Fish Shellfish Immunol 2022 Mar 01;122:170-180. doi: 10.1016/j.fsi.2022.02.012.

AMPK-mediated glutaminolysis maintains coelomocytes redox homeostasis in Vibrio splendidus-challenged Apostichopus japonicus.

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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.

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