Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061; Perdue AgriBusiness LLC, Salisbury, MD 21804.
Dairy Visions LLC, Chandler, AZ 85249.
J Dairy Sci. 2020 Oct;103(10):8948-8966. doi: 10.3168/jds.2020-18155. Epub 2020 Aug 26.
Understanding uptake of AA by mammary tissue as supply varies is critical for predicting milk component production. Our objective was to develop an in vitro method to quantify cellular uptake, efflux, and intracellular metabolism of individual AA that could be implemented for evaluating these factors when AA supply and profile are varied. Bovine primary mammary epithelial cells were grown to confluency and exposed to medium with an AA profile and concentration similar to lactating dairy cow plasma for 24 h. Cells were then preloaded in medium enriched with N-labeled AA for 24 h followed by removal of the N-labeled medium and incubation with medium enriched with C-labeled AA for 0, 15, 60, 300, 900, 1,800, and 3,600 s. Extracellular free AA and intracellular free and protein-bound AA were analyzed for concentrations and isotopic enrichment by gas chromatography-mass spectrometry. A dynamic, 12-pool model was constructed representing extracellular and intracellular free and protein-bound pools of an AA, and their respective N and C isotopes. Markov chain Monte Carlo simulation (n = 5,000) was conducted to evaluate prediction errors by deriving standard errors and posterior distributions for rate constants, fluxes, and pools. Cellular Ala influx and efflux were higher than Leu, reflecting Ala role in driving system L transport and the high capacity of sodium-dependent transport. The Ala and Leu turnover rates were 181 and 95, 580 and 857, and 74 and 157% per hour for extracellular, intracellular, and fast protein-bound pools, respectively. The intracellular and extracellular Ala to Leu ratios were quite different, meaning the blood AA profile is not the AA profile provided for protein translation. The high level of exchange and rapid turnover of pools provide a mechanism for matching the AA supplies to the precision necessary for translation. This also understates the importance of using experimental medium similar to what is observed in vivo given that some AA depend on other AA for influx (exchange driven). The average root mean squared prediction error across the isotope enrichments, pools, and concentrations was 9.7 and 14.1% for Ala and Leu, respectively, and collinearity among parameters was low, indicating adequate fit and identifiability. The described model provides insight on individual AA transport kinetics and a method for future evaluation of AA transport and intracellular metabolism when subjected to varying AA supplies.
了解乳腺组织对 AA 的摄取情况，因为供应是变化的，这对于预测乳成分的生产至关重要。我们的目标是开发一种体外方法来量化单个 AA 的细胞摄取、外排和细胞内代谢，当 AA 的供应和分布发生变化时，可以用这种方法来评估这些因素。将牛原代乳腺上皮细胞培养至汇合，并在与泌乳奶牛血浆相似的 AA 分布和浓度的培养基中孵育 24 小时。然后，细胞在富含 N 标记 AA 的培养基中预孵育 24 小时，然后去除 N 标记的培养基，并在富含 C 标记 AA 的培养基中孵育 0、15、60、300、900、1800 和 3600 秒。通过气相色谱-质谱法分析细胞外游离 AA 以及细胞内游离和蛋白结合 AA 的浓度和同位素丰度。构建了一个动态的 12 池模型，代表 AA 的细胞外和细胞内游离及蛋白结合池及其各自的 N 和 C 同位素。通过推导速率常数、通量和池的标准误差和后验分布，进行马尔可夫链蒙特卡罗模拟（n = 5000），以评估预测误差。Ala 的细胞内流入和流出高于 Leu，反映了 Ala 在驱动系统 L 转运中的作用和钠依赖性转运的高容量。细胞外、细胞内和快速蛋白结合池的 Ala 和 Leu 周转率分别为每小时 181 和 95、580 和 857、74 和 157%。细胞内和细胞外 Ala 与 Leu 的比值差异很大，这意味着血液 AA 分布不是提供给蛋白质翻译的 AA 分布。池的高交换水平和快速周转为匹配 AA 供应与翻译所需的精度提供了一种机制。鉴于某些 AA 依赖于其他 AA 进入细胞（交换驱动），因此使用与体内观察到的类似的实验培养基来评估 AA 转运和细胞内代谢的重要性就更低了。在所有同位素丰度、池和浓度中， Ala 和 Leu 的平均均方根预测误差分别为 9.7%和 14.1%，参数之间的共线性较低，表明拟合和可识别性良好。该模型提供了对单个 AA 转运动力学的深入了解，以及未来评估 AA 转运和细胞内代谢的方法，当 AA 供应发生变化时可以使用该方法。
