LPS-induced Dephosphorylation Of AMPK Potentiates Inflammatory Injury Via Repression Of Autophagy

Objective: AMP-activated protein kinase(AMPK)is a crucial metabolic regulator with profound modulatory activities on inflammation.Although the anti-inflammatory benefits of AMPK activators were well-documented in experimental studies,the pathological significance of endogenous AMPK in inflammatory disorders largely remains unknown.The present study investigated the phosphorylation status of endogenous AMPK and the potential roles of AMPK in mice with lipopolysaccharide(LPS)-induced lethal inflammation.Method: LPS was injected intraperitoneally to induce severe inflammation and acute lung injury in male BALB/c mice.To investigate the phosphorylation status of AMPK,vehicle or various doses of LPS(10 mg/kg,20 mg/kg)was injected,the mice were sacrificed by decapitation 3 h post LPS challenge and lung sample were harvested for determination of the phosphorylation level of AMPK and acetyl-CoAcarboxylase(ACC)with immunoblot analysis.To evaluate the potential roles of AMPK in inflammatory injury,the mice were treated with the AMPK activator A-769662(30 mg/kg)30 min before LPS(20 mg/kg)exposure.The mice were sacrificed at 3 h or 18 h.The plasma samples and lung sample were harvested for further experiments.To evaluate the potential effects of the AMPK activator A769662 on the mortality of LPS-challenged mice,the survival of experimental animals was assessed every 6 h for at least 7 days.To investigate the mechanisms underlying the potential roles of AMPK in inflammatory injury,the autophagy inhibitor 3-MA(15mg/kg),the mTOR activator 3-BDO(100mg/kg)or the ULK1 inhibitor MRT68921(50mg/kg)was co-administered with the AMPK activator A-769662.The mice were sacrificed at 18 h.The plasma samples and lung sample were harvested for further experiments.The histological abnormalities of the lung tissue were observed with H&E staining.The level of IL-6 of the harvested blood sample were detected by ELISA.The level of AMPK?,phosphorylated AMPK?(Thr172),?-actin,ACC,Phosphorylated ACC(Ser79),ULK1,phosphorylated ULK1(Ser757),4E-BP1,phosphorylated 4E-BP1,P70S6K1,phosphorylated P70S6K1,LC3,p62 in lung tissues were detected by Western blot.Results: The results indicated that LPS dose-dependently decreased the phosphorylation level of AMPK and its target protein acetyl-CoAcarboxylase(ACC).Reactivation of AMPK with the AMPK activator A-769662 suppressed LPS-induced elevation of interleukin 6(IL-6),alleviated histological abnormalities in lung and improved the survival of LPS-challenged mice.Treatment with A-769662 restored LPS-induced suppression of autophagy,inhibition of autophagy by 3-MA reversed the beneficial effects of A-769662.Treatment with A-769662 suppressed LPS-induced activation of mammalian target of rapamycin(mTOR),co-administration of mTOR activator abolished the beneficial effects of A-769662 and the suppressive effects of A-769662 on uncoordinated-51-like kinase 1(ULK1)phosphorylation.Inhibition of ULK1 removed the beneficial effects of A-769662.Conlusion: These data indicated that LPS-induced dephosphorylation of AMPK could result in weakened inhibition of mTOR and repression of ULK1-dependent autophagy,which might potentiate the development of LPS-induced inflammatory injury.These data suggests that pharmacological restoration of AMPK activation might be a beneficial approach for the intervention of inflammatory disorders.