The Long Noncoding RNA Blnc1 Protects Against Obesity-induced Insulin Resistance By Remodeling Mitochondrial Function In White Fat And Its Potential Mechanism

Obesity is a complex metabolic disorder associated with many serious chronic diseases,such as type 2 diabetes,non-alcoholic fatty liver and coronary heart disease.It is listed as one of the top ten diseases threaten human health by the World Health Organization.Thus,we need to further study the pathogenesis of obesity and discover the new therapeutic targets.Recent studies show that long noncoding RNA(lnc RNA),a type of noncoding RNA,plays a vital role in the occurrence and development of obesity.A previous study has reported that lnc RNA SRA(steroid receptor RNA activator)is the first lnc RNA that is found to regulate adipogenesis.It binds to the promoter region of PPAR? and promotes its transcription to affect adipogenesis.Current studies have shown that lnc RNA Blnc1(Brown fat lnc RNA1)expresses in high abundance of brown fat and plays an important role in the regulation of brown adipocyte differentiation and thermogenic genes.In our previous study,we found that Blnc1 was also expressed in white fat tissue.However,the regulatory role of Blnc1 on the mitochondrial function of white adipocytes is poorly understood.In this study,male C57BL/6 mice are fed a high-fat diet(HFD)to induce obesity,followed by injection of Blnc1-over adenovirus in the epididymal fat(e WAT).And we investigate the effect of Blnc1 on the systemic glucose and lipid metabolism and the effect on the e WAT mitochondrial biosynthesis.Then in vitro studies,mitochondrial biogenesis and function are analyzed in 3T3-L1 pre-adipocytes with Blnc1 over-expression or knockdown.Furthermore,we explore the specific molecular mechanism of Blnc1 regualating the mitochondrial biosynthesis and function in pre-adipocytes,and provides the experimental evidence for the new therapeutic targets of obesity.Part ?.The effect of e WAT-specific overexpression of Blnc1 on systemic metabolism in high-fat-diet induced obese miceObjectives We aimed to clarify the effect of e WAT-specific overexpression of Blnc1 on systemic glucose and lipid metabolism in HFD-induced obese mice,and to explore its effect on the mitochondrial biosynthesis of e WAT.Methods Male C57BL/6 mice were fed a high-fat diet(HFD)for 12 weeks to induce obesity,and was randomly divided into HFD-Ad-GFP(n = 5)and HFD-Ad-Blnc1 group(n = 5).In HFD-Ad-Blnc1 group,mice were multi-points injected with adenovirus particles carrying Blnc1(Ad-Blnc1)in the two sides of e WAT.And the HFD-Ad-GFP mice were injected with negative virus.After 5 weeks,intraperitoneal glucose tolerance test(IPGTT)and insulin tolerance test(ITT)were conducted to assess glucose tolerance and insulin sensitivity,respectively.The body weight,adipose tissue and liver weight of the different treatment groups were measured.In addition,Hematoxylin and eosin(H&E)staining was performed to characterize the structure and lipid accumulation in the liver and e WAT.And the mitochondrial biosynthesis in e WAT was detected by using mito-tracker staining,mitochondrial DNA(mt DNA)copy number,real-time quantitative PCR(q PCR)and western blotting.Results 1.Compared with the normal diet goup(NCD),mice fed a HFD had higher body weight(P < 0.001),impaired glucose tolerance,insulin resistance,hepatic lipid deposition,less e WAT mitochondrial quantity and the gene expression levels related to mitochondrial biosynthesis(P < 0.05).2.Compared with HFD-Ad-GFP group,Blnc1 was specifically overexpressed in e WAT of HFD-Ad-Blnc1 mice(P < 0.05).And e WAT weight was singnificantly reduced in HFD-Ad-Blnc1 mice(P < 0.001).3.After the e WAT-specific overexpression of Blnc1 in obese mice,plasma adiponectin level was significanatly increased,glucose tolerance was improved and insulin resistance was reduced(P < 0.05).4.Compared with the HFD-Ad-GFP mice,the lipid deposition and triglyceride content in liver of HFD-Ad-Blnc1 mice were significantly reduced(P < 0.05).The expression of hepatic adiponectin receptor and its downstream lipolytic genes(Pgc1?,dipo R2,APPL1,Acadl,Aox,PPAR?)were increased(P < 0.05).5.Through mt DNA copy number detection and mito-tracker staining,HFD-Ad-Blnc1 mice were observed to increase mitochondrial biosynesis in e WAT compared with HFD-Ad-GFP mice,and the gene expression related to mitochondrial biosynthesis and function(Pgc1?,Pgc1?,Adiponectin,electron transport chain-ECT)were significantly upregulated(P < 0.05).Conclusions HFD-induced obesity can cause insulin resistance,hepatic steatosis and impair mitochondrial biosynthesis in white fat adipose.The e WAT-specific overexpression of Blnc1 in obese mice can effectively increase plasma adiponectin level,alleviate obesity-induced insulin resistance and hepatic steatosis,by partly reversing the impairment of mitochondrial biosynthesis in e WAT.Part ?.The effect of Blnc1 on the mitochondrial biosynthesis and function of 3T3-L1 pre-adipocytesObjectives In the first part,we found that e WAT-specific overexpression of Blnc1 in obese mice significantly improved obesity-induced insulin resistance and hepatic steatosis,partly reversed the impairment of mitochondrial biosynthesis in e WAT.And we speculated that Blnc1 had a certain repair effect on mitochondrial function of e WAT.In order to clarify the role of Blnc1 on white fat mitochondrial biosynthesis and function,we overexpressed or knock down Blnc1 in 3T3-L1 pre-adipocytes and observed the effect of Blnc1 on mitochondrial biosynthesis and function.Methods To examine the role of Blnc1,3T3-L1 pre-adipocytes were overexpressed or knock down Blnc1 by infected the Blnc1-over adenovirus or the Blnc1-KD lentivirus.Stable Blnc1-KD cell lines were generated by puromycin screening.The number and shape of mitochondria were tested through mito-tracker staning,mt DNA copy number and transmission electron microscope.The oxygen respiration rate(OCR)and ATP production were detected to assess mitochondrial function.Moreover,the q PCR and western blotting were used to detect the expression of mitochondrial ETC complexes and key genes of biosynthesis and function.Results 1.Blnc1-over adenovirus and Blnc1-KD lentivirus were able to successfully overexpress or knock down Blnc1 in 3T3-L1 pre-adipocytes(P < 0.001).And the stable Blnc1 knockdown cell line was obtained.2.In 3T3-L1 pre-adipocytes overexpressing Blnc1,mito-tracker staining was deepened,mt DNA copy number was increased,mitochondrial cristae was increased under electron microscope and the gene expression related to mitochondrial biosynthesis(Pgc1?,Pgc1?,NRF1)was upregulated(P < 0.05).In terms of mitochondrial function,the gene expression of mitochondrial ETC complexes was significantly upregulated,oxygen respiration rate and total ATP production were increased in Blnc1-over 3T3-L1 pre-adipocytes(P < 0.05).3.After knocking down Blnc1,mirochondria were damaged in 3T3-L1 pre-adipocytes,which showed lighter mito-tracker staining,decreased mt DNA copy number,reduced mitochondrial cristae,and downregulated expression of genes related to mitochdrial biosynthesis(P < 0.05).In terms of mitochondrial function,the gene expression of mitochondrial ETC complexes was significantly downregulated,oxygen respiration rate and total ATP production were decreased in Blnc1-KD 3T3-L1 pre-adipocytes(P < 0.05).Conclusions In 3T3-L1 pre-adipocytes,Blnc1 is required for mitochondrial biosynthesis and function.Overexpression of Blnc1 can increase mt DNA copy number,upregulate mitochondrial biosynthesis and gene expression levels of ETC complexes,promote mitochondrial oxygen respiration and ATP production.However,knockdown of Blnc1 can decrease mt DNA copy number,downregulate mitochondrial biosynthesis and gene expression levels of ETC complexes,inhibit mitochondrial oxygen respiration and ATP production.Part ?.The potential molecular mechanisms of Blnc1 on the regulatory of mitochondrial biosynthesis and function in 3T3-L1 pre-adipocytes.Objective The mechanism of lnc RNA regulation of target genes is complex and diverse.One of the molecular mechanisms for lnc RNA in the nucleus is to activate or inhibit the transcription of target genes by recruiting some transcription factors or RNA binding protein.Therefore,we speculated that Blnc1 may regulate the mitochondrial biosynthesis and function by binding related proteins.The purpose of this part was to discover a RNA-binding protein that directly bound to Blnc1 and its specific molecular mechanism for regulating target gene.Methods Blnc1 binding protein was identified using RNA-protein pull down and RNA-binding protein immunoprecipitation(RIP)experiments.Then,the specific molecular mechanism of Blnc1 regulating Pgc1? through hn RNPA1 was explored by Chromatin immunoprecipitation(Ch IP)technology.Results 1.Through RNA protein pull down and RIP experimental techniques,it was confirmed that Blnc1 and hn RNPA1 were directly bound to each other.2.In 3T3-L1 pre-adipocytes,over-expressing hn RNPA1 could significantly reduce the expression level of Pgc1?(P < 0.001).Based on the alignment of the Pgc1? upstream promoter sequence and the common binding region of hn RNPA1,we found multiple binding positions of hn RNPA1 and Pgc1? promoter region.Ch IP experiment was used to verify that hn RNPA1 antibody could enrich the Pgc1? promoter region(P < 0.001).3.In 3T3-L1 pre-adipocytes overexpressing Blnc1,the above Ch IP experiment was performed,and it was observed that the binding of hn RNPA1 to the Pgc1? promoter region was significantly reduced after overexpression of Blnc1(P < 0.05).Conclusions Blnc1 binds directly to hn RNPA1 protein.Hn RNPA1 can specifically bind to the Pgc1? promoter region and inhibit the transcriptional activity of Pgc1?.One of the mechanisms by which Blnc1 regulates the mitochondrial biosynthesis and function of preadipocytes is to stimulate the transcription of Pgc1?(a key mitochondrial biosynthesis gene)via decoying hn RNPA1.