Effect Of High Concentrate Diet On Hepatic Proteomics And Function In Dairy Goats

High proportion of concentrate diet is commonly fed to lactating diary cows in order to meet the demands of high milk production.However,feeding an excessive amount of concentrate diet to lactating ruminants usually leads to disorders of ruminal fermention,which ultimately results in a negative effect on health and milk production.As a key organ for nutrients metabolism,liver also plays an important role in stress response and immune function in the body.In the present study,we investigated the effects of long-term feeding high concentrate diet on ruminal fermentation,milk quality and production,blood biochemical parameters as well as the proteomics profile in liver of lactating dairy goats.Furthermore,the effects of endogenous LPS and butyrate on hepatocyte glucose and lipid metabolism was investigated in primary hepatocytes cultured in vitro.1 Effect of high concentrate diet on ruminal parameters and milk composition12 mid-lactacting goats were randomly assigned to two groups fed with high concentrate(HC,F:C 35:65)or low concentrate(LC,F:C 65:35)diet for 12 weeks,respectively.Milk yields and conponsition were recorded weekly.Samples including rumen fluid,serum and liver tissues were collected at the end of experiment.Ruminal pH was detected using pH probes meter.The level of volatile fatty acids(VFA)including acetate,propionate,butyrate,valerate,isobutyrate,and isovalerate in ruminal fluids was determined by gas chromatography.Free LPS content in ruminal fluids was measured by a commercial assay kit.Biochemical parameters in serum were detected by automatic biochemical analyzer with commercial kits.Compared to LC goats,HC goats showed a significant decrease in ruminal pH(P<0.01),while the level of VFA including propionate(P<0.01),butyrate(P<0.01),isobutyrate(P<0.05),valerate(P<0.01),isovaletate(P<0.01),total VFA(P<0.05)as well as the ratio of A/P(P<0.01)was significantly increased.HC goats showed higher milk yield(P<0.01),milk lactose content(P<0.01),milk fat content(P<0.01)and milk protein content(P<0.01)than LC goats.Lactating time significantly affected the percentage of milk lactose(P<0.01),milk fat(P<0.05)and milk protein(P = 0.08).HC challenge also increased free LPS concentration in ruminal fluids(P<0.01),serum albumin(P = 0.07)and low-denisity lipoprotein cholesterin(LDLC)(P<0.05)concentrations.2 Effect of high concentrate on hepatic proteomics and lipid metabolismCompared with LC goats,HC goats showed higher ratio of body mass to liver weight(P<0.05).The expression of CRP(P<0.05)and SAA(P<0.05)gene was significantly up-regulated in liver of HC goats compared to LC,while TNF-α mRNA expression showed a tendency to decrease(0.05<P<0.1).2-DE and MALDL-TOF techniques were used to analyze proteomic profile in liver,and 20 proteins were differentially expressed in liver between LC and HC goats,and these proteins were identifed successfully.The functions of these 20 proteins are involved in oxidative stress,mitochondrial function and retinoic acid(RA)metabolism.Total antioxidative stress capacity(P<0.05)were decreased in HC goats compared to LC.In addition,high concentrate also increased hepatic mitochondrial ATP8(0.05<P<0.1)and COX3(P<0.05)gene expression.The expression of genes involved in lipid metabolism was also evaluated and compared between LC and HC goats.The resulsts demonstrated that the level of cholesterol content in liver was decreased in HC compared to LC goats(P<0.05).mRNA expression of SREBP2(P<0.05),SREBP1c(P<0.05),ACAA2(P<0.05),ABCA1(P<0.01),ABCG1(P<0.01),ACSS1(0.05<P<0.1)、FASN(P<0.05)、PPARα(P<0.05)、SREBP1c(P<0.05)、DGAT1(P<0.05),DGAT2(P<0.05),LXRα(P<0.01)and LXRβ(P<0.05)in liver was significantly increased in liver of HC compared to LC goats.3 Effect of sodium butyrate and LPS on glucose metabolism in primary cultured hepatocytesForty-five day-old lambs with 2.42 kg average body weight were sacrificed and the liver tissues were collected immediately after slaughtered.The hepatocytes were isolated and purified,and then cultured for 4 h attachment period,and transfered to growth medium for 72h,and cultured for another 24h in serum-free medium before LPS or butyrate treatment.The hepatocytes were treated with 5 nmol/L sodium butyrate,100 ng/mL LPS or 5 nmol/L sodium butyrate combined with 100 ng/mL LPS for 24 h,respectively.Hepatocytes and the medium supernatant were collected for analysis.Cell viability was measured by CCK method.The level of glucose in the supernatant was detected with the commercial kit.Real-time PCR and western blot methods were used to measure mRNA and protein expression.The results showed that sodium butyrate significantly reduced glucose concent in the supernatant(P<0.01),and increased AMPK protein content(0.05<P<0.1).LPS not only increased total AMPK protein content(P<0.05)but also increased p-AMPK protein level(P<0.05).G6Pase mRNA expression was markedly decreased by sodium butyrate(P<0.01)or LPS treatment(P<0.01).Sodium butyrate down-regulated PCK2(P<0.05),PKLR(P<0.05),and COX1(P<0.05)genes expression.However,LPS treatment up-regulated ND4(0.05<P<0.1).Taken together,these results suggest that lactating goats fed high concentrate for a long term increased milk yield while rumen occurred acidosis and decrease the total antioxidative capacity which may lead to increase mitochondrial oxidative phosphorylation and faty acids oxidation;butyrate sodium decrease the gluconeogenesis via AMPK,but LPS activate mitochondrial oxidative phosphorylation via AMPK.