were used to detect the expression of related proteins in skeletal muscle and myocardium.The experimental results showed that insulin resistance in C57BL6 mice was successfully induced by continuously feeding them with 60% high fat diet for 12 weeks.Compared with the control group,the mice in the experimental group gradually became lethargic and unresponsive,their hair gradually changed from flat and glossy to vertical and matte,their movements became more sluggish,gradually tending to bow back,curl up,prefer to lie down,unwilling to exercise,and slow to move,with a significant increase in weight gain and Lee’s finger count;Insulin resistance index significantly increased,glucose intolerance;The weight of skeletal muscle increased significantly,but the increase was not significant around 12 weeks.Pathological changes such as loose muscle cell arrangement,muscle fiber atrophy,and marginal keratinization gradually occurred.The skeletal muscle cells gradually showed lipid deposition,mitochondrial damage,and muscle fiber atrophy,and the glycogen content in skeletal muscle cells gradually decreased;In the experimental group,the heart of mice showed a significant tendency to grow along the transverse axis,gradually developing into sparse myofilament arrangement,myofilament dissolution,and finally developing into myofilament fibrosis;The gradual appearance of nuclear shrinkage,increased lipofuscin in the myocardium,swelling of myocardial mitochondria,and fracture of the cristae eventually develop into myocardial fibrosis.At this time,the content of creatine kinase in the blood gradually increases,and the TGF-β in the myocardium gradually increases and P-Smad2/3protein expression gradually increased.By detecting the changes in insulin signaling pathways in tissues,it can be seen that the expression of PI3 K in skeletal muscle and myocardium of mice fed high fat diet is significantly lower than that of the control group,and the expression of PI3 K in the experimental model group has a continuous downward trend as the number of feeding weeks increases;The phosphorylation level of FOXO1 in the experimental group was significantly lower than that in the control group,and FOXO1 in the experimental group was located in the nucleus;The expression level of G6 Pase protein was significantly higher than that of the control group,and there was no difference in the expression of G6 Pase in skeletal muscle and myocardium of mice fed high fat diet for 10 and 12 weeks;The fusion degree of GLUT4 with cell membrane in the experimental group was lower than that in the control group;From the 8th week,the expression level of GLUT4 and the degree of fusion with the cell membrane no longer decreased;The expression level of phosphorylated form GSK-3β was significantly lower than that of the control group,but the expression level of unphosphorylated GSK-3β protein was higher than that of the control group;In the experimental group,the PFK and LDH activity in skeletal muscle and myocardium gradually increased,and the glycolytic pathway gradually strengthened.The detection results of energy metabolism related indicators in tissues showed that the contents of ATP,COX-Ⅰ,and COX-Ⅴ in the skeletal muscle and myocardium of mice in the experimental group were significantly lower than those in the control group,and gradually decreased with the prolongation of feeding time;AMPK,P-AMPK,PGC-1 in skeletal muscle of experimental group mice α The expression of SIRT1 and SIRT1 protein in the control group was significantly lower than that in the control group,and showed a downward trend with the prolongation of feeding time with high fat diet;The Mfn2 and Opa1 proteins in the skeletal muscle and myocardium of the experimental group mice decreased significantly,while the Drp1 protein increased significantly,showing a significant time-dependent decrease and increase;The expression of PINK1 protein in skeletal muscle and myocardium of experimental group mice did not significantly change,but the expression of Parkin protein showed a gradual downward trend.The results showed that continuous feeding of 60% high fat feed for 12 weeks successfully induced insulin resistance in C57BL6 mice.After insulin resistance,pathological damage and functional decline gradually occurred in skeletal muscle and myocardium;Insulin signaling pathway is inhibited,leading to decreased glucose transport and increased gluconeogenesis;In addition,mitochondrial dysfunction occurs,manifested as a decrease in ATP synthesis ability,weakened mitochondrial biogenesis,decreased mitochondrial fusion,increased division,and decreased autophagy.In addition,it was found that during the development of insulin resistance,the skeletal muscle and myocardium of C57BL6 mice mainly rely on glycolytic pathways for energy supply.In the early stages of insulin resistance,the glycogen synthesis pathway of C57BL6 mouse skeletal muscle is inhibited.This experimental study clarified the transformation of cellular energy sources,changes in mitochondrial dynamics within tissues,and changes in mitochondrial function within tissues during the development of insulin resistance in mouse skeletal muscles and cardiac mechanisms,providing some reference for further research. |
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