Julia Sánchez-Ceinos

BACKGROUND: Lysine methyltransferase SET7 activates gene expression through mono- methylation of histone H3 at lysine 4 (H3K4me1) and modulates protein function via mono- methylation of non-histone proteins (Kme1). However, its role and molecular targets in endothelial dysfunction associated with cardiometabolic disorders remain unknown. METHODS: Endothelial-specific Setd7 knockout ( Setd7 EC-KO) mice were generated and endothelial function assessed in WT and Setd7 EC-KO mice after high-fat diet (HFD). Human aortic endothelial cells (HAECs) were used to investigate SET7 expression and function under metabolic stress. SET7-dependent histone and non-histone targets were identified by proteomic and ChIP analyses. Insights from these datasets guided the design of bioinformatic, molecular, and functional studies to define their regulatory mechanisms. Clinical relevance was evaluated in human arteries. RESULTS: HFD selectively increased endothelial SET7 expression in WT mouse aortas. Despite comparable metabolic abnormalities, Setd7 EC-KO mice were protected from HFD- induced endothelial dysfunction, oxidative stress, and inflammation. In HAECs, high glucose emerged as the strongest inducer of SET7 expression, promoting pro-inflammatory and pro- oxidant gene expression, monocyte adhesion, and ROS generation. These effects were reproduced by overexpression of catalytically active SET7 and reversed by its inhibition or silencing. Proteomic and ChIP analyses revealed that SET7-dependent H3K4me1 activates transcription of spliceosome components, linking aberrant mRNA splicing to endothelial inflammation and oxidative stress. Moreover, Kme1-proteomics identified endothelial nitric oxide synthase (eNOS) as a direct SET7 substrate. Bioinformatic analyses and mutagenesis experiments demonstrated that SET7-mediated mono-methylation of eNOS at K494 disrupts calmodulin (CaM) binding and impairs NO synthesis. These molecular signatures were also observed in internal mammary arteries from patients with vascular disease and hyperglycemia. CONCLUSIONS: SET7 drives endothelial dysfunction through a dual mechanism: 1) H3K4me1-dependent activation of splicing machinery triggering inflammation and oxidative stress, and 2) eNOS mono-methylation at K494 reducing NO bioavailability. Targeting SET7 may therefore represent a promising avenue to safeguard endothelial homeostasis in cardiometabolic disease. GRAPHIC A graphic abstract is available for this article.

Bariatric surgery (BS) is the most effective treatment for obesity and its related comorbidities, resulting in significant adipose tissue (AT) loss and functional recovery. However, the molecular mechanisms in the AT that drive these health benefits remain poorly understood, and predictive factors for adequate weight loss are currently needed. This study aimed to identify the proteomic signature of subcutaneous AT (SAT) linked to BS-induced weight loss and metabolic improvement, and to determine potential predictors of individual response. Sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics was performed on paired abdominal SAT samples from individuals with obesity before and after BS. Bioinformatic and correlation analyses were conducted between proteomic data and changes in anthropometric and clinical parameters. In addition, the effect of sera from BS participants on subcutaneous adipocytes was assessed in vitro. The SAT response to BS was characterised by an up-regulation of mitochondrial metabolism and a down-regulation of inflammation, which correlated with different anthropometric and circulating markers. Notably, obesity duration was associated with BS-induced weight loss. Individuals with short-standing obesity (SSO) exhibited greater proteomic remodelling, weight reduction, and clinical improvement post-BS than those with long-standing obesity (LSO). Furthermore, a regulatory axis involving miR-223-3p, glucose metabolism, and cytoskeleton reorganisation in adipocytes might be connected to the positive effects of BS in LSO. Overall, our results indicate that obesity duration is related to BS-induced weight loss through SAT proteomic remodelling. Addressing this relationship could help develop new strategies to enhance weight management and metabolic health in people living with obesity.

The adipose tissue stores excess energy in the form of neutral lipids within adipocyte lipid droplets (LDs). The correct function of LDs requires the interaction with other organelles, such as the endoplasmic reticulum (ER) as well as with LD coat-associated proteins, including Rab18, a mediator of intracellular lipid trafficking and ER–LD interaction. Although perturbations of the inter-organelle contact sites have been linked to several diseases, such as cancer, no information regarding ER–LD contact sites in dysfunctional adipocytes from the obese adipose tissue has been published to date. Herein, the ER–LD connection and Rab18 distribution at ER–LD contact sites are examined in adipocytes challenged with fibrosis and inflammatory conditions, which represent known hallmarks of the adipose tissue in obesity. Our results show that adipocytes differentiated in fibrotic conditions caused ER fragmentation, the expansion of ER–LD contact sites, and modified Rab18 dynamics. Likewise, adipocytes exposed to inflammatory conditions favored ER–LD contact, Rab18 accumulation in the ER, and Rab18 redistribution to large LDs. Finally, our studies in human adipocytes supported the suggestion that Rab18 transitions to the LD coat from the ER. Taken together, our results suggest that obesity-related pathogenic processes alter the maintenance of ER–LD interactions and interfere with Rab18 trafficking through these contact sites.

Background: Obesity is characterized by adipose tissue dysregulation and predisposes individuals to insulin resistance and type 2 diabetes. At the molecular level, adipocyte dysfunction has been linked to obesity-triggered oxidative stress and protein carbonylation, considering protein carbonylation as a link between oxidative stress and metabolic dysfunction. The identification of specific carbonylated proteins in adipose tissue could provide novel biomarkers of oxidative damage related to metabolic status (i.e prediabetes). Thus, we aimed at characterizing the subcutaneous and omental human adipose tissue carbonylome in obesity-associated insulin resistance. Methods: 2D-PAGE was used to identify carbonylated proteins, and clinical correlations studies and molecular biology approaches including intracellular trafficking, reactive oxygen species assay, and iron content were performed using in vitro models of insulin resistance. Results: The carbonylome of human adipose tissue included common (serotransferrin, vimentin, actin, and annexin A2) and depot-specific (carbonic anhydrase and α-crystallin B in the subcutaneous depot; and α-1-antitrypsin and tubulin in the omental depot) differences that point out the complexity of oxidative stress at the metabolic level, highlighting changes in carbonylated transferrin expression. Posterior studies using in vitro prediabetic model evidence alteration in transferrin receptor translocation, linked to the prediabetic environment. Finally, ligand-receptor molecular docking studies showed a reduced affinity for carbonylated transferrin binding to its receptor compared to wild-type transferrin, emphasizing the role of transferrin carbonylation in the link between oxidative stress and metabolic dysfunction. Conclusions: The adipose tissue carbonylome contributes to understanding the molecular mechanism driving adipocyte dysfunction and identifies possible adipose tissue carbonylated targets in obesity-associated insulin resistance.

Preadipocytes are crucial for healthy adipose tissue expansion. Preadipocyte differentiation is altered in obese individuals, which has been proposed to contribute to obesity-associated metabolic disturbances. Here, we aimed at identifying the pathogenic processes underlying impaired adipocyte differentiation in obese individuals with insulin resistance (IR)/type 2 diabetes (T2D). We report that down-regulation of a key member of the major spliceosome, PRFP8/PRP8, as observed in IR/T2D preadipocytes from subcutaneous (SC) fat, prevented adipogenesis by altering both the expression and splicing patterns of adipogenic transcription factors and lipid droplet-related proteins, while adipocyte differentiation was restored upon recovery of PRFP8/PRP8 normal levels. Adipocyte differentiation was also compromised under conditions of endoplasmic reticulum (ER)-associated protein degradation (ERAD) hyperactivation, as occurs in SC and omental (OM) preadipocytes in IR/T2D obesity. Thus, targeting mRNA splicing and ER proteostasis in preadipocytes could improve adipose tissue function and thus contribute to metabolic health in obese individuals.

Preadipocytes are crucial for healthy adipose tissue expansion. Preadipocyte differentiation is altered in obese individuals, which has been proposed to contribute to obesity-associated metabolic disturbances. Here, we demonstrate that impaired alternative splicing and dysregulated endoplasmic reticulum (ER)- associated protein degradation (ERAD) represent marker pathways of dysfunctional preadipocytes in obese individuals with insulin resistance (IR)/type 2 diabetes (T2D). Down-regulation of a key member of the major spliceosome, PRFP8 /PRP8, as observed in IR/T2D preadipocytes from subcutaneous (SC) fat, prevented adipogenesis by altering both the expression and splicing patterns of adipogenic transcription factors and lipid droplet-related proteins, while adipocyte differentiation was restored upon recovery of PRFP8 /PRP8 normal levels. Adipocyte differentiation was also compromised under conditions of ERAD hyperactivation, as occurs in SC and omental (OM) preadipocytes in IR/T2D obesity. Thus, targeting mRNA splicing and ER proteostasis in preadipocytes could improve adipose tissue function and thus contribute to metabolic health in obese individuals.