The Roles And Molecular Mechanisms Of MiR-191 In Hepatic Insulin Resistance Induced By Cigarette Smoke

Diabetes is a major public health problem in China and even in the world.Diabetes refers to a group of metabolic diseases characterized by systemic destruction of glucose homeostasis,caused by genetic and environmental factors.Insulin resistance is a decrease in the sensitivity of insulin-sensitive tissues and cells(including liver,muscle,and fat)to insulin,which occurs mainly in the early stages of type 2 diabetes and plays an important role in the development of type 2 diabetes.There are many reports of increased risk of diabetes from smoking,and epidemiological studies have found that smoking is an independent risk factor for insulin resistance and diabetes.Smoking is one of the main causes of diabetes and has a dose-effect relationship.The more cumulative smoking,the higher the risk of diabetes is get.At present,the relationship between smoking and insulin resistance is relatively clear.Compared with non-smoking populations,smokers are more likely to develop insulin resistance.The mouse model supports the epidemiological findings: Cigarette smoke exposure can promote the development of insulin resistance and diabetes.In recent years,mi RNAs have been extensively studied.mi RNAs can regulate a variety of biological processes.A large number of studies have found that mi RNAs are also involved in the course of insulin resistance.mi RNA also plays an important regulatory role in the development of diabetes,but the molecular mechanisms need further studied.Therefore,we investigated the roles of mi R-191 in smoking-induced diabetes and insulin resistance,and provided scientific evidence for discovering emerging molecular markers of diabetes and insulin resistance,as well as therapeutic targets and preventive measures.Part Ⅰ The role of micro RNA-191 regulating IRS-1 in the insulin resistance of hepatic cells induced by cigarette smoke extractObjective To investigate the effects of cigarette smoke extract(CSE)on mi R-191 in hepatic L-02 cells and its role in hepatic L-02 cell insulin resistance induced by CSE.Methods In this study,human liver epithelial L-02 cells were used to study to establish the insulin resistance model of hepatic L-02 cells induced by CSE.The kit was used to detect the glucose uptake and intracellular glycogen content,and the protein level in hepatic L-02 cell was detected by Western blot;the levels of mi R-191 was detected by q RT-PCR;SC79,an activator of Akt signaling,was used;luciferase reporter gene was used to verify the specific binding of mi R-191 to IRS-1 in the 3’-UTR region;application of transient transfection techniques to investigate mi R-191 regulates the role of IRS-1 in CSE-induced L-02 cell insulin resistance.Results 1.For L-02 cells,treatment with CSE decreased the levels of glucose uptake and intracellular glycogen and increased the levels of mi R-191 in a dose-dependent manner.The results indicate that CSE induces insulin resistance and up-regulates mi R-191 levels in L-02 cells.2.The levels of insulin-stimulated IRS-1,p-IRS-1 and p-Akt were reduced in a dose-dependent manner which indicates that CSE induces inhibition of IRS-1and the Akt signaling pathway in L-02 cells.3.SC79 prevented the decrease of p-Akt,glucose uptake and intracellular glycogen in L-02 cells induced by CSE.These data indicate that Akt signaling pathway is involved in the decrease of glucose uptake and glycogen levels induced by CSE.4.Anti-mi R-191 reversed CSE-induced decrease of glucose uptake and intracellular glycogen levels.Indicating that mi R-191 is involved in the CSE-induced insulin resistance in L-02 cells.5.Anti-mi R-191 significantly rescued CSE-induced p-IRS-1 and p-Akt expression.These results indicate that mi R-191 plays an important role in the decrease of p-IRS-1 and p-Akt in L-02 cells induced by CSE.6.Target Scan analysis revealed alignment of mi R-191 with its target site in the 3’-UTR of IRS-1,which was confirmed by Dual luciferase reporter gene assay.These results indicate that mi R-191 targets IRS-1 3’-UTR through its binding site.7.Anti-mi R-191 reversed the decrease of glucose uptake and glycogen content,p-IRS-1,IRS-1,and p-Akt caused by CSE.Downregulating the expression of IRS-1 by si RNA after knockout of mi R-191 blocked the inhibition of the insulin resistance in L-02 cells caused by anti-mi R-191.These data suggest that mi R-191 participates in CSE-induced L-02 cell insulin resistance through IRS-1.Conclusions For L-02 cells,treatment with CSE caused the decrease of glucose uptake and intracellular glycogen levels in a dose-dependent manner.Moreover,the insulin signaling pathway was disordered,as characterized by reduced levels IRS-1,p-IRS-1 and p-Akt;CSE up-regulates the levels of mi R-191 in L-02 cells;mi R-191 via IRS-1 inhibiting the activating of Akt is involved in the CSE-induced insulin in L-02 cells.Part Ⅱ A population study on the relationship between mi R-191 and smoking-induced diabetesObjective To investigate the general characteristics of non-smokers and smokers,blood lipids,blood glucose and glycated hemoglobin,smoking in diabetics and non-diabetics,and blood glucose level,HOMA-β and HOMA-IR and serum mi R-191 levels in different smokers.Methods The Jiangsu Center for Disease Control and Prevention,a partner of this project,established a cohort of smoking and diabetes aged 18 to 70.This cross-sectional study selected 1,658 people and collected epidemiological data including population characteristics,smoking;collection of anthropometric,blood pressure,and diabetes-related blood parameters;q RT-PCR detection of blood mi R-191 levels.Results 1.There was no significant difference in average BMI,waist circumference,diastolic blood pressure,and systolic pressure between smokers and non-smokers.The average age of smokers was significantly higher than that of non-smokers,indicating that smoking had no significant effect on BMI,waist circumference,Diastolic pressure and systolic blood pressure,while the average age of smoking population was higher than that of non-smoking population.2.There was no significant difference in average total cholesterol,low-density lipoprotein,and high-density lipoprotein levels between smokers and non-smokers,and the average triglyceride level in smokers was significantly higher than the average triglyceride levels in non-smokers,indicating that smoking had no significant effect on total cholesterol,low-density lipoprotein and high-density lipoprotein levels.The average triglyceride level in smoking population was higher than that in non-smoking population.3.The fasting blood glucose level and the glycated hemoglobin level of smokers were significantly higher than that of non-smokers,indicating that smoking can elevate fasting blood glucose level and the glycated hemoglobin level.4.The daily smoking level and number of smoking packs in diabetic patients were significantly higher than those in non-diabetic populations,suggesting that smoking may be related to diabetes.5.According to the increasing smoking packages,the blood glucose level and HOMA-IR were increased and the HOMA-β index declined,indicating that with the increase of smoking,there is a tendency of abnormal glucose metabolism and insulin resistance.6.The serum mi R-191 levels in severe smokers were increased than those in non-smokers,suggesting that smoking may be related to the elevated levels of mi R-191 in the serum.Conclusion The daily smoking level and the number of smoking packages in diabetic patients were significantly higher than those in non-diabetic populations.With the increase of smoking,there is a tendency of abnormal glucose metabolism and insulin resistance,indicating that smoking can increase the risk of diabetes.Furthermore,the serum mi R-191 levels in smokers were significantly increased than those in non-smoking populations.In summary 1.For hepatocytes,treatment with CSE caused decreases of glucose uptake and intracellular glycogen levels in a dose-dependent manner.Moreover,the insulin signaling pathway was disordered,as characterized by reduced levels IRS-1,p-IRS-1 and p-Akt;CSE up-regulates mi R-191 levels;mi R-191 via IRS-1 inhibiting the activating of Akt is involved in the CSE-induced insulin in hepatocytes.2.The daily smoking level and the number of smoking packages in diabetic patients were significantly higher than those in non-diabetic populations.With the increase of smoking,there is a tendency of abnormal glucose metabolism and insulin resistance,indicating that smoking can increase the risk of diabetes.Furthermore,the serum mi R-191 levels in smokers were significantly increased than those in non-smoking populations.