This study significantly expanded the proteomic and acetylomic datasets of human adipose tissue compared to previous studies [10,11,12,13,14,15,16,17,18,19,20,21]. Functional alterations in WAT were closely associated with the progression of obesity and metabolic disorders. A positive energy balance leads to lipid accumulation in WAT, and excessive expansion is accompanied by structural remodeling and functional changes, including angiogenesis disorders, adipocyte hypoxia and necrosis, macrophage infiltration, inflammation, and fibrosis. Concurrently, excess lipids may accumulate ectopically in VA, the liver, and other organs [24, 25]. Previous studies have identified structural, functional, and metabolic regulatory differences between SA and VA [6, 26]. This study aimed to elucidate the distinct characteristics of SA and VA in MUO, explore underlying mechanisms, and identify potential intervention targets associated with the differential regulation between SA and VA through integrative proteomic and acetylomic analyses. While proteomic data suggested some degree of separation, the acetylomic results underscored nuanced differences between SA and VA. This highlighted the diversity and complexity of acetylation modifications, as well as the shared acetylated features across different samples. Subsequent functional enrichment analyses were conducted to explore the regulatory mechanisms of acetylation modifications in greater depth, aiming to better understand their roles in biological processes.

The ECM, a dynamic structure composed of macromolecules secreted by cells into the extracellular interstitium, remodels to accommodate lipid droplet growth during adipose tissue expansion [27, 28]. ECM remodeling is tightly regulated by focal adhesion, which mediates bidirectional physical communication between cells and the ECM [29]. During obesity, the overgrowth and altered secretion profile of adipocytes lead to and are influenced by ECM remodeling through: (1) adipose tissue fibrosis caused by excessive ECM synthesis and cross-linking, in which the TGF-β signaling pathway plays a key role [30], and (2) inflammation in adipose tissue, induced and exacerbated by ECM remodeling, resulting from local hypoxia and mechanical stress [27]. ECM remodeling is also intricately regulated by insulin and metabolism, with the PI3K signaling pathway playing a central role. Insulin binding to adipocyte receptors stimulates glucose uptake and glycolysis via the PI3K-Akt-mTORC1/GSK3 pathways and promotes lipid synthesis while inhibiting lipolysis via the PI3K-AKT-SREBP−1c/FOXO1 pathways. Dysfunctional adipocytes result in insulin resistance and metabolic disorders [31,32,33].

The upregulated differential proteins and acetylated proteins in SA were predominantly enriched in ECM remodeling-related pathways, while those in VA were enriched mainly in metabolic pathways. These findings confirmed the existence of differential expression and acetylation regulation patterns between SA and VA in MUO, highlighting the superior expansion and remodeling ability of SA and the poor adaptability of VA, which leads to rapid functional deterioration. Enhancing the ECM microenvironment in SA to sustain its energy storage capacity and compensatory role in obesity, while mitigating lipid toxicity by promoting remodeling regulation in VA, may represent potential intervention strategies for MUO. Notably, upregulated Kac sites in VA were significantly enriched in the COVID-19 pathway. Previous transcriptomic studies have shown that a history of COVID-19 can affect the transcriptome of WAT with depot-specific differences [34]. Our study provided protein-level evidence for the differential effects of COVID-19 on distinct WAT depots and underscored the critical role of acetylation modifications.

COL6A1, COL6A3, and ITGA5 were validated as potential targets for regulating the ECM remodeling process, as they were upregulated in SA compared to VA and overlapped with both our dataset and previous proteomic studies [23]. COL6A1 and COL6A3 are predominant ECM constituents, playing critical roles in regulating fibrotic changes, tissue rigidity, inflammatory responses, adipocyte expandability, and insulin sensitivity in WAT [28, 30, 35]. ITGA5, a member of the integrin family, acts as a fibronectin receptor in the ECM and is involved in adipocyte differentiation, ECM remodeling, inflammatory microenvironment modulation, and metabolic regulation in WAT [36, 37]. Elevated COL6 expression in SA has been reported in patients with type 2 diabetes (T2D) [23], while increased ITGA5 expression in SA has been observed in obese individuals [38]. Overlapping analyses reinforced the existence of differential regulation in ECM remodeling processes, primarily mediated by fibranexin and integrin, between SA and VA in obese individuals. Our findings further confirmed that these differential regulations were influenced not only by protein expression levels but also by acetylation modifications, emphasizing the critical role of post-translational Kac modifications in obesity and metabolic disorders.

Notably, this study identified 599 acetylated proteins in adipose tissue that were not present in a previous dataset from obese patients [23]. These acetylated proteins were enriched in inflammation- and immune-related pathways, further highlighting the critical role of inflammation in adipose tissue in the development of obesity-associated metabolic disorders, consistent with prior studies [5, 27]. Our study confirmed that inflammation in WAT is closely correlated with acetylation modifications and provided potential acetylation modification targets. Future studies could explore effective interventions for MetS in obese populations by focusing on Kac sites enriched in ECM remodeling and inflammation-inducing pathways in WAT.

Histone acetylation is critical for adipose tissue expansion, lipid metabolism, and insulin resistance, making it a potential target for preventing obesity and related metabolic abnormalities [21, 39, 40]. Our analysis revealed that differentially modified Kac sites on histones, including H1.2K63, H1XK90, and H3.7K80, were significantly upregulated in VA. H1.2K63 has been identified as a non-proteolytic ubiquitylation target triggered by DNA double-strand breaks, facilitating the recruitment of DNA repair factors [41]. In contrast, the Kac modification of H1X and H3.7 has not been comprehensively investigated. Glucosamine NDST1 was identified as a key enzyme associated with these differentially acetylated histone Kac sites. NDST1 may regulate intracellular lipid metabolism and autophagy by controlling heparan sulfate chain modifications [42], and reduce fibrosis in adipose-derived stem cells when overexpressed [43]. While NDST1 represents a potential intervention target for MUO, the role of NDST1-associated histone Kac sites, such as H1XK90, in chromatin remodeling and metabolic regulation requires further investigation.

Metabolic syndrome represents a cluster of interconnected metabolic risk factors, often varying significantly among individuals. Our findings suggested that adipose tissue-specific pathways were relatively robust and function independently of systemic metabolic variability, as evidenced by the high intercorrelation of protein abundance and acetylation profiles among subjects with distinct metabolic characteristics. Consistent with recent transcriptomic datasets of adipose tissue in MHO and MUO populations [44], our findings highlighted shared regulatory mechanisms within adipose tissue and emphasized the necessity for further analyses at the single-cell level. Integrating our findings with prior proteomic datasets [23, 45], we proposed that mitochondrial dysfunction related to non-acetylated proteins and inflammation dysregulation related to acetylated proteins were key molecular characteristics of MUO adipose tissue. These processes collectively contributed to metabolic dysregulation through impaired energy metabolism, oxidative stress, and chronic low-grade inflammation. Targeting these pathways, especially depot-specific acetylation modifications in adipose tissue, may represent a promising therapeutic approach for managing metabolic disorders in MUO patients.

This study has several limitations. A limited sample size of male patients was analyzed, avoiding the effects of gender but reducing the generalizability of our findings. Additionally, the lack of multiple testing corrections increased the risk of false positives but ensured that potential targets were not prematurely excluded. This study served as an initial exploration to identify potential targets and generate hypotheses for future research. Validation and mechanistic studies, though important, were not included. Future studies with larger, more diverse cohorts, including female participants, are needed to validate these findings and enhance their generalizability.