Study On The Expression Of Hexokinase-1 In The Development Of Goose Fatty Liver And The Regulation Of Its Function

Goose liver can deposit a lot of fat without any obvious pathological symptoms during overfeeding,suggesting there exists a tolerance mechanism in goose liver.Mitochondrion is an important organelle for aerobic respiration and ATP synthesis,and provides energy for cellular activities,thus it is also called the power plant of the cell.Evidence indicates the association of mitochondrial loss and dysfunction with mammalian non-alcoholic fatty liver diseases.Our preliminary data show that the expression of hexokinase 1(HK1),a mitochondrial out-membrane protein,is significantly induced in the development of goose fatty liver,suggesting its involvement in the formation of goose fatty liver.However,the underlying mechanism is unclear.This study was aimed to determine the expression of HK1 in different tissues at different time points in the overfed vs normally fed geese,the influence of the fatty liver-related factors and transcription factors on the expression of HK1,as well as the biological functions of HK1.The main methods and results are as follows:1.Study on spatiotemporal expression pattern of HK1 gene in the development of goose fatty liver.The mRNA abundance of HK1 in the liver was significantly increased in the late stage of overfeeding(day 19 of overfeeding)but not in the early and middle stages of feeding(day 7 to 14 of overfeeding);the mRNA abundance of mRNA in abdominal fat was significantly increased in the middle stage of overfeeding but not in the early or late stages of overfeeding;the mRNA abundance of HK1 in breast muscle was significantly decreased in the middle and late stages of overfeeding but not in the early stage of overfeeding.These results indicate that the role of HK1 gene in the development of goose fatty liver is influenced by tissue type and overfeeding time.2.Effect of fatty liver-related factors on the expression of HK1 in goose primary hepatocytes.In goose primary hepatocytes treated with fatty liver-related factors(high glucose,insulin and fatty acids)respectively,it was found that 50 mM glucose treatment could significantly induce the mRNA expression of HK1 gene;high insulin(50-100 nM)treatment could significantly inhibit the expression of HK1 gene;0.25 mM and 0.5 mM palmitic acid treatment could induce the expression of HK1 gene,and 0.125 mM oleic acid and linoleic acid treatment significantly inhibited the expression of HK1 gene.These results indicate that the enhanced expression of HK1 gene in goose fatty liver may be partly attributable to the increase of glucose and saturated fatty acids in the blood.3.Regulatoin of HK1 expressoin by transcription factors.Based on bioinformatics analysis,Pax-4 and HNF-1 were predicted as transcription factors of HK1.In the goose primary hepatocytes treated with transcriptional agonists(streptozotocin and retinoic acid),the expression of HK1 was significantly inhibited.On the other hand,knocking down of HNF-1 expression also resulted in a significant decrease in HK1 expression.These results indicate that Pax-4 and HNF-1 are probably transcription factors of HK1 gene.The inhibition of HK1 expression by the agonists may involve more complex mechanisms,which needs further investigation.4.Analysis on the biological function of HK1 gene.The HK1 gene was overexpressed or knocked down by siRNA in goose primary hepatocytes,followed by determining fat deposition in the cells using oil red staining method,by examing the amount of reactive oxygen species in the cells with ROS detecting kit,as well as by comparing transcriptomes of the cells transfected with overexpression vectors vs.empty vectors using RNA-seq technology.The data showed that the overexpression of HK1 led to the increase of fat deposition in goose primary hepatocytes and the decrease in the amount of ROS,whereas knocking down of HK1 expression led to the decrease of fat deposition in goose hepatocytes and the increase in the amount of ROS.There were 1609 differentially expressed genes in the goose primary hepatocytes transfected with overexpression vectors vs.empty vectors,including 933 genes upregulated and 676 genes downregulated.Through GO and KEGG pathway analyses on the differentially expressed genes,it was found that the difference genes were mainly enriched in the glycolipid metabolic pathway,followed by the tumor formation pathway and immune response pathway,indicating that HK1 may be involved in the formation of goose fat liver through these pathways.These findings are consistent with previously reported enrichment of the differentially expressed genes the the at metabolism signal pathway,cell growth and death signal pathway,and the immune response signal pathway,which was identified based on transcriptome analysis of goose fatty liver vs normal liver.In summary,HK1 gene may be involved in goose fat liver by affecting fat deposition,oxidative stress,cell growth,and immune response.The expression of HK1 is influenced by the fatty liver-associated factors as well as transcription factors Pax-4 and HNF-1.