Hou Pengfei, Wang Caihong, Zhou Miaomiao, Liu Hongyun
College of Animal Sciences, Zhejiang University, Hangzhou, China.
College of Agriculture, Liaocheng University, Liaocheng, China.
J Anim Physiol Anim Nutr (Berl). 2022 Jan;106(1):24-32. doi: 10.1111/jpn.13546. Epub 2021 Apr 9.
Intestinal absorption of peptides is vital for the overall health and productivity of dairy cows. This study investigated the regulation, uptake and transport of dipeptides in bovine intestinal epithelial cells (BIECs). We also evaluated the effects of time, pH, concentration of the dipeptides, temperature, presence of diethylpyrocarbonate (DEPC)-an inhibitor of PepT1, and other dipeptides (Met-Met, Lys-Lys or Met-Lys), on the uptake and transport of Gly-Sar-FITC, which was a common fluorophore-labelled dipeptide. Under controlled experiments, BIECs were treated with 25 μM LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor) and 25 μM Perifosine (a protein kinase B (AKT) inhibitor). The subsequent expression of PepT1 in the BIECs was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. It was found that the uptake and transport of Gly-Sar-FITC were significant high at 37℃ than that at 4℃. The optimal pH for transport and uptake of Gly-Sar-FITC was 6.0-6.5, whereas the two properties decreased significantly in the presence of DEPC, Met-Met, Lys-Lys and Met-Lys (p < 0.05). The apical-to-basolateral transport was also found to be significantly higher than the reverse transport (p < 0.05). PI3K and AKT inhibitors were found to significantly suppress the expression of PepT1, thus impairing uptake and transport of Gly-Sar-FITC. Findings of this study thus suggest that the uptake and transport of Gly-Sar-FITC in BIECs are mediated by PepT1, and the PI3K/AKT signalling pathway regulates the absorption of small peptides.
肽的肠道吸收对奶牛的整体健康和生产性能至关重要。本研究调查了牛肠上皮细胞（BIECs）中二肽的调节、摄取和转运。我们还评估了时间、pH值、二肽浓度、温度、焦碳酸二乙酯（DEPC）（一种PepT1抑制剂）的存在以及其他二肽（Met-Met、Lys-Lys或Met-Lys）对荧光素标记的常见二肽甘氨酰-肌氨酸（Gly-Sar-FITC）摄取和转运的影响。在对照实验中，BIECs用25μM LY294002（一种磷脂酰肌醇3激酶（PI3K）抑制剂）和25μM哌立福辛（一种蛋白激酶B（AKT）抑制剂）处理。随后通过逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法评估BIECs中PepT1的表达。结果发现，Gly-Sar-FITC在37℃时的摄取和转运显著高于4℃时。Gly-Sar-FITC转运和摄取的最佳pH值为6.0-6.5，而在DEPC、Met-Met、Lys-Lys和Met-Lys存在的情况下，这两个特性显著降低（p<0.05）。还发现从顶端到基底外侧的转运显著高于逆向转运（p<0.05）。发现PI3K和AKT抑制剂显著抑制PepT1的表达，从而损害Gly-Sar-FITC的摄取和转运。因此，本研究结果表明，BIECs中Gly-Sar-FITC的摄取和转运由PepT1介导，PI3K/AKT信号通路调节小肽的吸收。
