The Roles Of Sirt1 Gene In Lipid Metabolism Of Chicken Liver

The liver is an important organ for animal energy metabolism,and is responsible for various functions such as glycolysis,glycogen synthesis,gluconeogenesis,fatty acid beta oxidation,lipogenesis,lipoprotein production and secretion.It regulates metabolic pathways and produces energy in response to nutritional status and hormonal signals.In particular,it plays an important role in the metabolism of glycolipids.The liver promotes transformation of excessive glucose to fat,which deposits in human body.Excessive glucose intake for a long time may cause obesity,lipid liver and other related metabolic diseases.In the poultry breeding,excessive fat deposition is harmful to the poultry health,leading to reduction of feed conversion rate and the laying rate of laying hens.The fat deposition of triglycerides mainly comes from blood lipids,which are mainly produced by the liver through a de novo synthesis pathway and secreted into the blood in chickens.Therefore,it is extremely important to understand lipid metabolism and the underlying mechanism in order to reduce lipid deposition in the chicken body.SIRT1,a NAD(+)-dependent histone deacetylase,plays crucial roles in multiple biological processes including gene transcription,cellular metabolism and stress responses through regulating the activities of histones and non?histones by deacetylation.Although it has been well established that there are some differences in glucose and lipid metabolism between birds and mammals,SIRT1 effects on liver energy metabolism and the underlying mechanisms remain completely unknown in chicken.In this study,we firstly analyzed the expression profile of Sirtl in various tissues and liver developmental stages in Guangxi Sanhuang chicken.Next,we considered the expression of Sirtl and other metabolism-related genes under energy starvation in chicken liver.We also identified many key metabolic genes expression regulated by Sirtl in a LMH cell line and chicken liver in response to high glucose and high fat metabolic stress.Finally,we analyzed Sirtl promoter flinction and studied its transcriptional and post-transcriptional regulation in chicken liver.By these studies,we want to reveal the crucial roles and the underlying mechanisms of Sirtl in lipid metabolism in chicken liver?and therefore provide an important theoretical support for controlling the chicken's lipid deposition during the rapid breeding process.(1)Expression pattern of Sirtl in various tissues and developmental stages of chicken liversIn order to detect the mRNA expression patterm of Sirtl,we used RT-qPCR to analyze various tissues in 4-week-chickens and different developmental stages of chicken livers.The results showed that the expression of Sirtl in kidney was higher than that of other tissues.The expression of Sirtl showed no difference in the different developmental stages of chicken livers.(2)The expression of Sirtl and other metabolism-related genes is regulated by energy starvation in chicken liver.In order to clarify the effects of starvation on Sirtl expression in chicken liver we tested metabolic parameters and Sirtl levels after fasting the chicken for 24 h.We found that body's blood glucose level was slightly decreased after 24 h fasting.The concentration of blood lipids was significantly decreased,but cholesterol level remained constant.Interestingly,Sirtl mRNA and protein levels were increased significantly after 24 h fasting.In addition,mRNA level of Pgc-la and Mt3 were increased by 7-folds and 26.8-folds,respectively.And mRNA levels of Ppara,Foxol,Hnfla,Vnnl,and Atgl were increased by 0.36 to 3 folds,while mRNA level of Fasn was decreased to about 1/13 in the liver compared with that of the normal feeding group.After short?term refeeding for 2 hours,the level of blood glucose was slightly increased,and the level of blood lipid was markedly increased,although it was still lower than that of the normal feeding group.Importantly,the Sirtl mRNA level was rapidly decreased in the liver,even lower than that of the normal feeding group.The mRNA levels of Pgc-1?,Ppar?,Foxo1,Hnf1?,Vnn1,Mt3 and Atgl were also rapidly decreased to the level of the normal feeding group,and the mRNA level of Fasn was increased to 1/4 fold to that of the normal feeding group.These results indicate that these genes are regulated by energy stress and could play an important role in the maintenance of blood sugar and lipid metabolism in the chicken liver.(3)Sirtl effects on gene expression in chicken liver.To further clarify the roles of Sirt1 gene in chicken hepatic metabolism,we compared the different expression genes(DEGs)after Sirtl was knocked down by siRNA in primary chicken liver cells through RNA sequencing assay.The results demonstrated that a total of 86 DEGs were identified between Sirt1 knocked down and control hepatocytes,of which 63 genes were down-regulated and 23 genes were up-regulated after Sirt1 was knocked down.GO analysis showed that 70 DEGs were involved in various biological processes,58 DEGs were related to cell composition,and 77 DEGs were related to molecular functions.KEGG pathway analysis showed that DEGs were involved in metabolism,genetic replication and expression,cell function,external signal transduction and diseases,among which Adh6,H6pd,Hexa_b and Glms were related to carbohydrate metabolism,and Adh6,Acsl4,Lipa,and Gnpat were related with lipid metabolism.Especially,the expression of Vnnl and Mt3 was decreased and Frzb increased.These genes were related to liver carbohydrate and lipid metabolism and liver cell development.Importantly,the expression of Adh6,Acsl4,Vnn1,and Mt3 but not Lipa,Frzb and H6pd can be rescued by the ectopic Sirtl overexpression in LMH cells.These results suggest that Sirt1 roles on hepatic metabolism are mediated by regulating the expression of these downstream targets.(4)Establishment of the stable Sirtl knocked down cell lineNext,we generated Sirtl knocked down stable cell line to investigate the role of Sirtl in the response to glucose and lipid metabolism.Three miRNAs were designed targeting the 3'UTR sequence of chicken Sirtl gene and selected for the effective one.Stable LMH cell lines were established by the infection of LMH cells with the retroviral vectors expressing these miRNA sequences.The results showed that three miRNAs were able to effectively inhibit the luciferase activity of the reporter vector containing the corresponding target site.After 48 h of transient expression in LMH cells,the Sirtl mRNA levels were inhibited by 30%,indicating that these miRNAs can interfere with Sirtl mRNA expression.Next,LMH cells were infected with the virus particles encapsulated with gSmiR30 and one cell line that stably overexpressed gSmiR30 was quickly obtained through G418 resistance screening.The expression rate of GFP was more than 99%in this cell line.Importantly,Sirtl expression was significantly knocked down,as indicated by the markedly decreased levels of Sirtl mRNA and protein expression.These results demonstrate that a LMH-gSmiR30 cell line with stably knocked down Sirtl expression has been successfully constructed.(5)Sirt1 metabolic effects in response to high glucose and high fat in LMH cell lineTo clarify Sirtl metabolic effects,we treated LMH-pmirG(control)and LMH-gSmiR30 cells with high-sugar and high-lipid media.The results showed that the formation of lipid droplets was significantly increased by red 0 staining in LMH-gSmiR30 cells compared with controls,suggesting that glycolysis and fat degradation were significantly inhibited in these cells.Furthermore,the expression of two key metabolic genes,G6pc and Atgl,was markedly reduced after Sirtl was knocked down in LMH-gSmiR30 cells.The expression of SIRT1,H6pd,Fasn,and Ppara gene was significantly inhibited by high glucose and high fat treatment in both control and LMH-gSmiR30 cells.However,we did not observe significant difference between two groups.These findings indicate that Sirtl is implicated in LMH cell response under the metabolic stress to glucose and lipid.(6)Investigate Sirtl promoter function and transcription regulationIn order to study Sirtl transcription regulation,a 14 bps 5'UTR sequence was amplified by 5'RACE technology.The functional fragments from the sequence were predicted through bioinformatics analysis.Furthermore,we confirmed that 106-129 bp upstream of the transcription initiation codon plays an important role in regulating Sirtl transcription,suggesting that it could be a TATA box element.In addition,the element located between 129 bp and 165 bp promotes the transcription of Sirt1 gene.(7)The effect of methylation on Sirtl expression in chicken liver cellsNext,we analyzed the promoter methylation and its effect on Sirtl expression in chicken liver cells.A 1600 bps "CpG" island in the sequence from the first exon to the upstream sequence was predicted via the Methyl Primer Express V1.0 software.It contains the first exon of the Sirtl gene and the upstream 1200 bps sequence.Importantly,Sirtl mRNA levels were significantly unregulated by more than 60%in LMH cells treated with 5-azacytidine for 6 days,suggesting that the promoter methylation inhibits Sirt1 transcription in chicken liver cells.(8)Sirt1 post-transcriptional regulation by miRNAs in chicken liverFinally,we considered Sirt1 post-transcriptional regulation by miRNAs in chicken hepatocytes.The 3'UTR sequence of Sirt1 gene was amplified by 3' RACE technology from chicken liver.136 potential miRNAs targeting this fragment were predicted by miRanda and TargetScan.Twelve miRNAs were selected based on the principle of high miRNA and target site scoring and low free energy.The results showed that gga-miR-204 and gga-miR-302b-3p can effectively inhibit the activity of luciferase containing the targeting site.Furthermore,the effects were completely abrogated after the targeting sites were mutated.These findings indicate that Sirt1 mRNA is regulated by both siRNAs by targeting the specific sequence.The expression of gga-miR-302b-3p cannot be detected in the chicken liver.Sirtl mRNA and protein levels were inhibited by overexpressing gga-miR-204 in LMH cells and primary chicken hepatocytes.These findings indicate that gga-miR-204 inhibits Sirtl expression via the post-transcription regulation in chicken liver.