| Peripheral tissue insulin resistance caused by obesity is an important pathological basis of type 2 diabetes.This study showed that the content of mir-204 in serum of obese patients was significantly higher than that of normal weight people.However,the tissue origin of high serum levels of mir-204 in obese patients and its relationship with insulin resistance in obese patients are still unclear.Islet tissue is the main source of insulin and an important endocrine organ in the human body,with exotic function.Studies have confirmed that after the injury of islet function in obesity,a large amount of miRNA will be secreted into the blood in the form of exosomes[1],giving play to biological activity.In view of the high concentration of mir-204 in the islet tissue,the islet tissue has the secretion function,and the serum of obese patients is highly expressed,it is speculated that the serum is rich in mir-204 may be derived from the islet tissue.However,how mir-204 plays a role in inducing insulin resistance in obesity remains to be verified.Bioinformatics prediction showed that mir-204 directly inhibited the expression of SIRT1.SIRT1 is an important target protein for the regulation of insulin resistance,and it can directly or indirectly affect the insulin signaling pathway in skeletal muscle,the main effector of glucose uptake,to improve insulin resistance[2].To sum up,we proposed our hypothesis that pancreatic islets may release mir-204 into the serum in the form of exosomes in the obese state,and then induce skeletal muscle insulin resistance by inhibiting SIRT1 in skeletal muscle tissue and affecting the downstream insulin classical signal transduction pathway.The following below is the research contents of this paper.1.To investigate the correlation between mir-204 and insulin resistance induced by obesity in skeletal muscle.In this study,the C57BL/6J obese mouse model was established by collecting the serum of clinically obese children and feeding them with high-fat diet.The levels of mir-204 and blood glucose in the serum of clinical children and skeletal muscle tissue of obese mice were detected.The results showed that the levels of mir-204 in the serum of obese children were positively correlated with the levels of obesity and fasting blood glucose.Obese mice 4 weeks oral glucose tolerance test and insulin tolerance test results show that high-fat feeding symptoms of insulin resistance in mice,meanwhile the obese mice skeletal muscle miR-204 levels with high-fat feeding were significantly increased in 3 weeks,and in the process of preparing the model at a high level expression of miR-204 in serum and skeletal muscle increased along with the development of the obesity,and miR-204 in the increased obesity induce insulin resistance.These results suggest that mir-204 is associated with insulin resistance induced by obesity.2.To clarify the mechanism by which mir-204 induces skeletal muscle insulin resistance by inhibiting SIRT1In order to explore the role of elevated mir-204 and its target protein SIRT1 in the mechanism of insulin resistance in skeletal muscle,this study further overexpressed mir-204 in skeletal muscle cells of C2C12 mice and combined with SIRT1 activator resveratrol to detect the expression of glucose uptake and insulin signaling pathway protein in skeletal muscle cells.The results showed that,after overexpression of mir-204 in C2C12 skeletal muscle cells,SIRT1 expression was significantly decreased,which in turn resulted in significantly decreased expression of Akt,a key signaling molecule in the insulin signaling pathway,and Glut4,a downstream membrane transporter.In addition,the activation ability of p-akt protein under the stimulation of insulin was significantly decreased,as well as the cell glucose uptake ability.After the overexpression of mir-204 in C2C12 skeletal muscle cells combined with the SIRT1 activator resveratrol,SIRT1 expression was significantly up-regulated,the expression of glucose transporter Glut4 in the insulin signaling pathway of skeletal muscle was significantly up-regulated,the expression level of p-akt protein under the stimulation of insulin was up-regulated,and the cell glucose uptake capacity was significantly up-regulated.The above results showed that the level of skeletal muscle mir-204 was significantly increased in the obese state,and the overexpression of mir-204 can cause insulin resistance in skeletal muscle cells,and the potential mechanism may be by inhibiting SIRT1.3.Elucidated that mir-204 secreted by pancreatic islets can be absorbed by skeletal muscles and induce insulin resistance.In this part of the experiment,mir-204 levels of serum,skeletal muscle cells,insulin-islet cells and islet cell supernatant were first detected in the serum and palmitate-acid-treated mouse skeletal muscle C2C12 cells and islet cell models of clinically obese children,and exosomes of serum and cell supernatant were further extracted.Exosomes were identified by tem and labeled protein CD63,and mir-204levels were detected in exosomes.The serum exosomes and supernatant exosomes were co-cultured with skeletal muscle cells for immunofluorescence detection to verify that pancreatic islet cells could release mir-204-containing exosomes and be absorbed by skeletal muscle cells.Further silencing of mir-204 in islet cells.The islet cell supernatant treated with palmitic acid after silencing of mir-204 was used to extract the exoskeleton weight suspension for incubation of skeletal muscle cells.After incubation for 48 h,the changes in glucose uptake and glucose consumption of skeletal muscle cells were detected to verify that the uptake of mir-204-containing exosomes secreted by islets by skeletal muscle could cause skeletal muscle insulin resistance.The results showed that there was no significant difference in the content of mir-204 in skeletal muscle cells under different concentrations of palmitic acid treatment,suggesting that the elevated mir-204 level in skeletal muscle may not be synthesized by itself under the condition of insulin resistance.The level of mir-204 in serum of obese children was significantly increased,and the level of mir-204 in serum exosomes showed the same trend.The increased level of mir-204 in the supernatant of islet cells treated with palmitic acid suggested that islet cells could secrete mir-204-containing exosomes under the condition of obesity,and both serum of clinical children and the supernatant exosomes of islet cells treated with palmitic acid could be absorbed by skeletal muscle cells.Skeletal muscle cells co-cultured with palmitate-treated islet cell exosomes showed significantly decreased glucose uptake,and the above situation was significantly improved after the silencing of mir-204.The results showed that pancreatic secretion of mir-204 exosomes can be absorbed by skeletal muscle cells and induce skeletal muscle insulin resistance.In summary,this paper proposed 1.Mir-204 inhibits the obesity-induced skeletal muscle insulin resistance induced by SIRT1;2.Pancreatic islet cells secrete mir-204in the form of exosomes and can be absorbed by skeletal muscle cells;3.The secretion of mir-204 exosomes by islet cells can induce skeletal muscle insulin resistance,and the mechanism may be that mir-204 inhibits the target gene SIRT1 to affect skeletal muscle glucose uptake.This experiment provides a new mechanism for the tandem pathway of islet and skeletal muscle tissue in the obesity model,and a new direction for subsequent drug intervention and treatment. |
PDF Full Text Request