Regulation Of Expression Of Key Related Protein Gene For Assembly And Secretion Of Very Low Density Lipoprotein In Liver Of Periparturient Dairy Cows

Ketosis and fatty liver based on energy metabolism disorder is a nutritionmetabolic disease related to hepatic lipid metabolism and develop in periparturientdairy cows generally. Assembly and secretion of very low density lipoprotein is one ofthe main component element that develop metabolic disorder in the peripartum.ApoB100,ApoE,MTP and LDLR are the key constitutive protein and regulatoryprotein for assembly and secretion of VLDL. Therefore, healthy multiparousperiparturient dairy cows, primary hepatocyte culture in vitro, and quantitive RT-PCRin this study were used to probe the regulation effects and mechanism of energy state,neuroendocrine and metabolites on key related protein gene expression for assemblyand secretion of very low density lipoprotein in liver of periparturient dairy cows.In the animal raising trial, thirty Chinese peripartum Holstein cows, with milkyield exceeding 5000Kg, 5-8yrs and 3-5 parities,were randomly assigned into threedifferent energyintake treatments, each of 10, fed with 100% energy diet (100%energyintake of nutrition needs of China Dairy Cow Raise Standard 2000, controlgroup), 120% energy diet and 80% energy diet at 28 days before parturition,respectively. After parturition, all the cows were offered criteria lactating dayprovisions until 56 days postpartum. All cows were fed curved hay and corn silagefirstly, then concentrates daily at 6:00,12:00 and 19:00. The cows were fedindividually and had free access to tap water. During the experimental period, plasmaglucose and NEFA concentrations, serum VLDL, LDL and HDL contents, liver TGand glycogen contents, and expression level of ApoB100 mRNA, ApoE mRNA, MTPmRNA and LDLR mRNA in liver were determined from 28d and 14d antepartum to1d, 14d, 28d and 56d postpartum. The results were as follows: The concentrations ofplasma glucose in all treatments decreased during the peripartum, especially in thecontrol group and high energy group , indicating that peripartum dairy cows fed withhigh energy diets during the dry period was more prone to develop hypoglycemia thanthose fed with low energy diets. The concentrations of plasma NEFA in all treatmentsincreased during day 1 to day 28 postpartum, and in low energy group wassignificantly lower than those of control group and high energy group, indicating lowenergy diet during the dry period may reduce the extent of postpartum fat mobilization.After parturition, the concentrations of serum VLDL in all cows decreasedsignificantly and the contents of liver TG in all cows increased significantly, and themean concentration of serum VLDL in cows fed with high energy diet was lower andthe mean content of liver TG in cows fed with high energy diet was higher than that ofcows fed with low energy diet, indicating that the export of liver TG in all cowsreduced after calving, and high energy diet during the dry period may interfere theassembly and secretion of VLDL and then promote the deposit of liver lipid afterparturition. The contents of liver glycogen in all cows decreased significantly aftercalving, and the mean contents of liver glycogen in control and high energy treatmentswere lower than that of low energy group, indicating that the reserve of liver glycogenwas mobilized in all cows, and low energy diet during the dry period may relieveenergy deficit after parturition in a certain degree. The abundance of ApoB100mRNAin liver decreased in all cows after calving, and was upregulated significantly in cowsfed with low energy diet during the dry period. The abundance of liverApoB100mRNA close to calving was upregulated in cows fed with high energy dietduring the dry period, but was downregulated significantly after parturition. LiverApoEmRNA abundance after parturition in cows was affected by differentenergyintake during the dry period. The relative expression of ApoEmRNA in cowsfed with low energy diet was higher than that of control group and high energy groupfrom 14d before calving to 28d after parturition. The results indicated that theabundance of ApoB100mRNA and ApoEmRNA in the peripartum was upregulated incows fed with low energy diet during the dry period, and may promote the assemblyand secretion of VLDL in liver of periparturient dairy cows. The liver MTPmRNAabundance in all cows was significantly upregulated in the peripartum, and there is nosignificant interblock difference. This indicated that different energyintake during thedry period had no effect on the gene expression of MTP in liver of periparturient dairycows, but the assembly and secretion of VLDL was regulated through promoting theexpression of MTPmRNA by increased liver TG content after calving in cows. Thelevel of liver LDLRmRNA in the peripartum was upregulated significantly in cowsfed with low energy diet during the dry period. The mean abundance of liverLDLRmRNA after calving was upregulate significantly in cows fed with standard andhigh energy diets during the dry period, and was significant lower than that of lowenergy diet. 14d after parturition, the liver LDLRmRNA abundance in all cows wasdecreased significantly by increased liver TG content after calving. The expressionlevel of liver LDLRmRNA may be one of the major regulation factors of the assemblyand secretion of VLDL in liver of dairy cows during the early lactation, but the causeof upregulated LDLRmRNA abundance in the peripartum in cows fed with low energydiet during the dry period needs to be further investigated.In the primary hepatic cell culture in vitro trial, new born calves were killed bycarotid artery bloodletting. Under the condition of asepsis, liver posterior lobe wascollected, then sheared and digested for separating hepatocytes. Cells were seeded(1×106) onto custodite cell culture dishes with polylysine. After attachment,fluorescent quantitative and semiquantitative RT-PCR were employed to determinethe expressing level of ApoB100 mRNA,ApoE mRNA,MTP mRNA and LDLRmRNA in primary hepatocytes which were treated with different concentrations ofinsulin (0 nmol/L,1 nmol/L,10 nmol/L,100 nmol/L,1000 nmol/L), glucagons (0nmol/L,1 nmol/L,10 nmol/L,100 nmol/L,1000 nmol/L) and leptin (0 ng/mL,2.5ng/mL,5 ng/mL,10 ng/mL,50 ng/mL). The results showed that the expression levelof ApoB100 mRNA, ApoE mRNA and MTP mRNA in hepatic cells was graduallydecreased with increasing concentration of insulin in cell culture media, and theexpression level of LDLR mRNA in hepatocytes was gradually increased. Thisindicated that the depressed effect of insulin on the assembly and secretion of VLDLmay be related to decreased expression of ApoB100 mRNA,ApoE mRNA and MTPmRNA and the increased expression of LDLR mRNA by insulin. The expression levelof ApoB100 mRNA and ApoE mRNA in hepatic cells was increased with lowerconcentration of glucagons and then decreased with increasing concentration ofglucagon in the media indicating that low dose of glucagon increased the assemblyand secretion of VLDL by promoting ApoB100 mRNA and ApoE mRNA expression,and high dose of glucagon may increase concentration of glucose, and increasedsecretion of insulin which inhibited ApoB100 mRNA and ApoE mRNA expression.Depressed expression of MTP mRNA and LDLR mRNA in hepatocytes wasenhanced with increasing concentration of glucagons in the media. With increasing theconcentration of leptin in culture media, the expression level of ApoB100 mRNA inhepatic cells was decreased significantly. And leptin showed the dual effects, firstlythe expression level of ApoE mRNA and MTP mRNA in hepatocytes was increasedunder low dose and then decreased with increasing concentration of leptin in theculture media, while the expression level of LDLR mRNA in hepatic cells was notobviously affected.In order to determine the effects of different concentration of NEFA (0 mmol/L,0.5 mmol/L,1.0 mmol/L,1.5 mmol/L,2.0 mmol/L), BHBA (0 mmol/L,0.5 mmol/L,1.0 mmol/L,1.5 mmol/L,2.0 mmol/L) and glucose (0 mmol/L,1.5 mmol/L,2.8mmol/L,5 mmol/L) in the cell culture media on the expression level of ApoB100mRNA,ApoE mRNA,MTP mRNA and LDLR mRNA in primary culturedhepatocytes of new born calves, fluorescent quantitation and semiquantitative RT-PCRwere applied. The results showed that the expression level of ApoB100mRNA andApoEmRNA in primary cultured hepatocytes was increased under low dose and thendecreased with increasing concentration of NEFA in the culture media, indicating thatlow dose of NEFA may increase the assembly and secretion of VLDL by promotingthe expression of ApoB100mRNA and ApoEmRNA, and high dose of NEFA mayreduce the assembly and secretion of VLDL by inhibiting the expression ofApoB100mRNA and ApoEmRNA, resulting TG accumulation in hepatocytes, whichmay influence hepatic function. Furthermore, the expression level of MTP mRNA inhepatic cells was gradually increased and on the contrary, the expression level ofLDLR mRNA in hepatocytes was gradually decreased with increasing concentrationof NEFA in the culture media. With increasing concentration of BHBA in culturemedia, the expression level of ApoB100mRNA and LDLRmRNA in hepatic cells wasgradually decreased. The expression level of MTPmRNA and ApoEmRNA inhepatocytes were gradually increased under lower dose and then decreased withincreasing concentration of BHBA in the media. In addition, the expression levels ofApoB100mRNA and ApoEmRNA in hepatic cells were gradually increased firstly andthen decreased as increasing concentration of glucose in the media, and peaked atphysiological concentration. This indicated that dairy cows with glycopenia orhyperglycosemia may inhibit the assembly and secretion of liver VLDL by reducingthe expression of ApoB100mRNA and ApoEmRNA by a certain mechanism. And theexpression levels of MTPmRNA and LDLRmRNA in hepatocytes were upregulatedwith increasing concentration of glucose.As above-mentioned, cows fed with high energy diet during the dry period canintensify negative energy balance postpartum, and the reason of hepatic lipiddeposition is related to downregulated expression of key constitutive protein and/orregulatory protein for assembly and secretion of VLDL. But cows fed with low energydiet during the dry period can upregulate the expression of key constitutive proteinand/or regulatory protein for assembly and secretion of VLDL, influence the assemblyand secretion of VLDL, decrease fatty liver development in periparturient dairy cows.The experiments in vitro verified that insulin, leptin, NEFA, BHBA are the inhibitionfactor of gene expression of key constitutive protein and/or regulatory protein forassembly and secretion of VLDL, but glucagons and glucose can promote itsexpression.