The Roles And Mechanisms Of MiR-191 In Arsenic-induced Hepatic Insulin Resistance And The Combined Intervention Of Vitamin B₁₂ And Folate

People are mainly exposed to relatively high concentration of arsenic through drinking water and burning coal.Arsenic pollution threatens about 220 million people in the world,of which about 19.6 million people in China are threatened by arsenic-contaminated water.Therefore,arsenic poisoning is one of the serious public health problems in the world and also in China.Chronic arsenic poisoning is related to a variety of diseases,including tumors,skin damage,cardiovascular diseases,and diabetes.However,the mechanism of damage to a variety of tissues caused by arsenic is not yet fully understood,and there are no effective prevention and treatment measures for arsenic poisoning.Therefore,further study of the molecular mechanism of arsenic poisoning is of great theoretical significance and application value for the prevention and treatment of arsenism.Diabetes is characterized by high blood glucose,whose main pathogenesis are insulin deficiency and/or insulin resistance.It has become the third serious disease after tumors and cardiovascular and cerebrovascular diseases,and also one of the current major public health problems.Population epidemiological studies and experimental studies have shown that arsenic poisoning causes imbalance of blood glucose homeostasis by damaging the intracellular insulin signaling pathway,which ultimately leads to diabetes.Micro RNAs（mi RNAs）have been reported to be involved in the occurrence and development of various metabolic syndromes including insulin resistance,but the specific mechanism is still unclear.The liver is the main metabolic organ of exogenous chemicals including arsenic.Besides,the liver is a major tissue for glycogen synthesis in response to insulin stimulation,and it plays an extremely important role in the arsenic-induced damage of the body and in the maintenance of blood glucose homeostasis.Therefore,exploring the effects of arsenic poisoning on hepatic insulin resistance and its specific mechanism is of great significance to the prevention and treatment of diabetes caused by arsenic poisoning.Vitamin B₁₂（VB12）and folate（FA）play important roles in the body in the form of coenzymes.Studies have found that the deficiency of VB12 and FA is more common in elderly diabetic patients compared to non-diabetic elderly,and supplementation of VB12 and FA improves symptoms of hyperglycemia and decreased insulin sensitivity.Therefore,we paid attention to the ameliorating effects of combined supplementation of VB12 and FA on insulin resistance and diabetes caused by arsenic.This study observed the effects of sodium arsenite（Na As O₂）exposure on insulin resistance in mice and human normal hepatic L-02 cells,and explored the roles and molecular mechanisms of mi R-191 in hepatic insulin resistance caused by arsenic poisoning.In addition,on the basis of the above research,we further investigated the effects of the combined intervention of VB12 and FA on systemic and hepatic insulin resistance of mice induced by chronic arsenite exposure.This study provides a scientific basis for exploring the molecular mechanisms and for finding early biomarkers of arsenic-induced diabetes.ObjectiveTo explore the effects of arsenic poisoning on systemic and hepatic insulin resistance,and the roles of mi R-191 by inhibiting glucose transporter 4（GLUT4）membrane translocation through insulin receptor substrate 1（IRS1）/protein kinase B（AKT）pathway in arsenite-induced hepatic insulin resistance,and the effects of combined intervention of VB12 and FA on it and its mechanism underlying it.MethodsThere are two parts of animal experiments.In the first part,male C57BL/6J mice were randomly divided into two groups（12 in each group）,and exposed to drinking water containing 0 or 20 ppm Na As O₂,respectively;in the second part,male C57BL/6J mice were randomly divided into 3 groups（12 in each group）,and they were given ordinary drinking water,drinking water containing 20 ppm Na As O₂,or drinking water containing VB12,FA and 20 ppm Na As O₂,respectively.At the end of animal modeling,glucose tolerance tests（GTTs）and insulin tolerance tests（ITTs）were performed to evaluate the systemic insulin resistance of mice.After treating hepatic L-02 cells with 0,1,2,4,or 8μM Na As O₂,the glycogen and glucose content assay kits was used to assess the cellular insulin resistance.In addition,after pretreated with AKT activator SC79,anti-mi R-191 and/or IRS1 si RNA,respectively,hepatic L-02 cells were treated with 0 or 4μM Na As O₂ for 24 h.Hepatic insulin resistance was detected by Periodic Acid–Schiff staining（PAS staining）and glycogen content assay kit;cell viability were detected by Cell Counting Kit 8（CCK-8）;mi R-191 levels were detected by quantitative real-time PCR（q RT-PCR）;the binding of mi R-191 to 3’UTRs of IRS1 was determined by luciferase reporter gene assay;IRS1,p-IRS1,AKT,and p-AKT（Ser473）levels in total protein and GLUT4 levels in membrane and cytoplasm protein were detected by Western blots;the levels of GLUT4 membrane translocation were detected by immunofluorescence staining;the levels of reduced glutathione（GSH）,oxidized glutathione（GSSG）and catalase（CAT）vitality were determined by the corresponding kits.Results1.The effects of chronic arsenite exposure on the systemic and hepatic insulin resistance,mi R-191 levels,IRS1/AKT pathway and GLUT4 translocation in livers of mice.Results showed that chronic exposure of arsenite caused glucose intolerance and decreases in insulin sensitivity and hepatic glycogen levels in mice.Besides,chronic arsenite exposure increased mi R-191 levels and decreased the levels of IRS1,p-IRS1,and p-AKT（Ser473）proteins,and the levels of GLUT4 in membrane protein in livers of mice.These indicate that chronic exposure of arsenite induces systemic and hepatic insulin resistance in mice,promotes the expression of mi R-191,inhibits the IRS1/AKT pathway,and reduces GLUT4 levels in the membrane protein in livers of mice.2.The effects of arsenite exposure on insulin resistance,mi R-191 levels,IRS1/AKT and GLUT4 translocation in hepatic L-02 cells Results showed that,high concentration（≥10μM）of arsenite inhibited the cell viability of hepatic L-02 cells,while≤8μM arsenite had no significant effect on the cell viability;arsenite exposure decreased glucose consumption and glycogen levels,increased mi R-191 levels,and decreased the levels of IRS1,p-IRS1,and p-IRS1 in total protein,showing a dose-response relationship.Moreover,as the concentration of arsenite gradually increased,the levels of GLUT4 in membrane protein gradually decreased,while the levels of GLUT4 in plasma protein did not change significantly;and immunoconfocal results showed that,the levels of membrane translocation of GLUT4 also gradually decreased.These indicate that in insulin-treated hepatocytes,arsenite induces insulin resistance,promotes the expression of mi R-191,inhibits IRS1/AKT activation,and inhibits translocation of GLUT4 to the plasma membrane.3.The roles of AKT in arsenite-induced insulin resistance and the inhibition of GLUT4 translocation in hepatic L-02 cellsResults showed that after pretreated with AKT activator SC79,the decrease in p-AKT（Ser473）levels,glucose consumption levels and glycogen levels induced by arsenite were reversed.Moreover,AKT activator SC79 reversed the decrease of GLUT4 translocation levels caused by arsenite.These indicate that for insulin-stimulated hepatocytes,the inhibition of the AKT pathway plays an important role in arsenite-induced insulin resistance and GLUT4 translocation inhibition.4.mi R-191 regulates the expression of IRS1 by binding to the 3’UTRs of IRS1Results showed that,mi R-191 was predicted to bind to IRS1.Moreover,the dual luciferase reporter gene assay confirmed that mi R-191 bound to the 3’UTRs of IRS1.5.The roles of mi R-191 in arsenite-induced insulin resistance and the inhibition of IRS1/AKT and GLUT4 translocation in hepatic L-02 cellsResults showed that the inhibition of mi R-191 by anti-mi R-191 reversed the decreases of glucose consumption,glycogen accumulation,and the levels of IRS1,p-IRS1 and p-AKT（Ser473）induced by arsenite in hepatic L-02 cells.Moreover,anti-mi R-191 reversed arsenite-induced the inhibition of GLUT4 translocation.These indicate that in insulin-treated hepatocytes,mi R-191 is involved in arsenite-induced insulin resistance,the inhibition of the IRS1/AKT pathway and the inhibition of GLUT4 translocation.6.The roles of mi R-191 by regulating IRS1 in arsenite-induced insulin resistance and the inhibition of IRS1/AKT and GLUT4 translocation in hepatic L-02 cellsResults showed that low expression of mi R-191 alleviated the decreases in glucose consumption and glycogen level,as well as the decreases in the levels of IRS1,p-IRS1 and p-AKT（Ser473）in total protein induced by arsenite,and these alleviations were restored by low expression of IRS1.Moreover,arsenite-induced GLUT4 translocation inhibition was alleviated by low expression of mi R-191,and this alleviation was restored by knockdown of IRS1.These indicate that in insulin-treated hepatocytes,mi R-191 is involved in arsenite-induced insulin resistance,the inhibition of the IRS1/AKT pathway,and the inhibition of GLUT4translocation through IRS1.7.Effects of combined intervention of vitamin B₁₂ and folate on systemic and hepatic insulin resistance,the increase of mi R-191 levels and the inhibition of IRS1/AKT and GLUT4 translocation induced by chronic arsenite exposureResults showed that the combined supplementation of VB12 and FA significantly improved the glucose intolerance and the decrease of insulin sensitivity in mice induced by chronic arsenite exposure;the combined supplementation of VB12 and FA alleviated hepatic glycogen levels decrease in mice induced by chronic arsenite exposure.Moreover,the combined supplementation of VB12 and FA significantly improved the increase of mi R-191 levels,the decrease of IRS1,p-IRS1and p-AKT（Ser473）levels,and the decrease of membrane GLUT4 levels induced by chronic arsenite exposure in livers of mice.These indicate that the combined supplementation of VB12 and FA alleviates the systemic and hepatic insulin resistance,the increase of mi R-191 levels,and the inhibition of IRS1/AKT pathway and GLUT4 translocation induced by chronic arsenite exposure.8.Effects of combined intervention of vitamin B₁₂ and folate on oxidative stress caused by chronic arsenite exposure in livers of miceResults showed that the combined supplementation of VB12 and FA improved the increase of GSSG levels and the decrease of GSH levels in livers of mice induced by chronic arsenite exposure.These indicate that the combined supplementation of VB12 and FA alleviates the oxidative stress induced by chronic arsenite exposure in livers of mice.ConclusionBy inhibiting the IRS1/AKT pathway,mi R-191 blocking the membrane translocation of GLUT4 is involved in arsenic-induced hepatic insulin resistance.The combined supplementation of VB12 and FA alleviates the insulin resistance,the increase of mi R-191 levels and the inhibition of IRS1/AKT pathway and GLUT4translocation induced by arsenic poisoning in livers of mice,which may be related to the effects of anti-oxidative stress.In summary,the novel findings of this study are as follows:1.Chronic arsenic poisoning induces systemic and hepatic insulin resistance;2.By inhibiting the IRS1/AKT pathway,mi R-191 blocking the membrane translocation of GLUT4 is involved in hepatic insulin resistance caused by arsenic poisoning;3.The combined supplementation of VB12 and FA alleviates systemic and hepatic insulin resistance caused by chronic arsenic poisoning,and the underlying mechanism may be related to the effects of blocking oxidative stress.