Study On Relationship Between Uncoupled Protein 2 And Diabetes Mellitus And Its Related Mechanism

As the population ages and people’s overall lifestyle changes,the incidence rate of type 2 diabetes is increasing rapidly,and has become one of the non-infectious diseases that jeopardize the global public health problem.Type 2 diabetes is a kind of disease caused by many factors.Its pathogenesis is complex and its etiology is difficult to identify.The most striking features of type 2 diabetes are insulin resistance and decreased islet function,which lead to absolute or relative insulin complement and insulin signaling pathways.Uncoupling protein 2(uncoupling protein 2)is a member of the uncoupling protein family,which is widely distributed in nervous tissue,liver,fat,and pancreas.Its main role is to affect the ATP/ADP ratio by transporting protons in mitochondria,resulting in a corresponding decrease in the number of ATP.At the same time,it can act on the respiratory chain,affect the production of reactive oxygen species,and play its biological role.A large number of studies have shown that uncoupled proteins play a role in improving oxidative stress and cell proliferation,and also plays a certain role in the treatment of diabetes and its complications.Single nucleotide polymorphism(SNP)is a widely studied single DNA base deletion,transformation,inversion and insertion in recent years,which can affect the function of related proteins.According to relevant statistics,there is one SNP for every 1000 nucleotides in the human genome.SNPs are widely distributed in the whole human genome,including coding region,non-coding region and intergene region.Studies have shown that SNPs lead to changes in protein function by affecting the bases in the coding region.There are also a large number of studies show that SNPs are associated with cerebrovascular diseases,type 2 diabetes and other diseases.However,due to ethnic and regional differences,SNPs vary greatly,especially in the study of type 2 diabetes.MicroRNAs belong to a class of endogenous gene noncoding RNAs with a length of 22 nucleotides.So far,a variety of miRNAs have been found in animals and viruses,which are involved in the regulation of gene expression at the post-transcriptional level in the body.Studies have shown that microRNAs are involved in a variety of biological functions in the body through their functions,and even play a certain role in the incidence of type 2 diabetes.However,due to the wide variety of microRNAs,the function and mechanism of action of most microRNAs are still unclear.This study aims to analyze the relationship between uncoupling protein 2SNP and type 2 diabetes through the clinical research,and to establish insulin resistance models in vivo(animals)and in vitro(cell),to further study the regulation of UCP2 upstream microRNAs,the effects and mechanism of UCP2 in improving insulin resistance,and explore the pathogenesis and new therapeutic targets of type 2 diabetes mellitus.Part one Association of uncoupled protein 2 gene polymorphism with type 2 diabetes mellitus and prediabetesObjective: The aim of this study was to investigate associations between the genetic of uncoupling protein-2 gene(UCP2)rs659366 polymorphism with prediabetes and type 2 diabetes(T2DM)in a northern Chinese population.Methods:This study is through cooperation and endocrinology clinic in our hospital medical center recruiting volunteers,all the volunteers have been routine physical examination and oral glucose tolerance test: finally confirmed470 people with pre-diabetes 470 newly diagnosed type 2 diabetes patients and 536 healthy control group in the determination of single nucleotide polymorphisms by ligase detection reaction(ligase detection reaction,LDR)technology USES the chi square test and single factor variance analysis to compare genotype and allele distribution differences,using Tukey test match analysis.Results:1.The frequency of AA genotypes was significantly higher than that of GG genotypes in prediabetic patients(odds ratio [OR]=1.833,95% confidence interval [CI]=1.164–2.888,P=0.008).In the diabetic population,GA and AA genotypes were significantly increased compared with the control group(GA:OR=1.716,95% CI=1.316–2.238,P=0.001;AA: OR=3.050,95%CI=1.970–4.720,P=0.001).2.The frequency of the A allele was significantly increased in the prediabetes and diabetes groups compared with the control group,and was significantly associated with an increased risk of prediabetes and diabetes(OR=1.327,95% CI=1.012–1.739,P=0.04;OR=1.719,95% CI=1.318–2.243,P=0.001,respectively).3.Furthermore,insulin levels of prediabetes and T2 DM diabetes groups were significantly decreased in those with the AA genotype compared with those with GG and GA genotypes.Summary: UCP2 rs659366 gene polymorphism may be involved in the pathogenesis of diabetes,leading to an increased risk of pre-type 2 diabetes and T2 DM.Part two Expression of miR-134-3p and UCP2 mRNA in pancreatic tissue of insulin resistant mice induced by high-fat dietObjective: To observe the effects of miR-134-3p on blood glucose,insulin,QUICKI,blood lipids and pancreatic lipid deposition in insulin resistance mice induced by high lipid,and its regulatory effect on UCP2.Methods:C57BL/6J mice aged 6 weeks were randomly divided into control group(CON,normal diet)with 10 mice and high-fat group(HFD,high ft diet)with12 mice.After 8 weeks,an intraperitoneal glucose tolerance test(IPGTT)was performed to evaluate the establishment of insulin resistance model in mice.The blood glucose values at the time periods of 0min,15 min,30min,60 min and 120 min were detected,and the area under the glucose curve(AUC)was calculated.Acquisition mice FPG,TG,HDL-C,LDL-C,method(ELLSA)by enzyme-linked immunosorbent experiment method in determination of fasting insulin level and insulin sensitivity index(QUICKI)calculated,and the detection of SOD,MDA,GSH,GSH-px.Pancreatic tissue samples were collected for H&E staining to observe the pathological changes of pancreatic tissue and lipid content.The expression levels of uncoupled protein 2 and miR-134-3p in pancreatic tissues were determined by real-time polymerase chain reaction(RT-PCR).Results:1.Establishment of insulin resistance model in mice induced by high-fat-dietFrom the second week of feeding,the body weight of mice in the HFD group was significantly higher than that in the CON group.The IPGTT test was performed at the end of the 8th week.Compared with the CON group,the blood glucose levels of HFD mice in 0min,15 min,30min,60 min and 120 min were significantly increased.Compared with the CON group,the area under the glucose curve of HFD group was significantly increased,which proved that the insulin resistance model was established successfully.2.Comparison of biochemical indexes levels between the two groups of miceCompared with the CON group,the levels of TG,TC and LDL-C in HFD group were significantly increased.The levels of SOD,GSH and GSH-Px in the CON group were significantly higher than those in the HFD group.Compared with the CON group,MDA level was significantly increased in the HFD group.3.Comparison of the UCP2 mRNA and miR-134-3p expression levels between the two groups of miceFluorescence quantitative RT-PCR results showed that compared with the CON group,the mRNA expression level of UCP2 in the pancreas of mice was significantly reduced.However,the level of miR-134-3p in HFD group was significantly increased.4.Comparison of pancreatic morphology and histology between the two groupsH&E staining showed that the pancreatic tissue structure in con group was clear and complete,and the cytoplasm was uniform red staining,with few lipid droplets vacuoles and no obvious steatosis;while in HFD group,the pancreatic tissue structure was chaotic and fuzzy,with lipid droplets of different sizes in the cytoplasm,and the lipid droplets squeezed the nucleus at the edge of the cell,showing obvious steatosis.Summary:1.High fat diet can cause insulin resistance and pathological changes of pancreas in mice.2.The mRNA expression of UCP2 was decreased in pancreatic tissue of insulin resistant mice,while the expression of miR-134-3p increased.Part three miR-134-3p inhibits UCP2 expression,relgulates PI3K/Akt signaling pathway and oxidative stress,and affects insulin resistant in MIN6 cellsObjective: TO observe the effects of miR-134-3p and UCP2 on PI3K/Akt pathway,insulin resistance and oxidative stress in MIN6 islet βcells.Methods:MIN6 cell insulin resistance model was established by palmitic incubation,and then cells were transfected with miR-134-3p mimic or inhibitor for over-expression or down-regulation of miR-134-3p level.This part is divided into two experiments,which are grouped as follows: normal control group(CON),palmitic acid incubation group(PA),palmitic acid +miR-134-3p simulation group(miR-134-p mimic),palmitic acid +miR-134-3p simulation control(mimic-negative control,mimic-NC);normal control group(CON),palmitic acid intervention group(PA),palmitic acid + miR-134 inhibitor group(miR-134-inhibitor),palmitic acid + miR-134 inhibitor control group(inhibitor-negative control,inhibitor NC).CCK-8 kit was used to determine the viability of cultured cells.Cell culture and glucose concentration of each group cell were determined by chemical method after transfection.The indexes related to oxidative stress(SOD,MDA,GSH,GSH-Px)were determined by Corresponding test kit.The miRNA and total RNA were extracted from cells of each group,and the miR-134-3p level as well as UCP2,PI3 K,Akt and oxidative stress pathway factors were detected by real-time quantitative PCR.The phosphorylation level of UCP2,PI3 K and Akt were detected by Western blot.Results:1.The insulin resistance model of MIN6 cells was constructed via PA invubation.24 hours after PA,the glucose content in the culture medium of MIN6 cells was measured.The glucose concentration in the PA group was significantly higher than that in the control group,and the difference was statistically significant(P < 0.05).Oil red O staining showed that lipid deposition in PA-treated MIN6 cells was significantly higher than that in the control group.These results suggested that the insulin resistance model of MIN6 cells after PA incubation was successfully established.2.Transfection efficiencyCompared with the control group,the expression level of miR-134-3p in miR-134-mimic group was significantly increased(P <0.05),while significantly decreased in miR-134-inhibitor group(P < 0.05).3.Effects of over-expression or down-regulation of miR-134-3p on UCP2,PI3 K,Akt and oxidative stress-related pathway factors.3.1 The effects of over-expression/down-regulation of miR-134-3p on UCP2 and PI3K/Akt mRNA levelsThe mRNA expression levels of UCP2,PI3 K and Akt in MIN6 cells in PA group were lower than those in CON group.After up-regulating the expression of miR-134-3p in MIN6 cells,the mRNA expression levels of uncoupled protein 2,PI3 K and Akt were further decreased compared with mimic-NC group(P < 0.05).After miR-134-3p mimic transfection in MIN6 cells,the mRNA expression levels of UCP2,PI3 K and Akt were increased compared with the inhibitor NC group(P < 0.05).3.2 The effects of over-expression/down-regulation of miR-134-3p on UCP2 and PI3K/Akt protein levelsThe total protein levels of PI3 K and Akt in the PA group were not significantly changed compared with those in CON group,but the expression levels of phosphorylated PI3 K,Akt and UCP2 were decreased,and the difference was statistically significant(P<0.05).After miR-134-3p mimic transfection,compared with mimic-NC group,the total protein of PI3 K and AKT protein in miR-134-mimic group did not change significantly.However,the expression levels of phosphorylated PI3 K,Akt and UCP2 were further decreased.After down-regulating the expression of miR-134-3p in MIN6 cells,PI3 K and total AKT protein in miR-134-3p inhibitor group was not significantly changed compared with that in inhibitor NC group.However,the expression levels of phosphorylated PI3 K,Akt and UCP2 were increased.4.The effects of over-expression/down-regulation of miR-134-3p on oxidative stressCompared with CON group cells,the levels of MDA,SOD and GSH-XP in PA-group were decreased(P < 0.05).After miR-134-3p mimic transfection,the levels of MDA,SOD and GSH-XP further decreased compared with mimic-NC group(P<0.05).After down-regulating the expression of miR-134-3p in MIN6 cells,the levels of MDA,SOD and GSH-XP in miR-134-3p group were increased compared with that in mimic-NC group(P< 0.05).Summary:1.PA can induce lipid deposition and insulin resistance in MIN6 isletβcells.2.miR-134-3p can inhibit the expression of UCP2,regulate PI3K/Akt pathway,and affect oxidative stress as well as insulin resistance of MIN6 isletβ cell.Conclusion:1.The polymorphism rs65936 of UCP2 gene is closely related to the occurrence and development of type 2 diabetes mellitus and its prediabetes mellitus.2.High-fat diet can induce insulin resistance and oxidative stress in mice,and increase the expression of miR-134-3p and decrease the expression of UCP2 in pancreas of mice.3.Mi R-134-3p can inhibit UCP2 expression,regulate of PI3K-Akt pathway,and affect oxidative stress and insulin resistance of MIN6 islet βcells.