The Expression Profile Of Long Non-coding RNA In Insulin Resistant Mouse Adipose Tissues And Its Effect On Insulin Sensitivity In 3T3-L1 Adipocytes

Obesity is a critical public health issue globally in developed and developing countries with numerous complications.Excess visceral fat accumulation is a major determinant of chronic metabolic disorders,including cardiovascular disease and type 2 diabetes mellitus?T2DM?.Insulin resistance is a pathological condition in which cells or tissues fail to respond appropriately to the normal actions of insulin.There are a handful of well-known causes of insulin resistance,such as obesity-mediated low-grade tissue inflammation,disturbed.lipid metabolism,and changes in the gastrointestinal microbiota.Ample researches have been carried out to unravel the correlation between insulin signal transduction and insulin resistance.Whereas,new evidence implies the potential involvement of transcriptional and epigenetic regulation in insulin resistance.Long non-coding RNAs?LncRNAs?are a class of RNAs with more than 200 nucleotides that mostly have no known protein-coding function.These RNAs were initially considered to be non-functional.However,enormous effort has been expended that LncRNAs are implicated in a number of biological processes,such as genome imprinting,chromatin modification,enhancer functions,RNA splicing,nuclear architecture.Several IncRNAs have been demonstrated to regulate adipogenesis by interacting with Peroxisome proliferator-activated receptor y?Ppary?,an adipocyte-enriched nuclear receptor.Moreover,PPARy is also involved in the insulin sensitivity with relevance to resistin and some pro-inflammatory cytokines.Thus we hypothesized that IncRNA may have an effect on the process of insulin resistance in adipose tissues.Therefore,male C57BL6 mice were fed a high-fat diet?HFD,6.0%kcal fat?for 12 weeks to induce insulin resistance.After the validation of insulin resistance model,we used deep RNA sequencing?RNA-Seq?to identify mRNAs and polyadenylated lncRNAs that are regulated during insulin resistance in mouse epididymal adipose tissues.To survey the functional contributions of IncRNAs during insulin resistance,we performed RNAi-mediated loss of function?LOF?experiments for candidate IncRNAs in 3T3-L1 adipocytes.Part 1:The expression profile of long non-coding RNAin insulin resistant mouse adipose tissuesObjectiveInvestigating the differential expression of lncRNAs between normal and insulin-resistant mouse epididymal adipose tissues?AE?.Screening IncRNAs associated with insulin resistance,thus providing a research basis for further functional studies.MethodsTwenty 5-week-old healthy male C57BL6 mice were adaptively fed for 1 week,and then randomly divided into normal chow diet?NCD,10 kcal%fat?and high fat diet?HFD,60 kcal%fat?group.After feeding for 12 weeks,oral glucose tolerance test?OGTT?and insulin tolerance test?ITT?were performed to assess the insulin sensitivity.Then the mice were sacrificed to measure the body weight and AE weight.Hematoxylin-eosin staining was executed to compare the adipocyte area between the two groups.The mRNA expression of interleukin-6?IL-6?and tumor necrosis factor-a?TNF-a?in adipose tissues was detected by Real-time PCR.Western blotting was used to detect the expression p-Akt?Ser473?and Akt protein.Deep RNA sequencing?RNA-Seq?was carried out to determine the differential expression of IncRNAs that are regulated during insulin resistance in mouse epididymal adipose tissues.Real-time PCR was used to validate the repeatability and reliability of RNA-seq data.For those differentially expressed IncRNA target genes,GO and KEGG pathway analysis were applied to seek the potential biological function.ResultsCompared with NCD group,the body weight and epididymal fat weight of the mice of HFD group were significantly increased?P<0.01?,the area of the adipocytes increased significantly,the glucose tolerance was abnormal and the insulin resistance was apparent in HFD group.Real-time PCR detected that the expression of IL-6 and TNF-? in epididymal adipose tissue of HFD group was significantly higher than that of NCD group?P<0.01?.Deep RNA sequencing identified 71 IncRNAs and 2985 coding genes that were significantly up-regulated while 145 lncRNAs and 2472 coding genes that were significantly down-regulated greater than twofold during insulin resistance in mouse epididymal adipose tissues.As assessed by Realtime-PCR,12 of 15 selected lncRNA genes up-regulated greater than threefold were confirmed as up-regulated during insulin resistance.GO enrichment analysis revealed that the differentially expressed lncRNAs target genes were mainly implicated in cellular component like membrane-bounded organelle,protein binding,catalytic activity,compound and RNA metabolic process.KEGG pathway analysis displayed that differentially expressed IncRNAs target genes participated in signaling pathways involved alcoholism,immunological inflammation,transcriptional misregulation in cancer and metabolic pathways,which are related to insulin resistance.ConclusionsCompared with normal white adipose tissue,IncRNAs expression in insulin resistant adipose tissues changed significantly,suggesting that IncRNAs were implicated in the development of insulin resistance in adipose tissues.Part 2:Effect of LncRNA on insulin sensitivity in mouse adipocytesObjectiveInvestigating the expression of the lncRNAs screened above between normal and insulin resistant 3T3-L1 adipocytes.Observing the effect of the differentially expressed IncRNAs on glucose uptake,mRNA expression of PPAR?,GLUT4 and IL-6,protein expression of pAkt/Akt.MethodsMouse 3T3-L1 preadipocytes were induced to differentiate into mature adipocytes by.lmg/ml insulin,lOmmol/L dexamethasone,0.5mmol/L 3-isobutyl-1-methylxanthine?IBMX?.Oil red O?ORO?staining was used to find the fat droplets in mature adipocytes.To further explore the biological validity of the adipocytes,the mRNA expression of adipogenesis markers,such as Fabp4 and PPARy,was measured by Real-time PCR.The adipocytes were cultured with 400 ?mol/L palmitic acid?PA?for 48 hours to establish a cellular insulin resistance model.Glucose-peroxidase method was executed to measure the concentration of glucose in the supernatant.The mRNA expression of PPARy,GLUT4 and IL-6 was quantified by Real-time PCR.The protein expression of pAkt/Akt were detected by western blotting.The differential expression of the IncRNAs screened above was examined by Real-time PCR between control?CON?and palmitic acid?PA?group.As assessed by Real-time PCR,the differentially expressed IncRNAs were chosen to undergo RNAi-mediated LOF.An unsilencing siRNA was used as a negative control?NC?.According to absence or presence of PA,the adipocytes were divided into 4 groups,NC group,siRNA group,NC+PA group and siRNA+PA group.Glucose uptake,mRNA expression of PPARy,GLUT4,IL-6 and protein expression of pAkt/Akt were measured after treatment.ResultsAs shown in ORO staining that fat droplets were dyed orange,the mouse 3T3-L1 preadipocytes were successfully induced to differentiate into mature adipocytes.And the Real-time PCR presented that mRNA levels of Fabp4 and PPAR? were significantly increased after adipogenesis.After the administration of PA,the glucose uptake of adipocytes decreased,the expression of PPAR?,GLUT4 mRNA and Akt phosphorylation declined,IL-6 mRNA increased.As assessed in Real-time PCR,9 of 15 selected lncRNAs were significantly up-regulated in PA group compared with CON group.The LOF assay for Gm26870,Gpr137b-ps,TCONS₀1864186 showed that silence of Gm26870 contributed to the improvement of glucose uptake and the expression of PPAR?,GLUT4 mRNA and Akt phosphorylation impaired by insulin resistance,together with alleviation of IL-6 expression.ConclusionPalmitic acid induces insulin resistance in 3T3-L1 adipocytes.LncRNA Gm26870 may have an influence on regulating insulin resistance.