Comparative Phosphoproteomic Analysis Demonstrates That AMPKα2 Knockout Exacerbates Hepatic Aging in Mice

Aging accompanies metabolic dysregulation, wherein the liver exhibits high sensitivity to age-related changes. AMP-activated protein kinase α2 (AMPKα2), a key energy metabolism regulator, lacks investigation regarding germline knockout effects on aged liver phosphoproteins. This study investigated germline AMPKα2 knockout effects on aged mouse liver through morphological analysis, Western blot (WB), and phosphoproteomics. AMPKα2 knockout significantly exacerbated glucose-lipid metabolism dysfunction, inflammatory responses, and age-related morphological changes, with enhanced senescent phenotypes validated by WB. Data-independent acquisition (DIA) phosphoproteomic analysis identified 4,448 specific phosphopeptides, among which 316 significantly differentially modified peptides. AMPKα2 knockout enhanced phosphorylation of glucose-lipid metabolism proteins, such as acetyl-CoA carboxylase 1 (Acaca) at S118, S80, S79, S157, and S117 sites, and genomic instability proteins, such as HSP90β (Hsp90ab1) at S255. Conversely, stress response protein HSP27 (Hspb1) phosphorylation at S86 was significantly reduced, validated by WB. This study revealed novel molecular signatures of AMPKα2 knockout in exacerbating hepatic aging and metabolic dysfunction, suggesting HSP27 as a potential AMPKα2 downstream effector through site-specific phosphorylation. This work first delineated the phosphoproteomic landscape of aged liver in AMPKα2 knockout mice, establishing foundations for targeting specific protein phosphorylation sites as therapeutic targets for age-related liver diseases.