The Effect Of ATP Synthase Inhibitor 1 (ATPIF1) Gene Knockout On Mouse Macrophage Function

BackgroundATPase inhibitory factor 1(ATPIF1)is a protein found in bovine heart mitochondria and is a physiological inhibitor of ATP synthase,which inhibits the F1 domain of H+-ATP synthase,thereby inhibiting the synthesis and hydrolysis of ATP synthase,and subsequently regulate ATP levels when intracellular ATP fluctuates in a timely manner.The activity of ATPIF1 is regulated by histidine protonation and serine 39(S39)phosphorylation.In the deprotonation and dephosphorization state of histidine,ATPIF1 binds to H+-ATP synthase,inhibits enzyme activity,and regulates ATP Levels.Studies have shown that the fluctuation of intracellular ATP levels is related to the occurrence and development of various diseases,so it is of great significance for the study of ATPIF1.Our previous studies have found elevated ATP levels in fibroblasts of ATPIF1 knockout mice and an increase in their differentiation into adipocytes.However,the function of ATPIF1 in bone marrow macrophages remains unclear.Here we aimed to study the effect of ATPIF1 knockout on macrophages.ObjectiveThe ATPIF1 systemic knockout mouse model was constructed by CRISPR/Cas9 to study the effects of ATPIF1 knockout on the function of mouse bone marrow macrophages under normal and high-fat diet conditions.Method(1)Identification of ATPIF1 knockout mice: Genotyping,q RT-PCR and Western Blot techniques were used to verify the knockdown of ATPIF1 gene,respectively.(2)Detection of ATP synthesis function: ATP/protein level was detected by ATP detection kit;Cell oxidative phosphorylation level and glycolysis ability were detected by Seahorse instrument;q RT-PCR was used to detect the m RNA relative expression of the tricarboxylic acid cycle(TCA)related enzymes including pyruvate kinase(PKm),isocitrate dehydrogenase(IDH),citrate synthase(CS),α-ketoglutarate dehydrogenase(OGDH)in macrophages.(3)Identification of macrophage immune function: Flow cytometry(FACS)was performed to detect the differentiation of M1 after LPS stimulation for 24 hours;q RT-PCR was used to detect m RNA relative expressions of cytokines in macrophages including tumor necrosis factor-α(Tumor Necrosis Factor-Alpha,TNF-α),Interleukin-1β(IL-1β),Interleukin-10(IL-10),Interleukin 1 receptor antagonist(IL-1RA).(4)Detection of mitochondrial autophagy levels: Western Blot was used to detect the protein levels of mitochondrial internal reference protein VDAC,autophagy-related proteins PINK1 and Parkin in macrophages.Result(1)Under normal and LPS stimulation,the ATP level,oxygen consumption and the m RNA expression of TCA-related enzyme in bone marrow macrophages of KO group were notably higher than those in WT group in mice with normal diet,indicating that ATPIF1 gene knockout can promote ATP synthesis in macrophage through increasing the function of TCA and respiratory chain.(2)Macrophages of mice with normal diet were stimulated with LPS for 24 h in high glucose environment.The M1 ratio in WT group was significantly lower than that cultured in low glucose environment;M1 ratio from KO mice cultured with high glucose after LPS stimulation is of no difference with that in low sugar,but was significantly higher in WT mice with high sugar.Under normal conditions,the m RNA expression levels of proinflammatory factors TNF-α and IL-1β in macrophages from normal diet KO mice were lower than those in WT group,while the m RNA expression levels of anti-inflammatory factors IL-10 and IL-1RA were remarkably higher.Under LPS stimulation,the m RNA expression levels of cytokines including TNF-α,IL-1β,IL-10 and IL-1RA in KO group were higher than those in WT group.This indicates that ATPIF1 knockout can increase the antistress ability and help to improve the innate immunity of macrophages.(3)The results of high-fat mice were consistent with the result from cell culture in vitro:Compared with WT group,ATP levels,basal and maximum oxygen consumption of bone marrow macrophages in KO group were much higher while the basic and maximum glycolysis levels were obviously lower;Compared with WT group,the expression levels of PKm,IDH,CS and OGDH in KO group were significantly increased as well as the cytokines including TNF-α,IL-1β,IL-10 and IL-1RA.(4)In the normal diet mice,the expression levels of autophagy-related proteins PINK1 and Parkin in the bone marrow macrophages of KO mice were higher than those in the WT group at baseline or after LPS stimulation for 2 h;In the high-fat group,the expression levels of PINK1 and Parkin were also enhanced in bone marrow macrophages from KO mice as compared with the WT group.ConclusionATPIF1 knockout can augment mitochondrial ATP synthesis by promoting tricarboxylic acid cycle and oxidative phosphorylation activities,enhance macrophage function by promoting M1 differentiation and cytokine secretion and increase mitochondrial function by elevating autophagy.