Comprehensive Proteomics and β-Hydroxybutyrylation Profiling in Starvation-Induced Gastrocnemius Muscle Remodeling

Starvation elicits profound metabolic adaptations in skeletal muscle, enabling survival during nutrient scarcity. While global proteomic changes underpinning muscle atrophy have been studied, the role of lysine β-hydroxybutyrylation (Kbhb), a novel metabolite-derived post-translational modification linked to ketone metabolism, remains largely unexplored. In this study, we subjected mice to 72 h of food deprivation and performed integrative quantitative proteomics and Kbhb-modified peptide profiling on gastrocnemius muscle. Starvation induced significant body weight and muscle mass loss, accompanied by increased systemic β-hydroxybutyrate levels and widespread Kbhb modification of muscle proteins. Proteomic analysis revealed extensive downregulation of ribosomal and translation-associated proteins, coupled with upregulation of autophagy and lipid catabolism pathways, highlighting a coordinated shift from anabolic processes to catabolic and oxidative metabolism. Deep Kbhb profiling identified over 7500 modified lysine sites across 2000 proteins, with starvation triggering a global increase in Kbhb on key metabolic enzymes involved in glycolysis, TCA cycle, fatty acid β-oxidation, and amino acid metabolism. Notably, starvation-enhanced Kbhb preferentially targeted evolutionarily conserved lysines proximal to catalytic or cofactor-binding domains, implicating a regulatory role in enzymatic activity modulation. Conversely, Kbhb on structural and contractile proteins was downregulated, suggesting functional reprioritization of muscle physiology during fasting. Our findings uncover lysine β-hydroxybutyrylation as a dynamic, metabolically responsive PTM mediating gastrocnemius muscle adaptation to energy deficiency, expanding the paradigm of potentially metabolite-driven epigenetic and non-epigenetic regulatory mechanisms in muscle metabolism.