The Functions Of MiR-200 In Fat Metabolism And MiRNA Expression Profiles In Cold-activated Adipose Tissue Of Mice

There are two classic adipose tissues in animals,white adipose tissue(WAT)and brown adipose tissue(BAT).WAT has the unique function of storing triglycerides while BAT is specialized for thermogenesis and energy expenditure as a defense against cold and obesity.Growing evidence has demonstrated the important role of micro RNAs in regulating a variety of biological processes,such as adipogenesis and brown fat thermogenesis.Our study focused on two topics in fat metabolism and miRNAs:1.The functions of miR-200 family in fat metabolism of miceThe miR-200b/a/429 cluster has been functionally characterized in mammalian reproduction.Our previous data showed that the miR-200b/a/429 cluster was strongly expressed in white adipose tissue.however,the potential role of the miR-200 family in adipocytes is poorly understood.We generated adipocyte-specific miR-200b/a/429knockout(ASKO)mice using a Cre-lox P system in which Cre expression was driven by the a P2 promoter.The ASKO and wild-type(WT)littermate mice were fed a normal or 60%high fat diet(HFD),and the changes in the growth rate,metabolic parameters,the response to cold exposure,and the expression of genes and proteins related to lipid metabolism were analyzed.Their putative target genes of miR-200 b were computationally predicted and validated via in vitro reporter assays.The main results are as follows:(1)Our data showed that the miR-200b/a/429 cluster was most strongly expressed in Gon-fat and least strongly expressed in BAT.Mi R-200b/a expression was significantly enhanced in primary brown differentiation and and decreased in 3T3-L1 differentiation.Moreover,Our results showed that the ASKO mice displayed significantly lower expression levels of miR-200b/a/429 in Gon-fat,Ing-fat,and BAT than the control littermates;however,the abundance of miR-200b/a/429 in other tissues,including liver,kidney and lung tissue,was not altered.So we successful generated the ASKO mice.(2)The ASKO mice displayed a similar growth rate to the WT mice when fed the CD but exhibited an increased body weight after 9 weeks of HFD induction.The total weight of adipose tissue from the ASKO mice are significantly increased with 34.6%.No difference was observed in the weight of other peripheral organs.HE staining of the WAT revealed the presence of enlarged adipocytes in the HFD-fed ASKO mice.(3)The GTT assay and ITT assay suggested that the HFD-fed ASKO mice exhibited glucose intolerance,and the decreased insulin sensitivity in the HFD-fed ASKO mice.Furthermore,we found that the liver and WAT represent the major insulin-responsive organs in the HFD-fed ASKO mice,as indicated by the remarkable decrease in Akt phosphorylation in these two organs compare with WT mice.(4)Compare with WT mice,The significantly decreased levels of the HSL and ATGL were observed in the WAT from the HFD-fed ASKO mice.Upon β3-adrenergic agonist CL-316,243 stimulation,significantly attenuated lipolysis was observed in the HFD-fed ASKO mice.(5)The plasma TAG levels of the HFD-fed ASKO mice were significantly higher than WT mice.And the total cholesterol levels of ASKO mice exhibited significantly lower under both diet conditions.The fasting insulin levels were significantly lower in the ASKO mice than in the WT mice under both CD and HFD conditions.(6)The results of metabolic cages revealed decreased oxygen expenditure in the HFD-fed ASKO mice.Under 4°C condition,the core body temperature decreased much faster in the HFD-fed ASKO mice than in the HFD-fed WT mice.(7)Dual-luciferase assays revealed the 3’UTR of Lhfp,Glis2,Eps8 or Rps6kb1 can bind with the seed region of miR-200 b.Furthermore,real-time PCR data revealed the significant elevation of Lhfp and Rps6kb1 in the WAT of HFD-fed ASKO mice.2.Mi RNA expression profiles in cold-activated white and brwon adipose tissueActivation of BAT and WAT can be induced by cold exposure and miRNAs have been shown to play the key role during this process.To identify the key miRNAs which are involved in the browning of the WAT and in the cold-induced thermogenesis in adipose tissue(including WAT and BAT),mice were housed at 6°C or 28°C for 10 days and deep sequencing of adipose miRNAs from control and treatment group was performed to profile adipose miRNAs expression.The main results are as follows:(1)We detected the expression level of Ucp1 in BAT and Ing-WAT from 28°C and6°C.The m RNA and protein of Ucp1 were significantly up-regulated by about 3.5 fold in the BAT and 143 fold in the Ing-WAT during prolonged cold treatment.(2)For cold-tread BAT,significant changes in miRNA expression were observed for27 DE miRNAs(fold change >2 and FDR <0.05),with 17 of those upregulated and 10 miRNAs downregulated.Significant changes were observed for 29 DE miRNAs in Ing-WAT,with 7 miRNAs upregulated and 22 miRNAs downregulated.Of these cold responsive DE miRNAs,nine were found overlapped in both BAT and WAT,including miR-144-3p,miR-203-5p,miR-6538,miR-3076-5p,miR-146a-5p,miR-146b-5p,miR-150-3p,miR-342-3p and miR-342-5p.(3)86 miRNAs were identified to be differentially expressed between BAT and WAT at 28°C.Mi R-499-5p,miR-883a-5p,miR-107-5p and miR-6481 were specificity high expressed in BAT while miR-196a-5p,miR-196b-5p,miR-196a-2-3p and miR-20b-5p were enriched in Ing-WAT.(4)RT-q PCR results demonstrated that miR-144-3p,miR-146a-5p,miR-146b-5p,miR-30e-5p and miR-200c-3p had statistically significant expression patterns which were similar to those seen in sequencing data.The results of BAT differentiation showed that miR-146a-5p,miR-146b-5p,miR-30e-5p and miR-200c-3p-3p were significant upregulation during adipogenesis,only miR-144-3p were downregulated.(5)The negative correlation analysis of different expression miRNAs and their predicted targets was analyzed basing on Microarray data(GSE51080).Negative correlation network of BAT was contained 14 miRNAs and 105 targeted genes,while there were 16 miRNAs and 55 targeted genes in ing-WAT.Five targeted genes(miR-146a-5p/Sphk2,miR-3076/Lonrf3,miR-144-3p/Bmpr1 b,mir-503-5p/Slc4a4 and miR-144-3p/Phlda1)were overlaped between BAT and ing-WAT network.In summary,the conclusions of this study are:(1)Our studies generated adipocyte-specific miR-200b/a/429 knockout mice using a P2-Cre/lox P system for the first time.(2)Our studies demonstrated the essential role of miR-200b/a/429 in adipose tissue in the regulation of HFD-induced whole-body metabolic changes,and suggest a new candidate gene for therapy of obesity and its associated diseases.(3)Our study identifies for the first time the crucial miRNAs involved in cold-activated white and brown adipose tissue by sequencing.