State Key Laboratory for Quality and Safety of Agro-products, Ningbo University, Ningbo, 315211, PR China; Yantai Marine Economic Research Institute, Yantai, 264034, PR China.
State Key Laboratory for Quality and Safety of Agro-products, Ningbo University, Ningbo, 315211, PR China; State-Province Joint Laboratory of Marine Biotechnology and Engineering, Ningbo University, Ningbo, 315211, China.
Fish Shellfish Immunol. 2022 Mar;122:170-180. doi: 10.1016/j.fsi.2022.02.012. Epub 2022 Feb 10.
Glutaminolysis has been proved to play an irreplaceable role in vertebrate immunity, including effects on cytokine production, bacterial killing, and redox homeostasis maintenance. Our previous metabolomics analysis indicated that glutaminolysis metabolic substrates glutamine (Gln) and metabolites glutamate (Glu) were significantly lower in Skin ulceration syndrome (SUS)-diseased Apostichopus japonicus. To further delineate the role of glutaminolysis, we assayed the levels of Gln and Glu. We found that their contents in coelomocytes were decreased, accompanied by an increase in glutathione (GSH) in pathogen-challenged Apostichopus japonicus. Consistently, the mRNA transcripts of three key genes in glutaminolysis (AjASCT2, AjGOT, and AjGCS) were significantly induced. Moreover, the increased MDA and NADPH/NADP levels in response to pathogen infection indicated that oxidative stress occurs during the immune response. The metabolic regulator AMPKβ could regulate glutaminolysis in vertebrates by inducing cells to take up extracellular Gln. To explore the underlying regulatory mechanism behind glutaminolysis that occurred in coelomocytes, the full-length cDNA of AMPKβ was identified from A. japonicus (designated as AjAMPKβ). AjAMPKβ expression was significantly induced in the coelomocytes after pathogen challenge, which was consistent with the expression of key genes of glutaminolysis. A functional assay indicated that AjAMPKβ silencing by siRNA transfection could increase the levels of Gln and Glu and depress the production of GSH. Moreover, the expression of glutaminolysis-related genes was significantly inhibited, and the reduction of redox homeostasis indexes (MDA and NADPH/NADP) was also observed. Contrastingly, AjAMPKβ overexpression promoted redox homeostasis balance. Intracellular ROS is mostly responsible for breaking redox homeostasis and leading to oxidative stress, contributing to cell fate changes in immune cells. Exogenous Gln and GSH treatments could significantly reduce ROS level while the AjAMPKβ silencing induced the level of ROS and accelerated the necrosis rate. All these results collectively revealed that AjAMPKβ could modulate cellular redox homeostasis by affecting the glutaminolysis in A. japonicus.
谷氨酰胺分解代谢在脊椎动物免疫中发挥着不可替代的作用，包括对细胞因子产生、细菌杀伤和氧化还原稳态维持的影响。我们之前的代谢组学分析表明，患有皮肤溃疡综合征 (SUS) 的刺参中的谷氨酰胺分解代谢底物谷氨酰胺 (Gln) 和代谢物谷氨酸 (Glu) 显著降低。为了进一步阐明谷氨酰胺分解代谢的作用，我们测定了 Gln 和 Glu 的水平。我们发现，它们在腔肠细胞中的含量减少，同时伴随着病原体攻击后的刺参中谷胱甘肽 (GSH) 的增加。一致地，谷氨酰胺分解代谢的三个关键基因 (AjASCT2、AjGOT 和 AjGCS) 的 mRNA 转录本显著诱导。此外，病原体感染引起的 MDA 和 NADPH/NADP 水平增加表明氧化应激发生在免疫反应过程中。代谢调节剂 AMPKβ 可以通过诱导细胞摄取细胞外 Gln 来调节脊椎动物中的谷氨酰胺分解代谢。为了探索腔肠细胞中发生的谷氨酰胺分解代谢的潜在调节机制，我们从刺参中鉴定了 AMPKβ 的全长 cDNA（命名为 AjAMPKβ）。病原体攻击后，腔肠细胞中 AjAMPKβ 的表达显著诱导，这与谷氨酰胺分解代谢关键基因的表达一致。功能测定表明，通过 siRNA 转染沉默 AjAMPKβ 可以增加 Gln 和 Glu 的水平，并抑制 GSH 的产生。此外，谷氨酰胺分解代谢相关基因的表达显著受到抑制，氧化还原稳态指标（MDA 和 NADPH/NADP）的降低也观察到。相反，AjAMPKβ 的过表达促进了氧化还原稳态平衡。细胞内 ROS 主要负责打破氧化还原稳态并导致氧化应激，导致免疫细胞中细胞命运的变化。外源性 Gln 和 GSH 处理可以显著降低 ROS 水平，而 AjAMPKβ 沉默诱导的 ROS 水平升高并加速了坏死率。所有这些结果共同表明，AjAMPKβ 可以通过影响刺参中的谷氨酰胺分解代谢来调节细胞内氧化还原稳态。
