Mechanism Of Glucocorticoid-Induced Fatty Liver In Chickens And The Relieving Effects Of Betaine

Fatty liver syndrome(FLS)is a common metabolic disease in laying hens and broiler chickens.Stress is an important factor that causes fatty liver in the chicken.Chronic stress will lead to increased plasma levels of glucocorticoid in the chicken,however,whether glucocorticoid receptor(GR)is involved in the mechanism of stress-induced chicken fatty liver remains unclear.Recently,it has been found that the increase of FTO gene expression and the decrease of RNA methylation m6A are usually associated with the accumulation of fat in the liver.However,the mechanism underlying the up-regulated FTO and the role of mRNA m6A motification in stress-induced fatty liver remain unknown.In addition,in human clinical practice,methyl donors,such as choline,betaine,methionine and vitamin B12,have been used effectively in the prevention and treatment of fatty liver caused by hyperlipidemia or stress.It has been shown that these nutrients can affect DNA methylation by regulating one carbon metabolism and thus regulate the trancription of lipid metabolic genes.Nevertheless,it remains unknown whether feeding betaine to laying hens may alleviate stress-induced fatty liver in offspring chickens.Therefore,in the present study,we established in vitro and vivo model of hepatic lipid accumulation,to investigate the role of GR and FTO-mediated m6A modification on CORT(stress)-induced chicken fatty liver and its mechanism.In addition,we want to investigate whether maternal dietary betaine supplementation can prevent CORT-induced fatty liver disease in juvenile chickens,and how such effects.1 Establishment of CORT-induced chicken fatty liverRugao yellow-feathered chickens(57.8±5.49 g)obtained from the Poultry Institute,Chinese academy of agricultural sciences,were randomly divided into two groups(15 hens per group).Hens were subjected to either vehicle(CON)or corticosterone(CORT)treatment with daily subcutaneous injection of solvent(15%ethanol)or corticosterone in a dose of 4.0 mg/kg body mass for 7 days(twice per day,8:00?9:30 and 16:00?17:30).Growth performance and daily feed intake were recorded.CORT injection significantly increased(P<0.05)the average daily feed intake but decreased(P<0.05)the final body weight.CORT-treated chickens had higher liver weight and liver index,together with significantly elevated plasma and hepatic TGs concentrations.The same phenomenon was seen in histological sections stained with Oil Red O showing higher hepatic lipid accumulation in CORT group(steatosis>50%of hepatocytes).Moreover,hepatic mRNA abundance of sterol regulatory element-binding protein-1(Srebf1),fatty acid synthase(Fasn),acetyl-CoA carboxylase(Acaca)and stearoyl-CoA desaturase(Scd)was significantly increased(P<0.05)in response to CORT challenge.Then we used western blot to detect lipogenic gene protein content in liver,and the results showed that the protein content of SREBF1 and SCD was significantly increased(P<0.05)after CORT treatment.Hepatic protein content of GR was also significantly increased(P<0.05)after CORT challenge.The above results showed that CORT treatment significantly increased hepatic TGs content and induced severe hepatic steatosis,indicating that the CORT-induced chicken fatty liver model was successfully established.2 The Changes of FTO and m6A in CORT-induced chicken fatty liverBoth FTO mRNA and FTO protein in the nuclear lysate were significantly increased(P<0.05)in the liver after chronic CORT administration.No significant alterations were detected for the methyltransferase like 3(METTL3)or wilms' tumour 1-associating protein(WTAP)in chicken liver nuclear lysate,yet methyltransferase like 14(METTL14)was significantly increased(P<0.05),in response to CORT challenge.Total RNA m6A level in chicken liver was significantly decreased(P<0.05)in response to CORT challenge.The pattern of m6A distribution was determined with Methylated RNA Immunoprecipitation sequencing(MeRIP-seq)that detected m6A peaks are mainly enriched around the CDS and 3' UTR of the transcripts.The classic consensus sequence,RRm6ACU(R=A or G),was found in most of the detected narrow peaks.No significant alterations were detected for m6A distribution pattern induced by CORT treatment.The lipogenic genes SREBF1,ACC,FASC and SCD mRNA m6A levels were significantly decreased(P<0.05)after CORT treatment.More importantly,to further validate the effect of m6A modification on lipogenic genes expression,targeted mutation of m6A methylation sites and FTO knockdown further validated the role of FTO and m6A on the 3' UTR of SREBF1 and SCD mRNA in the regulation of luciferase activities of these genes.The above results suggest that FTO-mediated m6A modification may play a regulatory role in CORT-induced chicken fatty liver.3 Role of GR and m6A on CORT-induced chicken fatty liver and its mechanismsTo further elucidate the molecular mechanisms of GR,FTO and m6A in CORT-induced chicken nonalcoholic fatty liver disease.We isolated the primary hepatocytes from eighteen-day-old chicken embryos liver.Dexamethasone combined with oleic acid(OA/DEX)was used to establish in vitro model of hepatic lipid accumulation.The results showed that OA/DEX treatment increased(P<0.05)TG accumulation and lipogenic genes expression in primary chicken hepatocytes.OA/DEX significantly increased(P<0.05)the expression of GR and FTO at both mRNA and protein level.RU486,an antagonist of GR,was able to prevent(P<0.05)OA/DEX-induced GR nuclear translocation,thereby decreasing GR protein in the nuclear lysates.Concurrently,OA/DEX-induced increase(P<0.05)of FTO,SREBF1 and SCD protein content in whole cell lysates of primary chicken hepatocytes was also restored by RU486.GR over-expression in DF-1 cells significantly activated(P<0.05)FTO expression at both mRNA and protein levels.A ChIP assay detected a significant increase(P<0.05)of GR binding to FTO gene promoter sequence that contains predicted glucocorticoid receptor element.Meclofenamic acid(MA),a FTO inhibitor,was able to prevent(P<0.05)OA/DEX-induced increase of total TG content in primary hepatocytes.Concurrently,OA/DEX-induced upregulation(P<0.05)of FTO,SREBF1 and SCD protein content was significantly prevented by FTO inhibition.However,FTO inhibition did not prevent the OA/DEX-induced upregulation of GR protein content in the nuclear lysate of primary hepatocytes.MeRIP-PCR results showed that MA treatment was able to alleviate(P<0.05)OA/DEX-induced m6A hypomethylation on Srebf1,Fasn,Acaca and Scd mRNAs in primary hepatocytes.The above results suggest that GR and FTO-mediated m6A modification play an important role in CORT-induced chicken fatty liver.4 Effect of in ovo betaine injection alleviates CORT-induced fatty liver in chickens and its mechanismsChronic stress or glucocorticoid exposure induces fatty liver in animal models.Betaine alleviates high-fat diet-induced fatty liver in rat and prenatal betaine programs offspring hepatic lipid metabolism in the chicken,yet it remains unknown whether and how feeding betaine to laying hens modulates the susceptibility of CORT-induced fatty liver later in life.In this study,fertilized eggs were injected with saline or betaine before incubation,and the hatchlings were raised at 8 weeks of age followed by 7 days of subcutaneous CORT injection.CORT-induced fatty liver was less severe in betaine-treated chickens,with significantly reduced oil-red staining and hepatic triglyceride content(P<0.05).The protective effect of prenatal betaine was associated with significantly up-regulated expression of PPARa and CPT1?,as well as mitochondrial DNA(mtDNA)-encoded genes(P<0.05).Moreover,betaine rescued CORT-induced alterations in methionine cycle genes,which coincided with modifications of CpG methylation on CPT1? gene promoter and mtDNA D-loop regions.In ovo injection of betaine was able to alleviate CORT-induced hypermethylation(P<0.05)on CPT1a promoter or mtDNA D-loop region.Furthermore,the elevation of hepatic GR protein content after CORT treatment was significantly reduced(P<0.05),while the reduction of GR binding to the control region of affected genes was significantly increased(P<0.05),in betaine-treated chickens.These results indicate that in ovo betaine injection protects the juvenile chickens from CORT-induced fatty liver.5 Effect of maternal betaine supplementation alleviates CORT-induced fatty liver in chickens and its mechanismsIn ovo betaine injection can prevent non-alcoholic fatty liver induced by CORT exposure in chickens.However,we still don't know whether dietary betaine supplementation may increase betaine deposition in the breeder eggs,and whether higher betaine content in the egg can prevent stress-or CORT-induced fatty liver disease in hatched chicks later in life.Therefore,here we use CORT treatment,a widely used stress model in birds,to investigate whether maternal dietary betaine supplementation can prevent CORT-induced fatty liver disease in juvenile chickens,and how such effects.In this study,One hundred and twenty Rugao yellow-feathered laying hens were randomly divided into control and betaine groups at 38 weeks of age.Hens were fed basal or 0.5%betaine-supplemented diet for 28 days before the eggs were collected for incubation.The hatchlings were raised at 7 weeks of age,male chickens were selected from each group and divided into two subgroups,subjected to either vehicle(VEH)or corticosterone(CORT)treatment with daily subcutaneous injection of solvent(15%ethanol)or CORT in a dose of 4.0 mg/kg body mass for 7 days.Chickens in the 4 groups(C-VEH,C-CORT,B-VEH and B-CORT)were killed at day 57.Betaine prevented CORT-induced rise in plasma TG level(P<0.05)and hepatic lipid accumulation(P<0.05).Lipogenic genes were significantly activated(P<0.05)at both mRNA and protein levels,whereas lipophagy and mitochondrial ?-oxidation genes were significantly(P<0.05)inhibited in the liver of CORT-treated chickens.These CORT-induced changes were significanly(P<0.05)allieviated by maternal betaine supplementation.Concurrently,betaine rectified CORT-induced alterations in methionine cycle genes,which was associated with modifications of CpG methylation and GR binding on promoters of major lipogenic and lipophagic genes.These results indicate that maternal betaine supplementation protects the juvenile chickens from CORT-induced fat accumulation in the liver via epigenetic modulation of lipogenic and lipophagy genes.