Atorvastatin Induced Liver Resistance Under Inflammatory Stress And The Underlying Mechanism

Objective: Statins as a primary or secondary medicine for cardiovascular diseases are used in clinic,which greatly reduce the incidence of cardiovascular disease and reduce the mortality of cardiovascular disease.However,more studies have shown that statins are associated with new onset of type 2 diabetes but its pathogenesis is not yet completely investigated.Inflammation as an independent factor is related with insulin resistance.Since statins may lead to insulin resistance in inflammatory state,is it possible that inflammation can become a susceptible factor of which pontentiates statins-induce diabetes.The liver insulin resistance plays a significant role in the pathogenesis of diabetes so our study focuses on the liver insulin resistance.Inflammation may induce insulin resistance by inducing excessive reactive oxygen species in the liver.This study explored whether statins might further increase the production of reactive oxygen species,especially mitochondrial reactive oxygen species,in the inflammatory background.Because statins can adjust the metabolism of cholesterol in liver cells and inflammation on cellular cholesterol metabolism balance function.Intracellular cholesterol accumulation especially abnormal mitochondrial cholesterol can lead to mitochondrial dysfunction.Mitochondria generated excess ROS and eventually induced insulin resistance.Our study aims to explore whether statins can induce liver insulin resistance in the inflammatory background.Methods: the first part,clinical data: Collecting the patients with type 2 diabetes in the Department of Endocrinology,the diagnosis of diabetes is more than 1 years and the blood glucose is stably controled.Patients diagnosed as chronic viral hepatitis,autoimmune liver disease,hereditary liver disease and acute infection were excluded.The daily alcohol intake of every selected patients was less than or equal to 20 g.The data(sex,age,etc.)and the results of liver ultrasound,weight,height,liver function,blood lipid,fasting blood glucose and glycated hemoglobin were collected.According to the CRP value,all patients were divided into inflammatory groups(CRP ? 10mg/L or hsCRP?3mg/L)and non inflammatory group(CRP<10mg/L or hsCRP<3mg/L.According to given statins or not,each group was divided into two subgroups: non inflammatory and not given statins group,non inflammatory and given statins group;inflammation and not given statins group,inflammation and given statins group.The second part: animal experiment: we established a hypercholesterolemia model by feeding Apo E-/-mice Western diet for eight months The chronic inflammation model was established by subcutaneous injection of casein every other day.The mice were randomly divided into 4 groups: control group(0.9% saline 0.5ml subcutaneous injection every other day),atorvastatin group(0.9% saline 0.5ml subcutaneous injection every other day+ atorvastatin 10mg/kg/d oral group.casein group(10% casein 0.5ml subcutaneous injection every other day).casein+ atorvastatin group(10% casein 0.5ml subcutaneous injection every other day+ atorvastatin 10mg/kg/d oral).The levels of serum inflammatory necrosis factor alpha(TNF?)and interleukin-6(IL-6)in mice were determined by enzyme-linked immunosorbent assay(ELISA).Serum total cholesterol(TC),low density lipoprotein cholesterol(FC)and triglyceride(TG)were measured by serum automatic biochemical analyzer.Liver tissue section HE staining was used to observe the liver lipid deposition.The determination of total cholesterol(TC)and free cholesterol(FC)in liver tissues were detected by enzymology.The content of triglyceride in liver tissue were determined by enzyme coupling colorimetry.Insulin receptor substrate of mouse liver-1(IRS-1),phosphoenolpyruvate carboxykinase(Pepck),hepatic glucose 6 phosphatase(G6Pase),phosphorylated protein kinase B/protein kinase B(p-Akt/Akt),START domain containing protein 1(Stard1)and START domain containing protein 3(Stard3)protein expression were determined by Western blot.The expression of stard1,stard3 to Stard6 gene was measured by RT-PCR.Fresh frozen section of liver tissue was stained with DHE to determine the superoxide anion in the cells.The third part: cell experiments: HepG2 cells were divided into 4 treatment groups: control group(0.5%BSA containing DMEM medium +LDL),atorvastatin group(0.5%BSA containing DMEM medium +LDL+ atorvastatin group(0.5%BSA),inflammation with DMEM medium +LDL+TNF ?)+ atorvastatin group,inflammation(0.5%BSA containing DMEM medium +LDL+TNF? + atorvastatin).the lipid deposition was determined by Oil red O staining under different treatment.Total cholesterol(TC)and free cholesterol(FC)in cells and mitochondria was detected by enzymology.The metabolism of cholesterol(synthesis,excretion and uptake)was determined.After cholesterol uptake(TopFluor cholesterol)was taken into cells the mitochondria were co-stained with cell mitochondria and the distribution of mitochondria cholesterol under different treatment conditions was analyzed.DHE staining was used to analyze the intracellular ROS.Mitochondrial potential was measured by membrane potential dependent mitotracker and mitochondrial ROS was measured by mitoSOX.After si RNA interference on HepG2 cells,the expression of mitochondrial cholesterol,mitochondrial ROS production and mitochondrial membrane potential were measured again.Results:The first part: In the non inflammatory group(CRP < 10mg/L or hsCRP<3mg/L),statins reduce serum TC and LDL significantly(P<0.05).However the difference of glycosylated hemoglobin,fasting blood glucose of patients and the incidence of fatty liver were not significant(P<0.05)while in the inflammation group(CRP ? 10mg/L or hsCRP?3mg/L),statins significantly increased glycosylated hemoglobin,fasting blood glucose level and the incidence of fatty liver(P<0.05).Although the absolute value of blood TC and LDL decreased there was no significant difference(P>0.05).In the second part,the serum inflammatory factors(TNF? and interleukin-6)in casein injection group and inflammatory + atorvastatin group were all higher than that in the control group(p<0.05).In the ITT and GTT tests,there was no difference between the control group and the atorvastatin group.Compared with casein group,the glucose tolerance decreased and insulin sensitivity decreased in casein+atorvastatin group.In inflammation state,atorvastatin impaired insulin signal(IRS-1,p-AKT/AKT)and increased liver G6 Pase and Stard3 protein expression,liver lipid deposition,hepatic triglyceride and free cholesterol(P<0.05).Atorvastatin also increased ROS on mice liver.Under non inflammatory conditions,atorvastatin had no such effect.Conclusions: Inflammation potentiated atorvastatin associated liver insulin resistance by enhancing free cholesterol accumulation in mitochondria with upregulated Stard3.