Table_1_Differential effect of two dietary protein sources on time course response of muscle anabolic signaling pathways in normal and insulin dysregulated horses.DOCX

The objective of the study was to characterize the temporal changes of phosphorylation patterns of mTOR signaling proteins in response to two dietary protein sources in insulin dysregulated (ID, n = 8) and non-ID (n = 8) horses. Horses were individually housed and fed timothy grass hay and 2 daily concentrate meals so that protein was the first limiting nutrient and the total diet provided 120% of daily DE requirements for maintenance. On sample days, horses randomly received 0.25 g CP/kg BW of a pelleted alfalfa (AP) or commercial protein supplement (PS). Blood samples were collected before and 30, 60, 90, 120, 150, 180, 210, 240, 300, 360, 420, and 480 min post feeding and analyzed for plasma glucose, insulin and amino acid (AA) concentrations. Gluteus Medius muscle samples were obtained before and 90, 180, and 300 min after feeding and analyzed for relative abundance of phosphorylated mTOR pathway components using western immunoblot analysis. There was no effect of protein source on postprandial glucose and insulin responses (P ≥ 0.14) but consumption of PS elicited a 2 times larger AUC for essential AA (EAA), greater peak concentrations of EAA and a shorter time to reach peak EAA concentrations compared to AP. Abundance of phosphorylated mTOR (P = 0.08) and rpS6 (P = 0.10) tended to be ~1.5-fold greater after consumption of PS at 90 min compared to AP. Dephosphorylation patterns differed between protein sources and was slower for AP compared to PS. ID horses had a 2 times greater (P = 0.009) AUC and 3 times higher postprandial peak concentrations (P < 0.0001) for insulin compared to non-ID horses after consumption of both treatment pellets, but EAA responses were similar between groups (P = 0.53). Insulin status did not affect rpS6 or mTOR phosphorylation after consumption of either protein source (P ≥ 0.35), but phosphorylated rpS6 abundance was twice as high in ID compared to non-ID horses (P = 0.007). These results suggest that the consumption of higher quality protein sources may result in greater postprandial activation of the mTOR pathway compared to equal amounts of a forage-based protein source. Moreover, ID does not impair postprandial activation of mTOR and rpS6 proteins in horses following a protein-rich meal.

本研究旨在解析胰岛素调节异常（insulin dysregulated, ID，n=8）与非胰岛素调节异常（non-ID，n=8）马匹在两种日粮蛋白质来源摄入后，雷帕霉素靶蛋白（mTOR）信号通路蛋白磷酸化模式的时序变化特征。试验马匹均采用单栏饲养，饲喂梯牧草干草与每日两次的浓缩日粮，使蛋白质成为第一限制性营养物质，且总日粮提供量达到马匹每日维持所需消化能（digestible energy, DE）的120%。在采样日，马匹随机接受0.25g粗蛋白（crude protein, CP）/kg体重的颗粒化苜蓿（pelleted alfalfa, AP）或商业蛋白质补充剂（commercial protein supplement, PS）。分别于饲喂前及饲喂后30、60、90、120、150、180、210、240、300、360、420、480分钟采集血液样本，检测血浆葡萄糖、胰岛素与氨基酸（amino acid, AA）浓度。同时分别于饲喂前及饲喂后90、180、300分钟采集臀中肌样本，采用蛋白质免疫印迹（western immunoblot）分析磷酸化mTOR通路组分的相对丰度。研究结果表明，蛋白质来源对餐后葡萄糖与胰岛素应答无显著影响（P≥0.14）；但与AP组相比，PS组的必需氨基酸（essential amino acid, EAA）曲线下面积（area under the curve, AUC）约为其2倍，必需氨基酸峰值浓度更高，且达峰时间更短。饲喂后90分钟时，PS组的磷酸化mTOR（P=0.08）与rpS6（P=0.10）的丰度呈现升高趋势，约为AP组的1.5倍。两组间蛋白质去磷酸化模式存在差异，AP组的去磷酸化速度慢于PS组。相较于非ID马匹，ID马匹在饲喂两种颗粒日粮后，胰岛素的曲线下面积（AUC）约为其2倍（P=0.009），餐后峰值浓度为其3倍（P<0.0001），但两组间必需氨基酸应答无显著差异（P=0.53）。胰岛素调节异常状态对两种蛋白质来源饲喂后的rpS6或mTOR磷酸化水平无显著影响（P≥0.35），但ID组的磷酸化rpS6丰度为非ID组的2倍（P=0.007）。上述结果提示，与等量饲草基蛋白质来源相比，高品质蛋白质来源的摄入可更强地激活餐后mTOR信号通路。此外，在富含蛋白质的日粮饲喂后，胰岛素调节异常并不会损害马匹体内mTOR与rpS6蛋白的餐后激活水平。