Zhu Yuhua, Lin Gang, Dai Zhaolai, Zhou Tianjiao, Li Tiantian, Yuan Taolin, Wu Zhenlong, Wu Guoyao, Wang Junjun
State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, 100193, China.
Department of Animal Science, Texas A&M University, College Station, TX, 77843, USA.
Amino Acids. 2015 Oct;47(10):2185-97. doi: 10.1007/s00726-014-1785-0. Epub 2014 Jul 5.
L-Glutamine (Gln) is an essential amino acid for intestinal growth and integrity. However, the underlying molecular mechanisms are not fully known. In the present study, porcine intestinal epithelial cells (IPEC-1) were used to test the hypothesis that autophagy is induced by Gln deprivation and inhibited by Gln supplementation. After a 2-day period of growth in normal medium, IPEC-1 cells were transferred to a Gln-free custom-made DMEM. Cell numbers, the distribution of autophagosomes, the abundance of the protein for an autophagy marker LC3B, as well as abundances of the mTOR and MAPK proteins during an 8-h period were determined. Furthermore, the rescue effect of 5 mM Gln was evaluated. Our results showed that Gln deprivation reduced the cell number, while enhancing the accumulation of autophagosomes and the expression of LC3B-II in IPEC-1 cells within 8 h. The concentrations of Glu, Asp, Cit, Arg, Leu, Ile, Val, Ala, β-Ala, Orn, Phe, Met and Ser in the culture medium were altered by Gln deprivation. Further analysis revealed that Gln deficiency inactivated, but Gln supplementation activated, the mTOR and MAPK/ERK signaling pathways. Collectively, our findings support the notion that Gln deficiency induces autophagy and disturbs amino acid metabolism in intestinal epithelial cells, as well as attenuated their mTOR and MAPK/ERK signaling pathways to inhibit protein synthesis and cell proliferation.
L-谷氨酰胺（Gln）是肠道生长和完整性所必需的氨基酸。然而，其潜在的分子机制尚不完全清楚。在本研究中，使用猪肠上皮细胞（IPEC-1）来验证以下假设：谷氨酰胺缺乏诱导自噬，而谷氨酰胺补充则抑制自噬。在正常培养基中生长2天后，将IPEC-1细胞转移至定制的无谷氨酰胺的DMEM中。测定8小时内的细胞数量、自噬体分布、自噬标志物LC3B蛋白丰度以及mTOR和MAPK蛋白丰度。此外，评估了5 mM谷氨酰胺的挽救作用。我们的结果表明，谷氨酰胺缺乏减少了细胞数量，同时在8小时内增强了IPEC-1细胞中自噬体的积累和LC3B-II的表达。谷氨酰胺缺乏改变了培养基中Glu、Asp、Cit、Arg、Leu、Ile、Val、Ala、β-Ala、Orn、Phe、Met和Ser的浓度。进一步分析表明，谷氨酰胺缺乏使mTOR和MAPK/ERK信号通路失活，而谷氨酰胺补充则激活了这些信号通路。总体而言，我们的研究结果支持以下观点：谷氨酰胺缺乏诱导肠道上皮细胞自噬并扰乱氨基酸代谢，同时减弱其mTOR和MAPK/ERK信号通路以抑制蛋白质合成和细胞增殖。
