| Insulin resistance(IR)is a pathophysiological state in which the sensitivity of insulin-responsive organs or parts to insulin is reduced.The liver is one of its target organs.To compensate the reduced efficiency of insulin-dependent glucose absorption and utilization in Type 2 diabetes,more insulin will be secreted,which induces hyperinsulinemia.Decreased hepatic glycogen synthesis,increased hepatic glucose output and enhanced hepatic gluconeogenesis are main manifestations of IR.Therefore,reducing insulin resistance and increasing liver insulin sensitivity are important means to treat diabetes.miR-122 is a liver-specific miRNA involved in pathways of lipid metabolism,oxidative stress,inflammation,tumorigenesis,viral infection an so on.The effect of miR-122 on lipid metabolism has been confirmed by many literatures.Silencing miR-122 can inhibit fat synthesis,promote fatty acid degradation,and effectively lower blood lipid levels in diabetic animals.In patients with Type 2 diabetes and impaired glucose tolerance,miR-122 was elevated,suggesting a correlation between miR-122 and Type2 diabetes.In this study,a rat model of Type 2 diabetes was induced by high-fat and high-sugar diet and subsequent injection of low-dose streptozotocin,in which the expression of miR-122 was monitored and the changes of glucose and lipid metabolism was investigated.The present study would shed a light on further study of the pathological mechanism of diabetes and provide novel target for prevention and treatment of diabetes.Part ? Expression of miR-122,IGF-1R,Akt/p-Akt in liver of Type 2 DM RatsObjective: Type 2 diabetic rat model was established by intraperitoneal injection of low-dose STZ and fed with high-sugar and high-fat diet.By measuring indexes in glycolipid metabolism and quantifying the expression of miR-122 and key proteins in insulin signaling pathway,the present study would provide the theoretical basis on underlying mechanism of miR-122 in T2 D.Method: Male healthy SD rats were randomly assigned into control group(ND)and high fat diet(HFD)group.The ND group was fed with normal diet,the HFD group was fed with high sugar and high fat diet.Six weeks later,the rats in HFD group were fasted for 16 hours.Streptozotocin was injected into the left lower abdominal cavity.Two days later,fasting blood glucose(FBG)in HFD group was measured.The rats with FBG concentration greater than 11.1 mmol/L were included in Type 2 diabetic group(DM).The general index,biochemical index,glucose tolerance and insulin sensitivity index of the two groups were measured.Total miRNAs were extracted from serum and liver.The m RNA expression level of miR-122 was detected by real-time quantitative PCR.Total proteins were extracted from liver.Protein expression of IGF-1R,p-Akt and Akt were detected by Western blotting.Results:1.The rat model of Type 2 diabetes was successfully established(1)Compared to ND group,DM rats showed significantly increased drinking and daily food intake and decreased body weight(P<0.05);the liver index in DM group was slightly increased but with no significant difference(P>0.05).(2)Compared with ND group,DM rats showed significant increase in FPG,serum fasting insulin level,TG,TC and FFA(P<0.05).Compared with ND group,QUICKI value of DM group was significantly decreased(P<0.05),suggesting decreased insulin sensitivity of Type 2 diabetic rats.(3)The blood glucose and serum insulin levels of DM group at 0 min,30 min,60 min,90 min and 120 min were significantly higher than those of ND group(P<0.001).Compared with ND group,the area under the glucose curve of DM group was also significantly higher(P<0.01).(4)In DM group,the central area of lobule was obviously affected,the arrangement of hepatocytes was disordered,the arrangement of hepatic cord and hepatic blood sinuses was disordered,the volume of hepatocytes increased and became round,the cytoplasm was loose,there were more vacuoles in the cytoplasm,showing obvious fatty degeneration.There was no abnormal morphology and fatty degeneration in ND group.(5)Compared with ND group,PAS staining in DM group was lighter,suggesting decreased glycogen content in liver,the difference of staining was statistically significant(P<0.001).2.Comparison of expression level of miR-122 in liver between two groupsReal-time PCR was used to quantitative analysis of the expression of miR-122 in rat liver.Compared with the ND group,DM rat showed significantly increased miR-122 in liver(P< 0.001).3.Comparison of serum miR-122 levels between two groups of ratsReal-time PCR was used to quantitative analysis of the expression of miR-122 in rat serum.Compared with the ND group,DM rat showed significantly increased serum miR-122(P< 0.001).4.Changes of protein expression of key proteins in insulin signaling pathway in two groups of ratsCompared with ND group,DM rat showed no significant difference in Akt total protein expression(P > 0.05)but significantly decreased phosphorylated Akt and IGF-1R(P<0.05,P<0.001).Part ? The effect of down regulating miR-122 expression on insulin resistance and glucose metabolism of Hep G2 cellsObjective: By down-regulating miR-122 in insulin resistant Hep G2 cells,the effect of miR-122 on insulin signaling pathway and gluconeogenesis of hepatocytes was further investigated,so as to explore its mechanism in insulin resistance.Method: The model of insulin resistance was established in Hep G2 cells by glucosamine.Expression of miR-122 was detected by real-time quantitative PCR.IGF-1R/PI3K/Akt signaling pathway related proteins were analyzed by Western blotting.miR-122-inhibitor was used to down-regulate the miR-122 level in insulin resistant Hep G2 cells.The changes of glucose uptake and expression of key enzymes of glucose metabolism in Hep G2 cells were further evaluated.Results:1.The IR model of Hep G2 cells was successfully establishedAfter treatment with glucosamine for 12 hours,the concentration of 2-DG6 P in Hep G2 cells was significantly lower than that in normal cells(P<0.001),suggesting reduced insulin sensitivity in glucosamine treated cells.2.Changes of miR-122 expression in glucosamine treated cellsCompared to control group,the expression levels of miR-122 in IR group was significantly higher(P<0.01).3.Effects of glucosamine treatment on IGF-1R/PI3K/Akt signaling pathway related protein expressionCompared with the control group,the expression of IGF-1R was significantly decreased in the IR group,the expression of Akt had no significant change,but the phosphorylation level of Akt protein was decreased,and the difference was statistically significant(P<0.05).4.Effect of inhibition of miR-122 level on IGF-1R/PI3K/Akt signaling pathway related protein expression in Hep G2 cellsCompared with control group,IR group showed significantly reduced expression of IGF-1R and p-Akt(P<0.001).After down regulating the expression of miR-122,the expression levels of IGF-1R and p-Akt was significantly increased than that in control group(P<0.001).Insulin sensitivity of Hep G2 cells was improved after down-regulating miR-122 expression.5.The effect of miR-122 down regulation on glucose metabolism(1)Compared with control group,the concentration of 2-DG6 P in IR group decreased significantly(P<0.001).So did the glucose uptake rate.After down regulating the expression of miR-122,the concentration of 2-DG6 P in the medium of miR-122-inhibitor group increased significantly(P<0.01),indicating that the glucose uptake rate increased.(2)Compared with control group,the expression of G6 PC and PEPCK genes in IR group was significantly higher(P<0.001).After down regulating miR-122,both G6 PC and PEPCK m RNA decreased significantly(P<0.05).Part ? Prediction and verification of potential targets of miR-122Objective: Aiming to clarifying the regulatory effect of miR-122 on liver insulin sensitivity and glucose metabolism,bioinformatics methods were used to screen potential targets of miR-122.The predicted candidates were experimental verified in vitro.Method: Target Scan,Pictar and MICROCOSM softwares were used to scan the target gene of miR-122.Hep G2 cells were assigned into NC mimics group,miR-122 mimics group,inhibitor control group and miR-122 inhibitor group.The expression level of candidate protein was analyzed by Western blotting.Binding sites of miR-122 with targets were identified by luciferase reporter gene system.Results:1.Changes of miR-122 level in Hep G2 cells after miR-122 mimics transfectionReal-time quantitative PCR showed significantly higher miR-122 in miR-122 mimics group than that in NC mimics group and parent group(P<0.001).No significant difference was observed in NC mimics and parental group(P> 0.05).2.Prediction of potential targets of miR-122Target scan(http://www.targetscan.org/)was used to predict the potential target genes of miR-122.IGF-1R is a potential target of miR-122.Potential binding sites of miR-122 were identified by sequence alignment.3.The effect of miR-122 on the expression of IGF-1RAfter transfected by miR-122 mimics,the expression of miR-122 in Hep G2 cells was up-regulated.Compared with the control group,the protein expression of IGF-1R decreased significantly.After transfected by miR-122 inhibitor,the expression of miR-122 was down regulated.Compared with control group,the expression of IGF-1R in inhibitor NC group increased.4.3'-UTR reporter gene validation of miR-122 prediction target IGF-1RHep G2 cells were transferred by IGF-1R-3'UTR-wt vector and miR-122 mimics,the value of fluorescein emission was significantly lower than that of NC mimics(P<0.05).However,compared with NC mimics,the fluorescein luminescent value of Hep G2 cells transfection with IGF-1R-3'UTR-mut and miR-122 mimics showed no significant difference.Part? Effects of IGF-1R silencing on insulin resistance and glucose metabolism in Hep G2 cellsObjective: To clarify the role of IGF-1R in the influence of miR-122 on insulin resistance.Method: The insulin resistance model of Hep G2 cells was established.The IGF-1R gene expression of Hep G2 cells was interfered by si RNA,and then the endogenous miR-122 expression was inhibited.assigned into control si RNA group(NC si RNA),IGF-1R si RNA group(IGF-1R si RNA),inhibitor control group(NC inhibitor + IR),miR-122 inhibitor group(miR-122 inhibitor + IR),miR-122 inhibitor combined with control si RNA group,miR-122 inhibitor combined with IGF-1R si RNA group.The m RNA and protein expression of IGF-1R in these groups was respectively detected by real-time PCR and Western blot.Results:1.The transfection effect of IGF-1R si RNA in Hep G2 cellsWestern blot analysis showed that the protein expression level of IGF-1 was significantly lower than that in control group.The difference is statistically significant(P<0.01).2.Effect of IGF-1R si RNA transfection on IGF-1R m RNA expression in insulin resistant Hep G2 cellsThe insulin resistance model of Hep G2 cells was established by fructose induction.Fluorescence quantitative PCR results shows that,compared with the NC inhibitor + IR group,the expression of IGF-1R m RNA in the miR-122 inhibitor + IR group was significantly increased,and the difference was statistically significantly(P<0.01).Compared with the miR-122 inhibitor + NC si RNA + IR group,the expression of IGF-1R m RNA in the miR-122 inhibitor + IGF-1R si RNA + IR group was significantly decreased,and the difference was statistically significantly(P<0.05).3.Effects of IGF-1R silencing on glucose uptake in insulin resistant Hep G2 cellsCompared with NC inhibitor+IR group,2-DG6 P and glucose uptake in miR-122 inhibitor+IR group were significantly increased(P<0.01).Compared with miR-122 inhibitor+NC si RNA + IR group,2-DG6 P and glucose uptake of miR-122 inhibitor+IGF-1R si RNA+IR group were significantly decreased(P<0.05).4.Effects of IGF-1R silencing on the expression of G6 Pase and PEPCK m RNA in Hep G2 cellsCompared with NC inhibitor+IR group,the expression of G6 Pase and PEPCK m RNA in miR-122 inhibitor+IR group significantly decreased in insulin resistance Hep G2 cells(P<0.01).Compared with miR-122 inhibitor+NC si RNA+IR group,the expression of G6 Pase and PEPCK m RNA in miR-122 inhibitor + IGF-1R si RNA + IR group increased significantly after silencing IGF-1R(P<0.01).Part? Study on the relationship between serum miR-122 level,HOMA-? and HOMA-IR in different glucose tolerance populationObjective: To provide theoretical reference for clinical diagnosis and pathogenesis of Type 2 diabetes,the relationship between serum miR-122 and insulin resistance,glycolipid metabolism,and function of islet ? cell was investigated in different glucose tolerance population,Method: 261 subjects aged 35-65 years were recruited from the physical examination center of the people's Hospital.All subjects were tested with oral glucose tolerance test(OGTT).According to the results of glucose tolerance test,subjects were assigned into normal glucose tolerance group(NGT),impaired glucose regulation group(IGR),Type 2 diabetes group(Type 2diabetes Mellitus,T2DM).Age,gender,BMI,WHR and other general parameters were recorded.Venous blood was collected to detect Hb A1 c,FBG,PBG,fins,2h INS,TC,TG,HDL-C,LDL-C and other biochemical indicators HOMA-IR and HOMA-? values were calculated.The expression level of serum miR-122 were measured.The general indicators,biochemical indicators and the correlation between miR-122 and these indicators were analyzed by SPSS21.0 software.The study was approved by the hospital ethics committee,and all the subjects signed informed consent.Results:1.Comparison of general data and blood biochemical indexes among groupsThere was no significant difference in age,gender,BMI and WHR among the three groups(P>0.05).PBG,fins,2 hours INS,Hb A1 c were gradually increased in NGT group,IGR group and T2 DM group,and the difference was statistically significant(P <0.05).HOMA-IR of IGR group was higher than that of NGT group(P<0.05),while HOMA-IR of T2 DM group was significantly higher than that of IGR group and NGT group(P<0.01).HOMA-? in NGT group,IGR group and T2 DM group decreased gradually,among which,HOMA-? in T2 DM group decreased significantly,and the difference between groups was statistically significant(P<0.01).The level of TC,LDL-C and TG in the group of IGR and T2 DM was higher than that in NGT group.The level of TC,LDL-C and TG in the group of IGR and T2 DM was significantly higher than that in the group of NGT and IGR(P<0.05).2.Comparison of serum miR-122 expression levels among three groupsThe serum miR-122 level of each group was compared,and the statistical results showed that there was significant difference among the three groups(P<0.01).The level of miR-122 in T2 DM group was significantly higher than that in NGT group and IGR group(P<0.01,P<0.05).The level of miR-122 in IGR group was higher than that in NGT group(P<0.05).3.Correlation analyiss between serum miR-122 level and metabolic indexesSpearman linear correlation analysis results showed that the expression level of serum miR-122 was significantly correlated with BMI,WHR,FBG,PBG,Hb A1 c,TC,TG,LDL-C,2h INS,FINS,HOMA-IR,HOMA-?(P<0.05).4.Influencing factors of serum miR-122 levelThe results showed that HOMA-IR and HOMA-? were the important factors affecting serum miR-122.5.Correlation analysis between serum miR-122 level and T2 DM incidenceAccording to the expression level of miR-122,the subjects were divided into four groups: Q1(0.78?1.23),Q2(1.24?1.87),Q3(1.88?2.23),Q4(2.24?3.46).The results showed that the incidence of T2 DM at different levels of miR-122 was 15.36%,26.74%,49.34%,62.23%,respectively.Conclusion:1.The expression level of miR-122 in liver is closely related to insulin resistance and the development of diabetes.2.miR-122 regulates liver insulin signaling pathway and glucose metabolism by targeting IGF-1R expression.3.Quantification miR-122 in human serum provides clinical significance for evaluation of insulin resistance and development of diabetes. |
PDF Full Text Request