| In most of dairy farms,high-yielding dairy cows are often fed high-concentrate diets to maintain the energy supply needed for their milk production.However,long term high concentrate diet feeding can induce subacute gastric acidosis(SARA)in dairy cows.Similarly,mastitis induced by exogenous E.coli infection can also cause systemic inflammatory responses in dairy cows in dairy cows.LPS is the major pro-inflammatory factor that causes these responses and primarily induces acute phase response in the liver.However,the downregulation of SCD1,a key enzyme of lipid metabolism,is also an important signal of lipid metabolism disorder.In this study,the SARA Holstein cows fed with high concentrate diet and the mastitis model experimentally induced with intramammary E.coli were applied for the research.Our results reveal that both models elicit inflammatory responses and the downregulation of SCD1 expression,latter is resulted from epigenetic regulation.Moreover,sodium butyrate is suggested that can attenuate the inflammatory response and modulate the fatty acid metabolic enzymes expression in LPS-stimulated bovine hepatocytes.1.Grain challenge affects systemic and hepatic molecular biomarkers of inflammation and metabolic responses to a greater extent in Holstein than Jersey cowsLong-term feeding of high-grain diets to dairy cows often results in systemic inflammation characterized by alterations in acute-phase proteins and other biomarkers,both in plasma and immune-responsive tissues like the liver.The molecular and systemic changes that characterize an acute grain feeding challenge remain unclear.The current study involved 6 Holstein and 6 Jersey cows in a replicated 2×2 Latin square.Periods(10 d)were divided into 4 stages(S):S1,d 1 to 3,served as baseline with total mixed ration(TMR)ad libitum;S2,d 4,served as restricted feeding,with cows offered 50%of the average daily intake observed in S1;S3,d 5,a grain challenge was performed,in which cows were fed a TMR ad libitum without(CON)or with an additional pellet wheat-barley(1:1;HIG)at 20%of dry matter intake top-dressed onto the TMR;S4,d 6 to 10,served as recovery during which cows were allowed ad libitum access to the TMR.Among the 28 biomarkers analyzed in blood 12 h after grain challenge on d 5,the concentrations of fatty acids and bilirubin increased in HIG Holstein but not Jersey cows.In Holsteins,feeding HIG also increased total protein and albumin while decreasing ceruloplasmin,myeloperoxidase,and alkaline phosphatase concentrations.At the molecular level,hepatic genes associated with inflammation(IL1B,IL6,TNF,TLR4,MYD88,and NFKB1)were upregulated in Holstein cows fed HIG versus CON.Despite such response,expression of the acute-phase proteins SAA and HP in Holsteins fed HIG compared with CON was markedly downregulated.In Holsteins fed HIG versus CON,the marked downregulation of SCD,ELOVL6,and MTTP along with upregulated CPT1A,ACOX1,and APOA5 indicated alterations in fatty acid and lipoprotein metabolism during grain challenge.Genes related to ketogenesis(HMGCS2 and ACAT1)were upregulated in Jerseys,and gluconeogenic genes(PDK4 and PCK1)were upregulated in Holstein cows fed HIG,suggesting alterations in ketone body and glucose production.Expression of phosphorylated p70S6K1,RPS6,and 4EBP1 proteins,as well as total mechanistic target of rapamycin(mTOR)protein,decreased in Holsteins fed HIG,whereas phosphorylated mTOR and 4EBP1 proteins increased in Jerseys fed HIG.From a metabolic and inflammatory biomarker standpoint,data indicate that Jersey cows better tolerated the acute grain challenge.Alterations in mTOR signaling proteins in both Jerseys and Holsteins fed HIG suggest a potential role for exogenous AA in the hepatic adaptations to grain challenge.It remains to be determined if these acute responses to a grain challenge can elicit long-term liver dysfunction,which could negatively affect welfare of the cow.2.Lipopolysaccharide derived from the rumen down-regulates SCD 1 expression and alters fatty acid composition in the liver of dairy cows fed a high-concentrate dietStearoyl-CoA desaturasel(SCD1)participates in fatty acid biosynthesis in the liver of lactating ruminants.Here,we conducted this study to investigate the impact of lipopolysaccharide derived from the rumen on SCD1 expression and on fatty acid composition in the liver of dairy cows fed a high-concentrate diet.Eight multiparous mid-lactating Holstein cows(455±28 kg)were randomly assigned into two groups in the experiment and were fed a low-concentrate diet(LC)or high-concentrate diet(HC)for 18 weeks.The results showed that the total volatile fatty acids and lactic acid accumulated in the rumen,leading to a decreased rumen pH and elevated lipopolysaccharides(LPSs)in the HC group.The long chain fatty acid profile in the rumen and hepatic vein was remarkably altered in the animals fed the HC diet.The triglyceride(TG),non-esterified fatty acid(NEFA)and total cholesterol(TCH)content in the plasma was significantly decreased,whereas plasma glucose and insulin levels were increased.The expression of SCD1 in the liver was significantly down-regulated in the HC group.In regards to transcriptional regulators,the expression of sterol regulatory element binding transcription factors(SREBFlc,SREBF2)and SREBP cleavage activating protein(SCAP)was down-regulated,while peroxisome proliferator-activated receptor a(PPARa)was up-regulated.These data indicate that lipopolysaccharide derived from the rumen down-regulates stearoyl-CoA desaturase 1 expression and alters fatty acid composition in the liver of dairy cows fed a high-concentrate diet.3.SCD1 expression is downregulated in liver and udder during E.coli mastitis through enhanced expression of repressive C/EBP factors and reduced expression of the inducer SREBP1ASCD1 expression is known to be stimulated under adipogenic conditions through a variety of transcription factors,notably SREBP 1 and C/EBPa and-(3.However,mechanisms downregulating SCD1 expression during illness related reprograming of the metabolism were unknown.Escherichia coli elicited mastitis is an example of such a condition and was found to downregulates milk and milk fat synthesis.This is in part mediated through epigenetic mechanisms.We analyzed here mechanism controlling SCD1 expression in livers and udders from cows suffering from experimentally induced E.coli mastitis.We validated with RT-qPCR that SCD1 expression was reduced in these organs of the experimental cows.They also featured decreased levels of mRNAs encoding SREBP1a but increased levels for C/EBPa and-3.Chromatin accessibility PCR(CHART)revealed that downregulation of SCD1 expression in liver was not caused by tighter chromatin compaction of the SCD1 promoter.Reporter gene analyses showed in liver(HepG2)and mammary epithelial(MAC-T)model cells that overexpression of SREBP la expectedly activated the promoter,while unexpectedly C/EBPα and-β strongly quenched the promoter activity.Abrogation of two from among of the three C/EBP DNA-binding motifs of the promoter revealed that C/EBPa acts in cis but C/EBPβ in trans.Overexpressing truncated C/EBPα or-β factors lacking their repressive domains confirmed in both model cells the direct action of C/EBPa,but not of C/EBPβ on the promoter.We found no evidence that epigenetic mechanism remodeling the chromatin compaction of the SCD1 promoter would contribute to downregulate SCD1 expression during infection.Instead,our data show for the first time that C/EBP factors may repress SCD1 expression in liver and udder rather than stimulating as it was previously shown in adipocytes.This cell type specific dual and opposite function of C/EBP factors for regulating SCD1 expression was previously unknown.Infection related activation of their expression combined with downregulated expression of SREBP1α explains reduced SCD1 expression in liver and udder during acute mastitis.4.Epigenetic mechanisms contribute to decrease SCD1 expression in the liver of dairy cows after prolonged feeding of high-concentrate dietIt is known that SCD1 expression is downregulated during SARA,but the underlying molecular mechanisms are unknown.To study these mechanisms,we enrolled 12 healthy multiparous mid-lactation Holstein cows into a diet-induced SARA experiment.Six cows were fed a high-concentrate diet for 18 weeks(60%content of high-concentrate to 40%forage;HC group),whereas the others received a low-concentrate diet ad libitum(40%high-concentrate content to 60%forage;LC group).Sustained low ruminal pH values(pH 5.6 maintained for 4 h/d)and reduced milk yield performance(2.07 kg/d less than LC cows)verified that SARA had been induced in the HC group.Results showed a significantly decreased concentrations of cis-9 monounsaturated long-chain fatty acids in plasma collected from hepatic but not portal veins.This was matched by reduced SCD1 mRNA and protein concentrations in HC livers.The expression levels of genes related to lipid formation(DGAT1 and PLIN2)were downregulated during SARA,whereas those of catabolic genes(CPT1A,CPT2,and ACOX1)and some inflammatory genes were upregulated.Expression of SCD1 was downregulated through reduced transcription and abundance of the transcription factor sterol regulatory element-binding protein 1(SREBPlc).This effect was augmented by local chromatin tightening and DNA methylation at and around the SREBP1c binding site in the SCD1 promoter.Chromatin immunoprecipitation assays confirmed that SARA reduced SREBPlc binding at the SCD1 promoter;hence,epigenetic mechanisms are involved in regulating the expression of genes related to long-chain fatty acid modification,partially through downregulation of both SCD1 and SREBP1c in the liver.Our results suggest that in addition to inflammatory genes,SCD1 is also involved in SARA-induced epigenetic regulation and its associated metabolic changes.This knowledge might help to provide a target for intervening against the detrimental metabolic effects of SARA.5.Sodium butyrate supplementation alleviates adaptive response to inflammation and modulates fatty acid metabolism in LPS-stimulated bovine hepatocytesSodium butyrate(SB)has been suggested that can ameliorate LPS-induced inflammation in dairy goats and maintain homeostasis,this study aimed to evaluate whether sodium butyrate exerts directly to attenuating the hepatic response to lipopolysaccharide-induced inflammation in primary bovine hepatocytes.Hepatocytes isolated from cows in～160 DIM postpartum were exposed to 0 or 0.5 mmol/L Sodium Butyrate(SB)for 18 h as pretreatment.Cells cultured without pretreating SB were then challenged with either 0(CON)or 4 μg/mL(LPS)of LPS for another 6 h.Cells pretreated with Sodium Butyrate were applied as SB group or 4 μg/mL LPS challenge as LSB group.Hepatocytes receiving LPS showed an inflammatory response with elevated TNF-α and IL-6 production in culture medium(P<0.05),which was attenuated by pretreatment with SB in LSB group(P<0.05).phospho-p65 and phospho-IκB protein expression and translocation into nuclear was suppressed by addition of SB as compared to LPS treatment cells.Genes(SREBF1,SCD1,and DGAT1)and proteins(SREBP1 and SCD1)related to fatty acid synthesis and desaturation were downregulated after LPS treatment(P<0.05).However,the expression of these genes and proteins were higher(P<0.05)in LSB cells than the LPS treated cells.The ratio of phospho-AMPKa to AMPKa and phospho-ACCa to ACCa were upregulated(P<0.05)in LSB group.SB pretreatment also reversed histone H3 deacetylation that was increased by LPS stimulation in bovine hepatocytes.Our results suggest that SB pretreatment suppresses bovine hepatocytes adaption of LPS-induced inflammatory response,accompanied with enhanced fatty acid synthesis,downregulated fatty acid oxidation and histone H3 deacetylation for neutralizing the negative effects from infection. |
