LncRNA SRA Promotes Hepatic Steatosis Through Repressing The Expression Of Adipose Triglyceride Lipase(ATGL) Via FoxO1 And PPARγ Pathway

BackgroundNon-alcoholic fatty liver disease(NAFLD)is a metabolic liver injury which characterized by hepatic steatosis and fat accumulation,and related to obesity,insulin resistance and type Ⅱ diabetes.In recent years,the prevalence of NAFLD increased both in developing and developed countries worldwide.The population survey in China found that the average incidence of NAFLD in adults is about 15%.Among them,males aged 40 to 49 are at higher risk and the incidence up to 28%.NAFLD can further develop into nonalcoholic steatohepatitis(NASH),cirrhosis and even hepatocellular carcinoma.It has become the second type of liver disease that seriously endangers human health.Hepatic lipid metabolism balance relies on the functional coordination of multiple physiological processes,including lipogenesis,free fat acid(FFA)β-oxidation,lipid uptake,very low density lipoprotein(VLDL)secretion and lipolysis.These processes are tightly controlled by genes and pathways.Adipose triglyceride lipase(ATGL)can hydrolyze the first ester bond of triglyceride(TAG),which is the rate-limiting enzyme in TAG hydrolysis.It is so important in liver for its function in TAG hydrolase which could promote FFA oxidation.Long non-coding RNA(lnc RNA)is a new type of RNA without coding function,which could regulate epigenetic,transcription and post-transcriptional,and plays an important role in the regulation of gene expression in the process of embryonic development,differentiation,liver lipid metabolism and the maintenance of homeostasis.Steroid receptor RNA activator(SRA)was initially characterized as a lnc RNA that functions as an RNA coactivator to enhance steroid receptor-dependent gene expression.SRA plays important roles in myogenesis,steroidogenesis and insulin signaling.In addition to reduced fat mass,SRAKO mice have decreased hepatic TAG levels and resistance to diet-induced obesity.Therefore,the further study of the regulatory role of SRA in the metabolism of glucose and lipid metabolism can provide new ideas and targets for the treatment of NAFLD.Content and objectives1.We analyzed the key genes related to lipogenesis,FFA β-oxidation and VLDL secretion in livers of WT and SRAKO mice by RT-q PCR.Chapter Ⅰ aims to find the target gene of SRA in regulation of hepatic lipid metabolism-ATGL.2.Previous studies have shown that Fox O1 is the upstream transcription factor of ATGL in adipocytes.However,the regulation of Fox O1 on ATGL in hepatocytes is unclear.In the chapter Ⅱ,we focus on whether SRA could affect the function of Fox O1 and its regulatory effect on ATGL in liver.3.It has been reported that PPARγ can induce ATGL transcription in adipocytes,and even its own transcription.The chapter Ⅲ aims to investigate whether SRA could affect the expression of ATGL through the PPARγ pathway and the role of SRA in the regulation of PPARγ.Methods and results 1.8-week-old WT,SRAKO mice were fed with normal chow or high-fat diet(HFD)to 20 weeks of age,ob/ob mice were fed with normal chow to 20 weeks of age,RT-q PCR was used to examine the expression of hepatic genes that regulate lipogenesis,FFA β-oxidation and VLDL secretion,to identify the key regulators which may prevent hepatic steatosis.2.Hepatocytes were isolated from WT and SRAKO mice,and Hepa1-6 cells were infected with sh SRA lentivirus or transfected with SRA expression vector.RT-q PCR was used to detect the SRA and ATGL m RNA levels,and ATGL protein levels were analyzed using western blot,ATGL enzyme activity and ketone body production were also examined with kits.3.The m RNA levels of SRA and ATGL in the liver of WT-Chow,WT-HFD and ob/ob mice were detected by RT-q PCR.The protein expression of ATGL was detected with western blot.Results1.SRAKO increase hepatic ATGL m RNA and protein expression in both normal chow and HFD compared with WT.It suggested that SRA knockdown can increase the level of hepatic ATGL expression.2.SRAKO hepatocytes have increased m RNA and protein levels of ATGL than WT,which consequently induce higher ATGL enzyme activity and ketone body production activity.Similar results were found in Hepa1-6 cells infected with sh SRA lentivirus.Overexpression of SRA inhibits ATGL m RNA and protein levels and ketone body production activity.It suggested that SRA knockdown can up-regulate ATGL expression,increase FFA β-oxidation activity.3.Under fasting conditions,hepatic SRA m RNA levles significantly increased in WT-HFD mice and ob/ob mice compared to WT-Chow;hepatic ATGL m RNA levels were siginifanlty reduced in WT-HFD and ob/ob mice compared to WT-Chow mice,but no change was found in fed conditions.In fast state,SRA negatively regulates ATGL transcription.SummarySRA negatively regulate ATGL transcription: SRA knockdown→ATGL upregulation→Increased FFA β-oxidation→decreased hepatic steatosis.Materials and Methods1.The hepatic nuclear protein of WT-Chow,WT-HFD and ob/ob mice,WT-Chow,SRAKO-Chow,WT-HFD and SRAKO-HFD was isolated to detect whether HFD and SRAKO affect Fox O1 nuclear translocation.2.Hepa1-6 cells were transfected with Fox O1 expression vector,and ATGL m RNA and protein expression levels were detected by RT-q PCR and western blot.Hepa1-6 cells were co-transfected with luci-ATGL,Fox O1 and SRA or infected with sh SRA lentivirus followed with Fox O1 inhibitor treatment,to investigate the effect of Fox O1 on ATGL expression and whether SRA affects the ATGL transcriptional activity through Fox O1 pathway.3.Hepa1-6 cells were transfected with SRA or infected with sh SRA lentivirus,and hepatocytes were isolated from SRAKO mice and stimulated with insulin to investigate the effects of SRA on insulin sensitivity and Fox O1 phosphorylation;PI3K/Akt pathway and MEK/ERK pathway inhibitors were involved to investigate the effects of PI3K/Akt and MEK/ERK signaling pathways on Fox O1 nuclear translocation and ATGL transcriptional activity.Results1.Either in fast or fed conditions,no change was found in WT and SRAKO mice hepatic nuclear Fox O1 expression lelves.Indicating that SRA deficiency did not affect the hepatic nuclear Fox O1 protein content.2.Overexpression of Fox O1 upregulate both the m RNA and protein levels of ATGL in Hepa1-6 cells.SRA inhibit and Fox O1 increase ATGL promoter driven luciferase,but SRA repress the Fox O1 mediated effect in dose-dependent manner.Fox O1 inhibitor induces the suppression of ATGL promoter transcription activity,and diminished the inhibitory effect of SRA on ATGL promoter activity.Indicating that in the hepatocyte,Fox O1 could up-regulate the transcriptional activity of ATGL,and SRA inhibits the expression of ATGL through Fox O1.3.Overexpression of SRA enhanced insulin signaling and the downstream pathways phosphorylation.SRA overexprssion increased Fox O1 nuclear retention in the absence of insulin,but insulin eliminated this effect and decreased nuclear Fox O1.The PI3K/Akt inhibitor wortmannin blocked the insulin-stimulated nuclear export of Fox O1,while the MEK/ERK inhibitor trametinib had no effect.Insulin reduced ATGL promoter driven luciferase activity,and wortmannin or trametinib did not completely reverse this inhibition.However,the Fox O1 inhibitor eliminated the suppressive effects of SRA and insulin on ATGL promoter luciferase.It suggested that SRA inhibit Fox O1 activity not through the nuclear translocation,and the inhibitory effect of insulin and SRA on the transcriptional activity of ATGL depends on the Fox O1 pathway,but the inhibition of SRA is independent of insulin.SummarySRA can inhibit the transcriptional activity of ATGL through Fox O1 pathway.SRA also can increase the phosphorylation of Fox O1 indcued by insulin,but not affect the nuclear translocation.Both SRA and insulin inhibit the ATGL transcription activity through Fox O1 pathway,but SRA function independent of insulin signaling.Materials and Methods1.Hepa1-6 cells were co-transfected with luci-ATGL luciferase reporter,PPARγ and SRA expression vectors,or infected with sh SRA lentivirus,followed with PPARγ agonists and inhibitors treatment to investigate the effect of PPARγ on ATGL transcription activity and whether SRA could affect the ATGL transcriptional activity through PPARγ pathway.2.The hepatic nuclear protein from WT-Chow,WT-HFD and ob/ob mice,WT-Chow,SRAKO-Chow,WT-HFD and SRAKO-HFD mice were isolated and the expression of PPARγ was detected by western blot.RT-q PCR was used to detect the WT-Chow,SRAKO-Chow,WT-HFD and SRAKO-HFD hepatic PPARγ m RNA expression levels.3.Hepa1-6 cells were co-transfected with SRA,PPARγ1 and PPARγ2 expression vectors,and treated with rosiglitazone.The m RNA levels of SRA,PPARγ1,PPARγ2 and its downstream lipid metabolism genes were detected using RT-q PCR.4.Hepa1-6 cells were co-transfected with SRA,PPARγ1 and PPARγ2 expression vectors,and co-immunoprecipitation was used to detect whether SRA could affect the interacted between PPARγ1/γ2 and RXRα through "scaffold" effect.Results1.Only in the presence of rosiglitazone,PPARγ increased ATGL promoter activity,overexpression of SRA blocked the ability of rosiglitazone/PPARγ to induce ATGL promoter activity.Conversely,SRA knockdown enhanced ATGL promoter activity mediated by rosiglitazone actived PPARγ.PPARγ inhibitor showed no effect on the ability of exogenous SRA to inhibit ATGL promoter activity.Indicating that SRA can block the ability of rosiglitazone actived PPARγ induced ATGL promoter activity,and this effect is not dependent on Fox O1.2.In the HFD and ob/ob livers,PPARγ protein levels are dramatically increased compared with that in normal chow mice.The increased m RNA and protein levels of hepatic PPARγ by HFD are substantially decreased in SRA deficiency.It suggested that SRA can inhibit the expression of PPARγ under HFD conditions.3.Both PPARγ1 and PPARγ2 could up-regulated the m RNA levels of themselves and the lipid metabolism genes in Hepa1-6 cells.Rosiglitazone could further up-regulated the expression of these genes.Overexpression of SRA further enhanced the expression of these genes.It suggested that SRA has transcription promoting effect on PPARγ1/γ2 and their downstream genes.4.The protein levels of PPARγ1/2 and RXRα are not affected by the overexpression of SRA in Hepa1-6 cells,but SRA can enhance the binding between PPARγ1/γ2 and RXRα.It suggested that SRA promotes the binding of PPARγ1/γ2 to RXRα through the “scaffold” effect.SummarySRA blocks the ability of rosiglitazone/PPARγ induced ATGL promoter activity,and SRA has a transcriptional promoting effect on both PPARγ1/γ2 and the downstream lipid metabolism genes,SRA also function as "scaffold" to promote the interaction between PPARγ1/γ2 and RXRα.