Post-translational modifications (PTMs) are the essential regulators of protein function and cellular homeostasis. Lysine acetylation, an evolutionarily conserved PTM present in all living organisms, was first identified on histones by Vincent Allfrey and colleagues six decades ago (Allfrey et al., 1964). Subsequent studies extended its presence to high-mobility group (HMG) proteins (R. Sterner et al., 1979) and tubulin (L'Hernault & Rosenbaum, 1983; R. Sterner et al., 1979). In the mid-to-late 1990s, lysine acetylation of the transcription factor p53 was discovered, marking a pivotal moment in understanding its role in cellular signaling (Gu & Roeder, 1997).

Over the past two decades, developments in mass spectrometry-based proteomics have vastly expanded the acetylome, offering valuable insights into the regulation and functional significance of site-specific protein acetylation (Abelin et al., 2023; Choudhary et al., 2014; Hansen et al., 2019; Kori et al., 2017; Xu, Shi, & Bao, 2022). These discoveries helped define the role of acetylation in controlling protein function, stability, subcellular localization, and interactions. In addition to acetylation, lysine residues can undergo various acylation modifications, including malonylation, succinylation, lactylation, glutarylation, crotonylation, benzoylation, propionylation, butyrylation, and β-hydroxybutyrylation (Hirschey & Zhao, 2015; H. Huang et al., 2018; Xie et al., 2016). Like acetylation, these modifications alter the charge of the modified residues, thereby impacting protein conformation and dynamics. While the repertoire of acylation marks continues to expand, the biological significance of many acylations remains poorly understood, underscoring the need for further investigation. Among these modifications, acetylation remains particularly noteworthy for its central role in regulating metabolic and epigenetic processes. It serves as a key mechanism for maintaining cellular homeostasis and facilitating adaptive responses to environmental cues.

Acetylation is broadly categorized into Nα-acetylation, Nε-acetylation, and O-acetylation, depending on the acetylation site and the amino acid modified (T. Y. Lee et al., 2010). This paper focuses on Nε-acetylation (hereafter referred to as acetylation), which occurs on the ε-amino group of lysine residues in proteins. Histone acetylation predominantly occurs on specific lysine residues in the N-terminal regions of core histones, which are rich in basic amino acids (Garcia et al., 2007). This modification neutralizes the positive charge of the lysine side chain, resulting in alterations to protein conformation, interactions, and function. Consequently, this structural alteration enhances nucleosome accessibility, facilitating gene expression and replication (Ali et al., 2018). Beyond histones, acetylation also modifies thousands of non-histone proteins, including metabolic enzymes, transcription factors, and signaling molecules, underscoring its broad impact on cellular function (Choudhary et al., 2009; Narita et al., 2019; Svinkina et al., 2015). As such, acetylation is essential for regulating many cellular processes, including metabolism, DNA repair, gene transcription, signal transduction, cell division, and proteostasis, which involves the synthesis, folding, and degradation of proteins (Narita et al., 2019). Dysregulated acetylation has been implicated in numerous diseases, including cancers (Lane & Chabner, 2009) and neurodegenerative disorders (Alrob et al., 2014; Finley & Haigis, 2012; Portillo et al., 2021). Furthermore, disrupted acetylation has been associated with aging (Eisenberg et al., 2014), various forms of dementia (Maze et al., 2015), alcoholic liver disease (Adhikari et al., 2023), and other metabolic and degenerative disorders (Arias-Alvarado et al., 2021). Despite these associations, the mechanisms underlying dysregulated acetylation in human diseases remain poorly understood, necessitating further research to clarify its causal roles and therapeutic potential.

Acetylation involves the transfer of an acetyl group from the central metabolic intermediate acetyl-CoA to the lysine side chain catalyzed by lysine acetyltransferases (KATs) and reversed by Zinc (Zn2+)- or nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacetylases (KDACs) (Fig. 1).

The availability of acetyl-CoA as an acetyl donor and NAD+ as a cofactor for sirtuins, type III histone deacetylases (HDACs), links protein acetylation to the cellular metabolic state. Consequently, acetylation serves as a critical metabolic sensor, modulating substrate utilization and enabling cellular adaptation to fluctuations in nutrient availability and energy demands. This adpative capacity, known as metabolic flexibility (Kelley et al., 1999), refers to the ability of cells and organisms to switch efficiently between substrates—such as glucose, fatty acids, amino acids, and ketones—depending on physiological conditions, thereby maintaining metabolic efficiency and homeostasis (Muoio, 2014; Muoio et al., 2012). Individuals with high metabolic flexibility can readily adapt to dietary changes or transitions between fed and fasted states, as well as rest and exercise states, without significant metabolic disruption.

Metabolic inflexibility, marked by nutrient overload and impaired substrate switching, disrupts energy homeostasis and is linked to insulin resistance and type 2 diabetes mellitus (T2DM) (Kelley & Mandarino, 2000; Neely & Morgan, 1974). It is often characterized by a persistent reliance on carbohydrate oxidation and an inability to shift to alternative fuels during metabolic transitions. Mitochondria are central to this process, as maintaining metabolic flexibility requires precise regulation of mitochondrial enzymes (Finck et al., 2005). Mitochondrial dysfunction contributes to aging, cancer, and metabolic syndrome, underscoring its role in the pathogenesis of metabolic inflexibility.

Protein acetylation has emerged as a pivotal regulator of core metabolic pathways, linking cellular metabolism and acetylation-mediated control mechanisms (Wang et al., 2010). Acetylation modulates metabolic processes by influencing transcriptional regulation and the functional activity of metabolic enzymes. It affects target proteins at multiple levels, including their abundance, enzymatic activity, and interactions with substrates or other proteins (Xiong & Guan, 2012). By modifying key metabolic enzymes, transcription factors, and histones, acetylation coordinates critical metabolic processes, including glycolysis, fatty acid synthesis and oxidation, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS). These acetylation-driven regulatory mechanisms are essential for maintaining metabolic flexibility and energy homeostasis, enabling cells to adapt to fluctuating nutrient availability and changing metabolic demands (Sivanand et al., 2018).

Acetylation, particularly histone acetylation, has been well-studied and highlighted in numerous high-impact review papers (D. E. Sterner & Berger, 2000; Struhl, 1998; Verdin & Ott, 2015), however its broader role in metabolic regulation is emerging. Despite significant advances, key gaps remain in understanding the site-specific functional role of acetylation and its precise role in disease pathogenesis. While therapies targeting HDACs and sirtuins hold promise, their clinical translation remains challenging due to the complexity of substrate selectivity and systemic effects. This review examines how compartmentalized acetyl-CoA metabolism and substrate-dependent availability regulate acetylation across subcellular compartments. We highlight the roles of acetyltransferases and deacetylases—particularly sirtuins as NAD+-dependent sensors—in modulating acetylation dynamics. In addition to enzymatic regulation, we also discuss non-enzymatic acetylation, which occurs under high acetyl-CoA conditions and contributes to metabolic feedback and proteome remodeling. To provide context for the regulatory role of acetylation, we first summarize current knowledge on metabolic flexibility and its underlying adaptive mechanisms. We then discuss how acetylation supports this metabolic flexibility, enabling cells to respond dynamically to energetic and environmental stress. Additionally, we examine the impact of acetylation on proteostasis, including its influence on protein synthesis, folding, degradation, and aggregation. Together, these perspectives highlight acetylation as a central node in the integration of metabolic signaling and protein quality control, with broad relevance to metabolic health and disease pathogenesis.