Obesity is a global health crisis linked to numerous chronic diseases, including type 2 diabetes, cardiovascular diseases, and cancer. It arises from an energy imbalance, involving dysfunctional adipose tissue accumulation. Adipose tissue comprises mature adipocytes and stromal vascular fractions (SVFs), including adipocyte progenitor cells (APCs). Adipogenesis, the formation of adipocytes from preadipocytes, is a crucial process in obesity development, regulated by transcription factors like PPARγ and SMAD4. Annexin A1 (ANXA1), an anti-inflammatory protein, is upregulated in the adipose tissue of obese individuals. While previous research suggested an anti-obesity effect through anti-inflammatory actions, the specific role of ANXA1 in adipogenesis remained unclear. This study aimed to elucidate the role of ANXA1 in adipogenesis and its impact on obesity.

Existing literature highlights the significance of adipogenesis in obesity and related metabolic disorders. Studies have explored the role of various transcription factors and signaling pathways in regulating adipogenesis. ANXA1's anti-inflammatory properties have been linked to its protective effects against various diseases. However, its specific role in adipogenesis and obesity, particularly concerning the regulation of adipocyte differentiation from preadipocytes in the context of SVFs, has not been fully understood.

The study utilized multiple approaches: Analysis of ANXA1 mRNA and protein levels in human and mouse adipose tissue (subcutaneous, visceral, brown) from obese and lean individuals and mice fed with high-fat diets (HFD) or normal chow diet (NCD). Generation of whole-body and adipose-specific ANXA1 knockout (*Anxa1^ko*, *Anxa1^ΔKO*) and transgenic (*Anxa1^Tg*) mice. Metabolic phenotyping of these mice, including body weight, energy expenditure, glucose tolerance tests, insulin tolerance tests, and lipid profile analyses. Isolation and analysis of stromal vascular fractions (SVFs) from adipose tissue. In vitro experiments involving SVFs included manipulation of ANXA1 levels (siRNA, overexpression), adipogenic differentiation assays (Oil Red O staining), and gene expression analysis (qPCR). Identification of ANXA1-interacting proteins using immunoprecipitation coupled with mass spectrometry (IP-MS). Co-immunoprecipitation (co-IP) and co-localization studies to investigate protein-protein interactions. Ubiquitination assays to examine SMAD4 ubiquitination levels. In vivo treatment with Ac2-26, an ANXA1-derived peptide, in db/db mice. In vivo treatment with GW9662, a PPARγ antagonist in ANXA1 knockout mice. Statistical analysis was performed using appropriate methods for each experiment.

1. ANXA1 mRNA and protein levels were significantly elevated in the subcutaneous adipose tissue (SAT) of obese humans and mice fed a HFD. 2. Whole-body or adipose-specific ANXA1 knockout in mice led to aggravated obesity, metabolic disorders, glucose intolerance, and insulin resistance compared to control mice. 3. ANXA1 overexpression in mice conferred resistance to HFD-induced obesity and improved metabolic parameters. 4. ANXA1 levels decreased during SVFs differentiation into mature adipocytes. 5. ANXA1 overexpression in SVFs suppressed adipogenesis and decreased the expression of lipogenesis-related genes. Conversely, ANXA1 knockdown enhanced adipogenesis. 6. ANXA1 promoted SMAD4 ubiquitination and degradation via the proteasome pathway. 7. ANXA1 interacted with PDLIM7, inhibiting the interaction between PDLIM7 and MYCBP2. 8. PDLIM7 inhibited MYCBP2-mediated SMAD4 ubiquitination. 9. Ac2-26, an ANXA1 peptide, mimicked the effects of ANXA1, inhibiting adipogenesis and obesity in mice. 10. In vivo treatment with the PPARγ antagonist GW9662 ameliorated obesity and metabolic dysfunction in adipose tissue specific ANXA1 knockout mice. 11. The interaction between ANXA1, PDLIM7, MYCBP2, and SMAD4 was confirmed in both SVFs and mature adipocytes as well as in the H5V cell line.

This study provides novel insights into the role of ANXA1 in obesity and its underlying mechanisms. The findings demonstrate that ANXA1 inhibits adipogenesis by regulating SMAD4 protein levels through a complex interplay of protein-protein interactions involving PDLIM7 and MYCBP2. The competitive binding of ANXA1 to PDLIM7 prevents the interaction of PDLIM7 with MYCBP2, leading to increased MYCBP2-mediated ubiquitination and degradation of SMAD4. Reduced SMAD4 levels subsequently downregulate PPARγ transcription, thus inhibiting adipogenesis and mitigating obesity. The efficacy of Ac2-26 in preventing obesity further supports the therapeutic potential of targeting the ANXA1 pathway. The study also highlights the importance of considering both the anti-inflammatory and anti-adipogenic actions of ANXA1 when assessing its role in obesity.

This research reveals a novel mechanism by which ANXA1 inhibits adipogenesis and prevents obesity. ANXA1's interaction with PDLIM7 disrupts the PDLIM7-MYCBP2 interaction, increasing SMAD4 degradation and reducing PPARγ activity. The efficacy of Ac2-26 suggests a potential therapeutic avenue. Future studies could explore the therapeutic implications of ANXA1 modulation and further elucidate the roles of PDLIM7 and MYCBP2 in adipogenesis and obesity.

The study primarily used mouse models. Further research is needed to confirm these findings in humans. The precise mechanisms of ANXA1 trafficking and its interaction with vesicles require further investigation. While the study focused on the K48-linked ubiquitination of SMAD4, additional research could explore other ubiquitination types and their roles in this pathway.