Study On The Expression Pattern,Transcriptional Regulation And Function Of MC5R Gene In The Development Of Goose Fatty Liver

Foie gras is a type of fatty liver induced by overfeeding in a short time.Interestingly,pathological changes such as inflammation do not occur in goose liver even in the case of severe steatosis,suggesting that goose has some protective mechanism to make the liver immune to the damage caused by severe steatosis.This tolerance may be related to fatty liver performance in geese.MC5R,a member of the G protein-coupled receptor family,has a variety of biological functions,including regulation on fat metabolism and inflammatory response.Therefore,MC5R is likely to be involved in formation of foie gras.However,there are few reports on the involvement of MC5R in fatty liver,especially goose fatty liver.This study aims to reveal the expression,regulation and mechanism of MC5R in the developmentd of goose fatty liver,lay a foundation for further elucidating the mechanism of goose fatty liver,and promote the production of fatty liver.Firstly,the expression level of MC5R in three tissues(liver,muscle and fat)closely related to fat metabolism during the development of goose fatty liver was detected by real-time quantitative PCR(RT-qPCR)and Western blot.Secondly,the regulation of MC5R expression in primary hepatocytes of goose was investigated by treating them with fatty liver associated factors(fatty acids,glucose,insulin)and the ligands of potential MC5R transcription factors(estrogen,thyroid hormone,troglitazone).Finally,the downstream genes and signaling pathways of MC5R were identified by MC5R overexpression in goose primary hepatocytes and transcriptome sequencing.The research contents and the results of this study are described in detail as follows:1.Study on the expression pattern of MC5R in the development of goose fatty liver:overfeeding significantly inhibited the mRNA expression of MC5R gene in the liver and abdominal fat,but did not significantly affect the mRNA expression of MC5R gene in pectoral muscle.The protein expression level of MC5R in the liver of the overfed geese on the 12th d of overfeeding was not significantly different from that of the control group(I.e.,the conventionally feeding group),but on the 24th d of overfeeding,the protein expression level of MC5R in the liver of the overfed geese was significantly decreased.The above results suggest that the role of MC5R in the formation of goose fatty liver is varied with tissue type and overfeeding time.2.Regulation of fatty liver associated factors on the expression of MC5R in goose hepatocytes:after 14 h of treatment of goose hepatocytes with fatty liver associated factors(glucose,insulin,fatty acid)with goose primary hepatocytes,MC5R expression level was determined.The results showed that high concentrations of glucose(125 mmol/L,250 mmol/L)significantly induced the mRNA expression of MC5R gene in goose primary hepatocytes with dose effect;insulin did not significantly affect the expression of MC5R gene.The mRNA expression of MC5R gene in goose primary hepatocytes was significantly induced by 0.25 mmol/L oleic acid3.Regulation of MC5R expression by ligands or activators of transcription factors:the upstream sequence of MC5R gene(2k bp)was used to predict the transcription factors that may be involved in fat/energy metabolisms,including estrogen receptor(ER),triiodothyronine receptor(T3R),and peroxidase proliferator-activated receptor(PPAR)They are potential transcription factors of MC5R gene.The mRNA expression level of MC5R in goose primary hepatocytes was detected after 14 h treatment with the ligands or activators(estradiol,thyroid hormone,and troglitazone)of the transcription factors The results showed that estradiol significantly inhibited the expression of MC5R at 2?mol/L concentration,thyroid hormone at all concentrations tested significantly inhibited the expression of MC5R,whereas troglitazone significantly induced the expression of MC5R at 1 ?mol/L concentration with dose effect4.Study on the potential mechanism of MC5R functioning in goose fatty liver:after 48 h of MC5R overexpression in goose primary hepatocytes,transcriptome sequencing analysis was carried out.The results showed that 1381 differentially expressed genes between overexpression group and control group were identified,including 838 unregulated and 543 downregulated.GO and KEGG pathway analyses indicated that the differentially expressed genes were enriched in the pathways that are associated with substance/energy metabolisms as well as immune response5.MC5R may affect the formation of goose fatty liver by regulating the expression of ATG5 and PDHB genes:the reliability of transcriptome data was verified by quantitative PCR of nine differentially expressed genes(CCNB2,CALML4,CDK1,BNIP3.ATG5,ELOVL7,ACAT2,PDHB and PEX13).The expression of ATG5 and PDHB genes in the livers of Landes geese overfed for 24 days was inhibited,suggesting that these two genes may mediate the regulation of MC5R on the formation of fatty liver.In summary,MC5R gene is involved in the formation of goose fatty liver,and its expression is regulated by glucose and fatty acids as well as by the potential transcription factors(ER,T3R,and PPAR).MC5R may be involved in the formation of goose fatty liver by affecting the metabolisms of substance/energy and immune response.These findings establish a foundation for further study on the role of MC5R in the formation of goose fatty liver and the related mechanism.