The Role Of SIRT4 In T Cell Metabolism And Function By Regulating AMPK/mTOR Signaling Pathway

BackgroundThe metabolism of naive T cells circulated in blood are a resting state with low energy requirements.When naive T cells are activated by a combination of T cell receptor（T cell receptor,TCR）-mediated antigen recognition and costimulatory signals such as CD28,anabolic metabolism is enhanced for cell proliferation and function.m TOR（Mammalian Target of Rapamycin,m TOR）is the downstream signal node of CD28.It’s activation can promote the expression of anabolic genes,which contribute to meet the demand of synthesis of intermediate products during cell proliferation.AMPK（Amp-activated protein kinase,AMPK）is considered to be an inhibitor of m TOR signaling,and it’s activation promotes catabolic gene expression,which contribute to product more ATP energy.AMPK-m TOR signaling pathway plays an important role in mediating T cell metabolism.SIRT4,a member of sirtuins family,has been reported to play an important regulatory role in the occurrence and development of a variety of metabolic diseases,such as diabetes.However,the mechanism of SIRT4 in T cell metabolism and function needs to be further clarified.ObjectiveTo study the role of SIRT4 in the function and metabolism of T cells mediated by AMPK-m TOR signaling pathway,and to explore the possible molecular mechanism.Methods1.In spleen of SIRT4 knockout mice constructed by CRISPR/Cas9 technology,the expression of SIRT4 gene were evaluated by western blotting glucose and insulin levels in peripheral blood,glucose tolerance test and pancreatic tissue GDH（Glutamate dehydrogenase,GDH）activity were detected for SIRT4-mediated functions.2.In SIRT4 knockout mice,the cell proliferation of CD4⁺T was measured by MTS after being cultured by CD3/CD28 and IL-2 in vitro.The cytokine IL-2 secretion of CD4⁺T cells was determined by ELISA after being cultured by CD3/CD28.Meanwhile,mitochondrial metabolism was measured by seahorse cell metabolism assay after CD3/CD28 and IL-2 stimulation,and the expression of SIRT1,the phosphorylation of m TOR and AMPKa were detected by western blotting3.The mitochondrial metabolism was determined by seahorse cell metabolism assay in SIRT4-overexpressing Jurkat cells after CD3/CD28 and IL-2 stimulation;Moreover,phosphorylation of m TOR and AMPKa were detected by western blotting4.SIRT4,the phosphorylation of m TOR and AMPKa were detected by western blotting in SIRT1 knockout Jurkat cells after PMA and ionomycin stimulation.The mitochondrial localization of SIRT4 in SIRT1 knockout Jurkat cells was detected by fluorescence confocal microscopy.Results1.The expression of SIRT4 in spleen of SIRT4 knockout mice was significantly lower than that of control group（p<0.05）,the glucose level in SIRT4 knockout mice was significantly lower than that in control group（p<0.05）,the insulin level and GDH activity in SIRT4 knockout mice were significantly higher than those in control group（p<0.05）.2.The proliferation capacity of SIRT4 knockout CD4⁺T cells was significantly lower than that of WT group（p<0.05）,IL-2 secretion level was significantly lower than WT group（p<0.05）.Moreover,the oxygen consumption level of mitochondrial oxidative phosphorylation and the phosphorylation of AMPKa was significantly decreased（p<0.05）in SIRT4 knockout CD4⁺T cells.However,SIRT1 and the phosphorylation of m TOR were significantly increased（p<0.05）.3.Compared with the control group,SIRT4 overexpression of Jurkat cells results in the phosphorylation of m TOR decreased（p<0.05）,the phosphorylation of AMPKa increased（p<0.05）.Meanwhile,the cell oxygen consumption increased significantly,mitochondrial oxidative phosphorylation increased（p<0.05）.4.After SIRT1 knockout in Jurkat cells,SIRT4 and the phosphorylation of AMPKa were significantly increased（p<0.05）,the phosphorylation of m TOR was significantly decreased（p<0.05）;Compared with the diffuse wild type,the localization of SIRT4showed punctate aggregation in mitochondria of SIRT1 knockout Jurkat cells.Conclusions1.During the activation metabolism of T cells,SIRT4 can promote T cell met-abolism and proliferation by activating AMPK signal.2.SIRT4 and SIRT1 antagonize each other in the regulation of AMPK signal in T cells.