Obesity-related metabolic disease is linked to impaired adipose tissue function, but the underlying molecular programs are difficult to assign to specific adipose-resident cell types, to mechanistically connect to inflammation, and to distinguish from alterations that normalize with weight loss. We integrated here a layered design combining untargeted proteomics and lipidomics to define obesity-associated, cell-type-resolved molecular phenotypes across isolated adipocytes and adipose microvascular endothelial cells, explore whether an obesity-like inflammatory milieu reproduces adipose-resident cell dysfunction, and identify molecular features that show evidence of recovery after surgery-induced weight loss. As expected, adipocytes from people with obesity show suppression of mitochondrial energy metabolism together with impaired lipid plasticity, as reflected by triglyceride remodelling. By mimicking an obesity-like inflammatory milieu with macrophage-conditioned media, we reproduced most of these changes in adipocyte cultures. Endothelial cells exhibited yet another, opposite trajectory in obesity, with reduced cell-cycle signalling and increased mitochondrial activation, which were recapitulated in vitro when these cells were exposed, respectively, to the secretions of inflamed macrophages and adipocytes. Bulk adipose tissue proteomes and lipidomes showed evidence of metabolic improvement after weight loss, with broad restoration of mitochondrial and substrate-handling pathways and reciprocal triglyceride remodelling. Alongside the inflammation-responsive adipocyte mitochondrial and lipid-handling dysfunction, our cell-type-informed framework probes macrophage and adipocyte-to-endothelial activation in obesity and delineates cross-context cellular programs that recover with weight loss. Additionally, we identified the elements that exhibit the strongest association with dyslipidaemia, hypertriglyceridemia and hyperglycaemia in individuals with obesity, confirming molecular signatures relevant to metabolic obesity in two cross-sectional samples. Vaishali Chaurasiya, Luyang Li, Aina Lluch participated equally.
Adipocyte depots throughout the body are physiologically and molecularly distinct. With age, adipocytes increase in and around aged thymi. However, thymic adipocytes completely lack molecular characterization. We developed and optimized methods to isolate adipocyte nuclei from mouse thymi of different ages and sexes. Single-nucleus multiomic analysis of male and female mice aged 4-9 months reveals that thymic adipocytes are heterogeneous, with at least two distinct populations. One subpopulation harbors a transcription and chromatin signature consistent with beige/brown fat. A larger subpopulation more strongly resembles classic white adipose tissue and expresses genes associated with epithelial-to-mesenchymal transition (EMT) and antigen presentation. Analysis of differentially open chromatin in the white compared to beige adipose population identifies binding sites for Foxn1 and HIF-1α/Arnt, consistent with a situation in which thymic white adipose cells emerge from thymic epithelial cells, possibly under hypoxic conditions. Immunofluorescence microscopy confirmed the expression of UCP1 protein in cells within the thymic parenchyma, most prominently in subcapsular cortical regions. This resource reveals a complex milieu of thymic adipocytes and identifies multiple avenues for directly probing their ontogeny, dynamics and functional significance.
The circadian clock maintains temporal control of metabolic processes and exerts a key role in adipocyte development. Discovery of clock-modulatory compounds may provide new avenues for metabolic disease therapy. Here we report the identification of flavonoid compounds, Quercetin and Fisetin, as clock-activating molecules with direct inhibitory action on adipogenesis and adipocyte lipid metabolism. Quercetin and Fisetin displayed robust RORα agonism that promoted clock oscillation with induction of clock genes. Treating preadipocytes with these compounds blocked their adipogenic differentiation. In mature adipocytes, Quercetin and Fisetin suppressed lipid accumulation by inhibiting lipogenic enzymes. Furthermore, activation of RORα by a synthetic agonist or ectopic expression were sufficient to inhibit adipogenesis. In mice treated with Quercetin or Fisetin, RORα was markedly induced in adipose depots with strong suppression of the adipogenic and lipogenic programs. While quercetin significantly attenuated lipid storage in adipose tissue in vivo accompanied with lowering of free fatty acids and improved insulin sensitivity, fisetin displayed a less robust effect with differential regulation of lipolytic pathway. Collectively, these findings uncovered the clock-activating properties of quercetin and fisetin that prevent adipocyte maturation and hypertrophy to limit adipose tissue expansion. These actions contribute, at least in part, to their beneficial effects on metabolic disorders.
Maladaptive interactions between adipocytes and immune cells drive obesity-associated inflammation, yet pharmacological interventions targeting this mechanism remain scarce. Here, we identify a disease-emergent, spatially organized, and conserved immunometabolic module within the obese white adipose tissue (WAT) microenvironment, reflecting the coordinated crosstalk between adipocytes and macrophages. By integrating single-nucleus RNA sequencing, spatial transcriptomics, and bulk RNA-seq across 247,406 nuclei from 489 individuals, we identified a transcriptionally distinct population of adipocytes with high end-of-trajectory signature (hEOS). This population, characterized by impaired adipokine secretion and defective insulin signaling, re-emerges in obesity and contributes to pathogenic WAT remodeling. These hEOS cells preferentially colocalize with a conserved macrophage subset (Mac3) within discrete niches, forming a co-adapted spatial unit that contributes to maladaptive inflammation. Mechanistically, HIF1A activation in hEOS under hypoxic conditions is associated with increased expression of the extracellular matrix protein LAMA4, which contribute to niche remodeling and is spatially associated with Mac3 macrophages. Our data further suggest that LAMA4 signals through an ITGB1-mediated pathway to promote NF-κB activation in Mac3 cells. The overall activity of this axis correlates with BMI, HbA1c, and insulin resistance in humans. Importantly, pharmacological intervention with the IKKε/TBK1 inhibitor amlexanox improved metabolic dysfunction in vivo, and this was accompanied by reduced adipocyte Lama4 expression and suppressed pro-inflammatory macrophage activation, consistent with attenuation of the hEOS-Mac3 module. Together, these findings define hEOS adipocytes as a disease-emergent, pharmacologically responsive population that is implicated in maladaptive adipocyte-macrophage crosstalk and prioritize the LAMA4-integrin-NF-κB axis as a potential therapeutic target.
The prevalence of obesity and its metabolic consequences have prompted researchers to pay more attention to adipose tissue biology, the mechanisms driving adipocyte proliferation, and the process of adipogenesis. The development of diverse in vitro cell models and molecular biology techniques has made it possible to investigate adipocyte commitment and differentiation, which are complex processes underlying adipose tissue function. These approaches enable a better understanding of adipocyte dysfunction and adipogenesis associated with obesity and its metabolic complications. To mimic white, beige, and brown adipocytes, researchers have established different cell models from animals and humans with adipogenic capacity. In this review, we have compiled and updated information on animal and human cell culture models used to study in vitro adipogenesis, including their main characteristics, protocols, and applications. We provide practical guidance on model selection for specific obesity-related questions such as insulin resistance, inflammation, browning, and depot-specific dysfunction. Additionally, we discuss co-cultures and three-dimensional culture systems, highlighting their value in creating a more physiological environment and elucidating the interactions between adipocytes and neighboring cells.
Adipose tissue plays a central role in metabolic homeostasis, and its properties are shaped during embryonic development through adipocyte differentiation. Embryonic preadipocytes, therefore, represent a relevant model to study early events in adipose tissue formation and lineage specification. This article describes a reproducible protocol for the isolation and in vitro differentiation of white and brown preadipocytes isolated from mouse embryos at embryonic day 15.5 (E15.5), a developmental stage at which adipose depots begin to form. The method provides detailed guidance for tissue microdissection, enzymatic digestion, primary culture, and lineage-specific differentiation conditions that support cell viability and adipogenic maturation. Representative results include lipid droplet accumulation and lineage-associated marker expression, confirming successful differentiation under defined culture conditions. Using this approach, embryonic preadipocytes can be directed toward white or brown adipocyte fates, enabling comparative analyses of developmental timing, lineage characteristics, and gene expression profiles. This protocol offers a practical and developmentally relevant tool for investigating adipose tissue formation and perinatal programming mechanisms in a controlled experimental setting.
The proteolytic cleavage of membrane-bound proteins, ectodomain shedding, functionally expands the reservoir of proteins/peptides available for endocrine crosstalk, and metabolic regulation. However, the functional understanding of secreted proteoforms, including whether they act synergistically or antagonistically with their membrane precursors, is often unknown. We aimed to develop a novel viral vector-based gene delivery platform enabling characterization of both membrane-bound and soluble proteoforms in adipocytes, independent of endogenous shedding. To this end, we elected amine oxidase copper-containing 3 (AOC3) as a target for validation. We describe a novel platform, termed 'EctoShed', achieving adipocyte-specific expression of different proteoforms of AOC3 - full-length and membrane-bound or a soluble AOC3 mimic (m-sAOC3) - by capitalising on the established lentiviral and adeno-associated virus gene delivery systems. In vitro transduction of primary white adipocytes induced significant expression of both isoforms, whilst retaining AOC3 enzymatic activity. In vivo delivery to inguinal white adipose tissue enabled depot-specific AOC3 expression and increased abundance of m-sAOC3 in serum. Mice expressing m-sAOC3 exhibited reduced fat mass and fasting glucose levels. Thus, EctoShed is a novel tool to dissect the functional roles of soluble proteoforms secreted from adipocytes, with broad in vitro and in vivo applications in cardiometabolic health.
Thermogenic brown adipocytes (BAs) are not only an energy sink but also regulate systemic metabolic health through secreted factors and extracellular vesicles (EVs). Here, we investigate the baseline profiles BA-EVs and how cold-induced thermogenesis alters the proteome of BA-EVs. We first established and validated transgenic mouse models expressing human CD63 (hCD63)-GFP for visualisation and isolation of EVs exclusively from adipocytes and BAs. We then used super-resolution microscopy to compare the subcellular localisation and maturation of EVs in adipocytes grown in culture or directly isolated from mice, revealing distinct features. We then used immunoprecipitation to enrich BA-EVs and employed mass spectrometry to define the EV-proteomes. We discovered that cold stimulation elevated secretion of mitochondrial and lysosomal proteins in BA-EVs. We validated the presence and activity of creatine kinase B (CKB) in BA-EVs. We finally showed that BA-EVs promote thermogenic gene expression in white adipocytes, but cold stimulation surprisingly blunted EV's effect in up-regulating Prdm16 expression. These data establish a novel system for analysing EV contents in vivo and provide insight into how EV proteomes dynamically adapt to non-shivering thermogenesis.
Obesity is recognised to be a risk factor for breast cancer since adipose tissue influences the tumour microenvironment. This study aims to investigate the effect of the secretome of 3T3-L1 adipocytes untreated or treated with liquorice root extract (LRE), containing flavonoids, phenolic acids, and saponins on MCF-7 breast cancer cells. By treating adipocytes with LRE, the secretion of certain pro-tumorigenic factors like IGFBP-6, resistin, and VEGF was reduced. MCF-7 cells exposed to conditioned medium from LRE-treated adipocytes exhibited an increase in reactive oxygen species levels, downregulation of the Nrf2 antioxidant pathway, and increased autophagy. Those conditions reduced cell viability, migration, and colony formation. Additionally, there was downregulation of genes associated with oestrogen signalling and tumour-related processes, including CYP19A1 (aromatase), ERα, Her2, and components of the renin-angiotensin system (RAS). These findings suggest that LRE can modulate the adipocyte secretome to influence breast cancer cell behaviour under obesity-related in vitro conditions.
Triple-negative breast cancer (TNBC) is characterized by highmetastatic potential and a lack of effective targeted therapies. Within the tumor microenvironment (TME) of TNBC, adipocyte can undergo transformation into cancer-associated adipocytes (CAA) through interactions with cancer cells; however, the specific role in the progress of TNBC is still not well described. This study aimed to investigate the impact of CAA on the malignant behavior of TNBC and its underlying mechanisms. CAA model was successfully established by co-culturing 3T3-L1-induced adipocytes with 4T1 cells, which exhibited characteristic features such as reduced lipid accumulation. Functional assays demonstrated that co-culture with CAA significantly enhanced the migration and invasion capabilities of 4T1 cells. In vivo experiments showed that co-injection of CAA with tumor cells accelerated primary tumor growth and promoted lung metastasis in mice. Mechanistic analysis revealed that in tumor tissues coexisting with CAA, E-cadherin expression was downregulated, accompanied by increased Ki67 expression and activation of the PI3K/AKT signaling pathway. Furthermore, CAA induces an immunosuppressive TME, characterized by elevated PD-L1 expression and reduced CD8+T cell infiltration. In conclusion, this study demonstrates that CAA promotes TNBC progression by activating epithelial-mesenchymal transition (EMT) and the PI3K/AKT pathway, as well as remodeling an immunosuppressive microenvironment, providing experimental insight into tumor-adipocyte interactions and identifying potential therapeutic targets.
Obesity and related metabolic disorders are often characterized by chronic adipose tissue inflammation, driving systemic insulin resistance and general metabolic dysfunction. Free Fatty Acid Receptor 2 (FFA2) has emerged as a potential modulator of adipocyte function, inflammation, and metabolism. To investigate the role of FFA2 expressed in the adipose tissue, we generated adipose-specific FFA2 knockout mice (Adipoq-F2-KO) and assessed metabolic outcomes under standard laboratory chow and high-fat, high-sugar Western diet conditions, with and without dietary fiber supplementation. We found that adipose-specific FFA2 deletion had minimal metabolic consequences under standard dietary conditions but significantly reduced body weight and adiposity when mice were fed a fiber (fructooligosaccharide)-supplemented Western diet. Subsequent fecal analyses and transcriptomic profiling indicated impaired intestinal lipid absorption as the primary driver of reduced adiposity, suggesting disrupted adipose-intestinal communication. Unexpectedly, the lighter Adipoq-F2-KO mice also exhibited heightened adipose inflammation, characterized by increased macrophage infiltration and pro-inflammatory cytokine expression. Furthermore, in vitro loss-of-function experiments in adipocytes revealed that FFA2 knockdown impaired adipocyte maturation, lipid storage, and anti-inflammatory signaling. Additional studies using intestinal epithelial cells exposed to adipocyte-conditioned media implicated adipose-derived signals in driving intestinal dysfunction. Collectively, our findings highlight adipose-specific FFA2 as critical in regulating adipose tissue inflammation, lipid metabolism, and inter-organ communication.
Background: The impact of overweight and adipocyte size on the development of type 2 diabetes mellitus (T2DM) remains unclear. Aim: We studied (1) the relationship between the state of adipocytes and/or overweight/obesity, the development of T2DM and its clinical and laboratory features; and (2) weight loss effect on glycemic level, endogenous hyperinsulinism (HI), insulin resistance (IR), and T2DM. Methods: We designed a systematic review by searching Web of Science, EBSCO, Scopus/ Science-Direct, Google Scholar, PubMed, Cochrane, and Wolter Kluwer for articles published in 26 years (2000-2026). The study was based on a systematic review of 3853 articles published worldwide. Results: In total, 142 full-text articles were assessed for eligibility. As overweight increases, the size of adipose tissue, adipocytes, and cell radius increase. The increase in cell size overloads intracellular transport and internal organs. The development of IR is a conformational change in cellular receptors caused by an excessive increase in cell size. The increase in cell size with overweight gradually leads to hyperglycemia and HI with the development of IR and T2DM. Any targeted intentional weight loss in patients with T2DM improves metabolic and cardiovascular health, reduces blood pressure and blood sugar, and decreases HI, IR, and T2DM. Conclusions: IR is a protective response of cells that prevents oversaturation and overflow. Overweight is an independent risk factor for the development of HI, IR, and T2DM. Targeted weight loss leads to the cure of HI, IR and T2DM.
Obesity markedly increases the risk of type 2 diabetes, highlighting the urgent need for novel therapeutic targets. The inter-alpha-trypsin inhibitor heavy chain 5 (ITIH5), which is predominantly produced by the adipose tissue, has emerged as a potential regulatory factor in obesity; however, the specific underlying mechanisms remain unclear. In this study, we identified ITIH5 as a key factor upregulated in obesity and closely associated with adipocyte differentiation and metabolic regulation. ITIH5 expression was significantly elevated in the adipose tissue of obese mice. In vitro experiments revealed that ITIH5 knockdown suppressed 3T3-L1 adipocyte differentiation, lipid accumulation, and inflammatory cytokine secretion, whereas ITIH5 overexpression markedly enhanced these effects. Mechanistically, activation of the PI3K/AKT signaling pathway was found to mediate ITIH5-induced adipogenic differentiation and lipid synthesis. Consistent with these findings, in vivo knockdown of ITIH5 in the inguinal white adipose tissue alleviated high-fat diet-induced obesity, reduced adipocyte hypertrophy, improved glucose tolerance, and increased energy expenditure. Conversely, overexpression of ITIH5 aggravated metabolic dysfunction. Collectively, these findings indicate that ITIH5 promotes adipogenesis and obesity progression via the PI3K/AKT pathway, providing a potential therapeutic target for obesity intervention.
Adipose tissue accumulation represents a significant challenge to both human metabolic health and agricultural productivity. While Ras and Rab Interactor 2 (RIN2) has been characterized as a Rab5 effector protein, its precise role in adipogenesis remains poorly defined. Here, we identified RIN2 as a novel critical regulator of adipogenesis in chicken. We found that RIN2 was widely expressed across various tissues and dynamic expression patterns during adipocyte differentiation. Through comprehensive functional analyses in both ICP-1 cells and primary preadipocytes, we identified that RIN2 overexpression enhances cellular proliferation, cell cycle progression, and adipogenic differentiation, whereas RIN2 knockdown produced contrasting inhibitory effects. Transcriptomic profiling uncovered that RIN2 functions through modulation of the cholesterol metabolic pathway and promotes fatty acid metabolism. Most notably, in vivo knockdown of RIN2 specifically reduced abdominal fat deposition without compromising other carcass characteristics. In summary, our findings identify RIN2 as a key regulator of fat accumulation and highlight its potential as a genetic target for improving poultry carcass composition.
Metabolic adaptation to both caloric excess and restriction promotes energy conservation by suppressing catabolic pathways via feedback mechanisms that remain incompletely defined. We identified TANK binding kinase 1 (TBK1) as a nutrient- and inflammation-responsive brake on AMPK signaling in adipocytes. Fasting or pharmacological AMPK activation induced Tbk1 transcription via a PGC1α/nuclear respiratory factor 1 axis, which, in turn, limited AMPK activity through a phosphorylation cascade to conserve energy. In obesity, this AMPK/TBK1 axis was disrupted due to chronically elevated basal TBK1, thereby restricting energy expenditure during fasting. Adipocyte-specific TBK1 deletion enhanced fasting-induced AMPK activation, mitochondrial function, and lipolytic gene expression in both lean and obese mice. Pharmacological TBK1 inhibition with amlexanox recapitulated these effects. Combined treatment of mice with amlexanox and the AMPK activator AICAR enhanced weight loss, improved glucose tolerance and insulin sensitivity, and suppressed inflammatory and lipogenic programs in adipose tissue, as well as fibrotic gene expression in the liver. Building on prior clinical observations linking TBK1 inhibition to metabolic health, these findings defined a nutrient-sensitive AMPK/TBK1 feedback loop that limited adipocyte catabolism and suggested that dual targeting of TBK1 and AMPK may help counteract metabolic adaptation and enhance the durability of obesity therapies.
Beige-adipocyte activity, mediated by CHRNA2, significantly influences adipose function and systemic metabolism. The CHRNB2 subunit forms a functional receptor with CHRNA2 and is essential for the response to nicotinic acetylcholine receptor agonists in beige adipocytes. Deletion of Chrnb2 in mice compromises the adaptive response to cold in subcutaneous adipose tissue and renders exacerbated metabolic dysfunction due to diet-induced obesity. This cholinergic signaling within subcutaneous adipose tissue declines with aging. CHRNB2 partial agonists, a family of drugs clinically used for smoking cessation, activate both murine and human beige adipocytes.
SERPINA12 is a member of the serpin superfamily that has been extensively studied in metabolic and inflammatory disorders. In recent years, increasing evidence has highlighted its emerging role in skin physiology and dermatological diseases. SERPINA12 is expressed in multiple skin cell types, including keratinocytes and dermal fibroblasts, where it participates in the regulation of inflammation, cellular proliferation, differentiation, and tissue homeostasis. Dysregulation of SERPINA12 has been implicated in several skin disorders. In psoriasis, altered SERPINA12 expression is associated with chronic inflammation, immune dysregulation, and abnormal keratinocyte proliferation, suggesting a potential modulatory role in psoriatic pathogenesis. Furthermore, emerging studies suggest a possible involvement of SERPINA12 in palmoplantar keratoderma, where it may contribute to aberrant keratinization and epidermal barrier dysfunction. This review summarizes current knowledge on the expression patterns, biological functions, and molecular mechanisms of SERPINA12 in the skin, with a particular focus on adipocytes, psoriasis, and palmoplantar keratoderma. Understanding the role of SERPINA12 in cutaneous biology may provide new insights into disease pathogenesis and identify potential therapeutic targets for skin disorders.
The progression of breast cancer is intricately linked to the dynamic crosstalk between tumor cells and stromal cells. Within this complex interplay, Cancer-Associated Adipocytes (CAAs) have emerged as pivotal stromal components driving breast cancer malignancy by establishing a unique "adipose-immune" interface-one that integrates adipose-derived metabolic cues with immune cell dynamics to create a niche that accelerates tumor invasion, angiogenesis, and treatment resistance. This review systematically analyzes the roles of CAAs in breast cancer pathogenesis, focusing on how CAAs regulate the Tumor Immune Microenvironment (TIME) and the Adipose Tissue Microenvironment (ATME) individually and how they influence therapeutic responses through their interplay. A particular emphasis is placed on the functional heterogeneity of CAAs across different breast cancer subtypes and metabolic contexts, and its implications for shaping immunosuppressive niches and immunotherapy resistance. Specific mechanisms include: reshaping adipokine and inflammatory cytokine profiles to foster a pro-tumorigenic secretory landscape; inducing metabolic reprogramming in tumor cells to sustain aggressive growth; mediating intercellular signaling via exosomes to propagate malignant traits; altering immune cell functional states to shift toward an immunosuppressive phenotype; and promoting the establishment of immune escape pathways. Based on these mechanisms, the review synthesizes CAA-targeted therapeutic strategies for breast cancer, including: disrupting key adipokine-mediated signaling cascades to interrupt tumor-stroma communication, modulating CAA-secreted factors to reorient immune cell activities toward anti-tumor functions, and rewiring lipid metabolic pathways in the TIME to enhance therapeutic sensitivity. In-depth dissection of CAA functional networks is crucial for elucidating their pathogenic significance in breast cancer and fueling precision immunotherapy innovation, as such insights may open avenues for rebalancing TIME immune interactions and boosting immunotherapeutic efficacy.
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