Postruminal choline supply during negative nutrient balance alters components of hepatic mTOR signaling and plasma amino acids in lactating Holstein cows

Choline requirements for dairy cattle are unknown. However, enhanced postruminal supply of choline may increase flux through the methionine cycle to spare Met for other functions such as protein synthesis and phosphatidylcholine (PC) synthesis during periods of negative nutrient balance (NNB). The objective was to investigate the effects of postruminal choline supply during a feed restriction-induced NNB on hepatic abundance and phosphorylation of mTOR (mechanistic target of rapamycin)-related signaling proteins, hepatic lipidome and plasma AA. Ten primiparous rumen cannulated Holstein cows (158 ± 24 DIM) were used in a replicated 5 × 5 Latin square design with 4 d of treatment and 10 d of recovery (14 d/period). Treatments were unrestricted intake with abomasal infusion of water, restricted intake (R; 60% of net energy for lactation requirements to induce NNB) with abomasal infusion of water (R0) or restriction plus abomasal infusion of 6.25, 12.5, or 25 g/d choline ion. Liver tissue was collected via biopsy on d 5 after infusions ended and used for Western blot analysis to measure proteins involved in mTOR signaling and untargeted lipidomics. Blood was collected on d 1 to 5 for plasma AA analysis. Statistical contrasts for protein and AA data were A0 versus R0 (CONT1), R0 versus the average of choline dose (CONT2) and tests of linear and quadratic effects of choline dose. Analysis of lipidomic data were performed with the web-based metabolomic processing tool MetaboAnalyst 5.0. Ratios of p -RPS6KB1: tRPS6KB1, p -EEF2: tEEF2, and p -EIF2: tEIF2 were greater with R (CONT1). Among those, supply of choline led to decreases in p -EEF2: tEEF2 (CONT2), p -EIF2: tEIF2 and tended to decrease p -EIF4BP1: tEIF4BP1. However, the effect was quadratic only for p -EEF2: tEEF2 and p-EIF2A: tEIF2A, reaching a nadir at 6.25 to 12.5 g/d choline ion. The ratio of p -RPS6KB1: tRPS6KB1 was not affected by supply of choline and was close to 2-fold greater at 25 g/d choline versus A0. Plasma Met concentration decreased with R (CONT1), but increased linearly with choline. Restriction also increased plasma 3-methyl-histidine (CONT1). The partial least squares discriminant analysis model of liver lipids distinguished treatments, with 13.4% of lipids being modified by treatment. One-way ANOVA identified 109 lipids with a false discovery rate ≤0.05. The largest group identified was PC species; all 35 detected decreased with R versus A0, but there were few differences among choline treatments. Overall, data suggested that dephosphorylation of EEF2 and EIF2A due to enhanced choline supply potentially helped maintain or increase protein synthesis during NNB. Enhanced postruminal choline had limited effects on the species of lipid produced during a period of NNB.

奶牛的胆碱（choline）需求量目前尚未明确。然而，在负营养平衡（negative nutrient balance, NNB）阶段，提升瘤胃后胆碱供给可增强蛋氨酸循环（methionine cycle）通量，从而节约蛋氨酸（Methionine, Met）用于蛋白质合成及磷脂酰胆碱（phosphatidylcholine, PC）合成等其他生理功能。本研究旨在探究饲粮限制诱导的NNB期间，瘤胃后胆碱供给对肝脏雷帕霉素机制靶标（mechanistic target of rapamycin, mTOR）相关信号蛋白的丰度与磷酸化水平、肝脏脂质组及血浆氨基酸（plasma amino acids, AA）的影响。试验采用重复5×5拉丁方设计，选用10头经瘤胃插管的荷斯坦奶牛，泌乳天数（days in milk, DIM）为158±24，每个试验周期包含4天处理期与10天恢复期（共计14天）。试验处理共设5组：自由采食并经皱胃灌注（abomasal infusion）生理盐水（A0组）、以60%泌乳净能水平限饲并灌注生理盐水（R0组，用于诱导NNB），以及限饲联合每日皱胃灌注6.25、12.5或25 g胆碱离子（R6.25、R12.5、R25组）。于灌注结束后第5天通过活体活检（biopsy）采集肝脏组织，用于蛋白质免疫印迹（Western blot）分析以检测mTOR信号通路相关蛋白，并开展非靶向脂质组学（untargeted lipidomics）检测；于灌注第1至5天采集血液样本用于血浆氨基酸分析。针对蛋白与氨基酸数据的统计对比包括：A0与R0组比较（CONT1）、R0组与各胆碱灌注组平均值比较（CONT2），以及胆碱剂量的线性与二次效应检验。脂质组学数据采用基于网页的代谢组学分析工具MetaboAnalyst 5.0进行分析。p-RPS6KB1:tRPS6KB1、p-EEF2:tEEF2及p-EIF2:tEIF2的比值在R组中更高（CONT1）。其中，胆碱供给可降低p-EEF2:tEEF2（CONT2）、p-EIF2:tEIF2的比值，并呈降低p-EIF4BP1:tEIF4BP1的趋势。不过，仅p-EEF2:tEEF2与p-EIF2A:tEIF2A的效应呈二次曲线，在胆碱离子灌注量为6.25~12.5 g/d时达到最低点。p-RPS6KB1:tRPS6KB1的比值不受胆碱供给影响，但在25 g/d胆碱灌注组中较A0组提升近2倍。血浆Met浓度在R组中降低（CONT1），但随胆碱灌注量增加呈线性升高。限饲还可提升血浆3-甲基组氨酸水平（CONT1）。肝脏脂质的偏最小二乘判别分析模型可区分不同处理组，其中13.4%的脂质分子受处理影响。单因素方差分析筛选出109个错误发现率（false discovery rate, FDR）≤0.05的脂质分子，其中占比最大的类别为PC类脂质：相较于A0组，R组中全部35种检测到的PC类脂质水平均降低，但各胆碱灌注组间差异较小。整体而言，试验数据表明，提升胆碱供给所介导的EEF2与EIF2A去磷酸化，可能有助于在NNB阶段维持或提升蛋白质合成效率。瘤胃后胆碱供给的提升对NNB阶段产生的脂质分子种类影响有限。