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

## TL;DR

The study shows how starvation changes muscle proteins and a specific chemical modification linked to ketone metabolism in mice.

## Contribution

The study reveals lysine β-hydroxybutyrylation as a dynamic PTM regulating muscle metabolism during starvation.

## Key findings

- Starvation increases β-hydroxybutyrate levels and widespread Kbhb modification in muscle proteins.
- Kbhb modification targets metabolic enzymes and reduces on structural proteins during fasting.
- Kbhb is linked to regulation of enzymatic activity and metabolic pathway shifts in muscle.

## Abstract

Starvation triggers profound metabolic reprogramming in skeletal muscle to sustain survival during nutrient scarcity, yet the role of lysine β-hydroxybutyrylation (Kbhb)—a metabolite-derived post-translational modification (PTM) linked to ketone metabolism—remains unclear. Here, we performed integrative quantitative proteomics and Kbhb profiling on gastrocnemius muscle from mice subjected to 72 h food deprivation. Starvation induced muscle atrophy, elevated systemic β-hydroxybutyrate levels, and widespread changes in Kbhb modification. Proteomic analysis revealed a shift from anabolic to catabolic/oxidative pathways, with downregulation of ribosomal proteins and upregulation of autophagy/lipid catabolism. Deep Kbhb profiling identified over 7500 modified sites across 2000 proteins, with starvation enhancing Kbhb on key metabolic enzymes (glycolysis, TCA cycle, fatty acid β-oxidation) at conserved residues proximal to functional domains, and reducing Kbhb on structural/contractile proteins. Our findings establish Kbhb as a dynamic PTM mediating gastrocnemius muscle adaptation to energy deficiency, expanding the role of metabolite-driven regulation in muscle metabolism.

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.