White Adipose Tissue Dysfunction Research Articles

Time-Dependent Progression of Metabolic Dysfunction in a Mouse Model of Polycystic Ovary Syndrome

Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women. PCOS is closely linked to metabolic disorders such as obesity, dyslipidemia, systemic insulin resistance (IR), adipose IR and white adipose tissue (WAT) dysfunction. Hyperandrogenism is a clinical hallmark of PCOS, and it plays a fundamental role in the development of metabolic perturbations associated with PCOS. Androgen excess is linked to metabolic dysfunction in multiple tissues, including WAT, in PCOS women. However, data on the disease onset and progression of WAT dysfunction and associated metabolic derangements in PCOS women are lacking. Therefore, we aim to test the hypothesis that the androgen dihydrotestosterone (DHT) induces time-dependent progression of metabolic dysfunction on both the molecular and functional levels in PCOS. This hypothesis will be tested using a well-characterized DHT-induced PCOS mouse model after 2, 4, 8 and 12 weeks of DHT administration. Methods: Three-week old female mice (C57BL/6N) were implanted with Silastic tubes filled with DHT (8 mg, s.c.) or vehicle (n=6/grp) for 2, 4, 8 and 12 weeks. Weekly body weight (BW, gravimetry), body composition (EchoMRI), retroperitoneal fat (RPF) mass (gravimetry) were assessed. Serum leptin, adiponectin and insulin were measured by ELISA, while cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and non-esterified free fatty acid (NEFA) were measured using a clinical chemistry analyzer. Adipose IR (insulin x NEFA) was calculated. RPF androgen receptor (AR), lipolytic marker adipose triglyceride lipase (ATGL), and adipogenesis marker peroxisome proliferator-activated receptor-γ (PPAR-γ) protein levels were assessed by Western blot. Results: DHT-treated mice showed significant (p<0.05) increases in BW (20.23±0.3 vs 17.63±0.1 g) and lean mass (17.77±0.5 vs 15.50±0.3 g) starting 2 weeks following DHT exposure where higher cholesterol (90.0±2.9 vs 70.0±5.8 mg/dL), insulin (0.51±0.08 vs 0.22±0.03 ng/mL) and adipo-IR (2.5-fold) as well as lower insulin-sensitizing adipokine adiponectin levels (4.9±0.2 vs 9.70±1.1 mg/mL) were also observed compared to vehicle. The increases in RPF mass (105.40±10.3 vs 32.78±1.6 mg) and total fat mass (2.43±0.5 vs 1.09±0.1 g) were only significant starting at weeks 4 and 8, respectively. DHT significantly increased NEFA (1.3- and 1.2-fold, 8- and 12-weeks post DHT, respectively) and leptin (1.5-fold, 12 weeks post DHT). Unexpectedly, DHT increased HDL-C (58.80±1.42 vs 41.40±1.2 mg/dL, 4 weeks post DHT), while decreasing triglycerides (76.17±5.4 vs 107.17±9.4 mg/mL) and LDL-C (6.5±0.8 vs 9.0±0.5 mg/mL) after 8 weeks post DHT. At the molecular level, DHT upregulated RPF AR expression (5-fold) and downregulated ATGL expression by 40% as early as 2 weeks post DHT, and downregulated PPARγ expression by 40% at 4 weeks post DHT indicating early onset of adipose tissue dysfunction. Conclusion and significance: Our findings suggest that some PCOS metabolic traits occur very early following the induction of hyperandrogenemia such as serum adiponectin, insulin and cholesterol, highlighting these markers as an early diagnosis potential biomarkers. The onset of WAT lipolysis and adipogenesis alterations as well as adipo-IR were also early events that may be drivers of disease progression. Therefore, actively screening and managing risk factors for metabolic derangements from early on in PCOS is critical for disease management. Supported by NIH-NIGMS P20GM121334 and NIH-NIMHD P50MD017338 grants and American Heart Association Predoctoral Fellowship 903804. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Unraveling Adipose Tissue Dysfunction: Molecular Mechanisms, Novel Biomarkers, and Therapeutic Targets for Liver Fat Deposition.

Adipose tissue (AT), once considered a mere fat storage organ, is now recognized as a dynamic and complex entity crucial for regulating human physiology, including metabolic processes, energy balance, and immune responses. It comprises mainly two types: white adipose tissue (WAT) for energy storage and brown adipose tissue (BAT) for thermogenesis, with beige adipocytes demonstrating the plasticity of these cells. WAT, beyond lipid storage, is involved in various metabolic activities, notably lipogenesis and lipolysis, critical for maintaining energy homeostasis. It also functions as an endocrine organ, secreting adipokines that influence metabolic, inflammatory, and immune processes. However, dysfunction in WAT, especially related to obesity, leads to metabolic disturbances, including the inability to properly store excess lipids, resulting in ectopic fat deposition in organs like the liver, contributing to non-alcoholic fatty liver disease (NAFLD). This narrative review delves into the multifaceted roles of WAT, its composition, metabolic functions, and the pathophysiology of WAT dysfunction. It also explores diagnostic approaches for adipose-related disorders, emphasizing the importance of accurately assessing AT distribution and understanding the complex relationships between fat compartments and metabolic health. Furthermore, it discusses various therapeutic strategies, including innovative therapeutics like adipose-derived mesenchymal stem cells (ADMSCs)-based treatments and gene therapy, highlighting the potential of precision medicine in targeting obesity and its associated complications.

Intermittent fasting promotes rejuvenation of immunosenescent phenotypes in aged adipose tissue.

The aging of white adipose tissue (WAT) involves senescence of adipose stem and progenitor cells (ASPCs) and dysregulation of immune cell populations, serving as a major driver of age-associated adipose dysfunction and metabolic diseases. Conversely, the elimination of senescent ASPCs is associated with improvements in overall health. Intermittent fasting (IF), a dietary intervention that incorporates periodic cycles of fasting and refeeding, has been reported to promote weight loss and fat mass reduction and improve glucose and insulin homeostasis in both murine and human studies. While previous studies have assessed the effects of IF on obesity-associated metabolic dysfunction, few studies have examined the aging-specific changes to ASPCs and immune cell populations in WAT. Here, we show that IF in 18-20-month-old mice reduced senescent phenotypes of ASPCs and restored their adipogenic potential. Intriguingly, IF-treated mice exhibited an increase in adipose eosinophils, which has been reported to be associated with improved WAT homeostasis and immunological fitness in aged mice. The observed cellular and metabolic changes suggest that IF may be a feasible lifestyle regimen to reduce cellular senescence which could result in attenuation of downstream aging-induced WAT dysfunction and metabolic diseases.

Obesity-induced PARIS (ZNF746) accumulation in adipose progenitor cells leads to attenuated mitochondrial biogenesis and impaired adipogenesis

White adipose tissue (WAT) is critical for whole-body energy metabolism, and its dysfunction leads to various metabolic disorders. In recent years, many studies have suggested that impaired mitochondria may contribute to obesity-related decline in adipose tissue function, but the detailed mechanisms remain unclear. To investigate these mechanisms, we carried out a comprehensive analysis of WAT from mice with diet-induced obesity. We discovered the transcription factor Parkin interactive substrate (PARIS or ZNF746), which suppresses the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a key regulator of mitochondrial biogenesis, to be accumulated in adipose progenitor cells from obese mice. Furthermore, we demonstrated that 3T3-L1 preadipocytes with overexpression of PARIS protein exhibited decreased mitochondrial biogenesis and impaired adipogenesis. Our results suggest that the accumulation of PARIS protein may be a novel component in the pathogenesis of obesity-related dysfunction in WAT.

THE INTERTISSUE COMMUNICATION BETWEEN THE HYPOTHALAMUS AND WHITE ADIPOSE TISSUE IN MAMMALIAN AGING AND LONGEVITY CONTROL

Abstract Recent studies in model organisms have demonstrated that the intertissue communications play a critical role in the regulation of aging and longevity. Our major goal is to understand the systemic regulation of aging and longevity in mammals and translate that knowledge into an effective anti-aging intervention that could make our later lives as healthy and productive as possible (“productive aging”). In mammals, we have previously demonstrated that the intertissue communication between the hypothalamus and adipose tissue, particularly mediated by extracellular vesicles-contained extracellular nicotinamide phosphoribosyltrasferase (eNAMPT), the rate-limiting NAD+ biosynthetic enzyme in mammals, functions to counteract age-associated physiological decline and promote longevity in mice. We have recently found that Ppp1r17-positive neurons in the dorsomedial hypothalamus (DMHPpp1r17 neurons) regulate white adipose tissue (WAT) function, including lipolysis and eNAMPT secretion, through the sympathetic nervous system (SNS), and the feedback loop between DMHPpp1r17 neurons and WAT plays a critical role in the regulation of aging and longevity in mice. Within DMHPpp1r17 neurons, the phosphorylation and subsequent nuclear-cytoplasmic translocation of Ppp1r17, regulated by cGMP-dependent protein kinase (PKG; Prkg1), affect gene expression regulating synaptic function, causing synaptic transmission dysfunction and impaired WAT function. Both DMH-specific Prkg1-knockdown, which suppresses age-associated Ppp1r17 translocation, and the chemogenetic activation of DMHPpp1r17 neurons significantly ameliorate age-associated WAT dysfunction, increase physical activity, decrease age-associated mortality rate, and extend lifespan. Thus, these findings clearly demonstrate the importance of the intertissue communication between the hypothalamus and WAT in mammalian aging and longevity control and provide critical insights into the development of effective anti-aging interventions.

Polymicrobial Infection Induces Adipose Tissue Dysfunction via Gingival Extracellular Vesicles

Recent studies have indicated that periodontitis promotes metabolic dysregulation and insulin resistance by affecting the function of white adipose tissue (WAT). However, the mechanisms linking periodontitis to adipose tissue dysfunction still need to be explored. Extracellular vesicles (EVs) deliver messages to distal sites and regulate their function. Also, recent studies have shown that periodontitis changes the composition of EVs in body fluids and that EVs might be one of the mechanisms underlying the relationship between periodontitis and insulin resistance. Herein, we explored the impact of polymicrobial oral infection with periodontal pathogens on the function of WAT and the role of gingival EVs (gEVs) in the process. Mice were subjected to oral inoculation with 109 Porphyromonas gingivalis and 108 Fusobacterium nucleatum every other day for 14 wk. This prolonged bacterial infection induced WAT dysfunction, characterized by reduced levels of AKT phosphorylation, adiponectin, leptin, and genes associated with adipogenesis and lipogenesis. We successfully isolated gEVs with satisfactory yield and purity. The RNA sequencing results showed that the differentially expressed microRNAs in the gEVs of mice with polymicrobial oral infection were involved in insulin signaling and adipose tissue function. Notably, our in vitro experiments and RNA sequencing results revealed the functional similarities between gEVs and plasma-derived EVs. Furthermore, intraperitoneal injection with gEVs derived from mice with oral infection induced the dysfunction of WAT in healthy mice. Overall, our findings provide evidence for the influence of polymicrobial oral infection on WAT function and propose gEVs as a novel pathway through which periodontal infection may exert its effects on WAT.

High-throughput proteomics uncovers exercise training and type 2 diabetes-induced changes in human white adipose tissue.

White adipose tissue (WAT) is important for metabolic homeostasis. We established the differential proteomic signatures of WAT in glucose-tolerant lean and obese individuals and patients with type 2 diabetes (T2D) and the response to 8 weeks of high-intensity interval training (HIIT). Using a high-throughput and reproducible mass spectrometry-based proteomics pipeline, we identified 3773 proteins and found that most regulated proteins displayed progression in markers of dysfunctional WAT from lean to obese to T2D individuals and were highly associated with clinical measures such as insulin sensitivity and HbA1c. We propose that these distinct markers could serve as potential clinical biomarkers. HIIT induced only minor changes in the WAT proteome. This included an increase in WAT ferritin levels independent of obesity and T2D, and WAT ferritin levels were strongly correlated with individual insulin sensitivity. Together, we report a proteomic signature of WAT related to obesity and T2D and highlight an unrecognized role of human WAT iron metabolism in exercise training adaptations.

Genetic deletion of MMP12 ameliorates cardiometabolic disease by improving insulin sensitivity, systemic inflammation, and atherosclerotic features in mice

BackgroundMatrix metalloproteinase 12 (MMP12) is a macrophage-secreted protein that is massively upregulated as a pro-inflammatory factor in metabolic and vascular tissues of mice and humans suffering from cardiometabolic diseases (CMDs). However, the molecular mechanisms explaining the contributions of MMP12 to CMDs are still unclear.MethodsWe investigated the impact of MMP12 deficiency on CMDs in a mouse model that mimics human disease by simultaneously developing adipose tissue inflammation, insulin resistance, and atherosclerosis. To this end, we generated and characterized low-density lipoprotein receptor (Ldlr)/Mmp12-double knockout (DKO) mice fed a high-fat sucrose- and cholesterol-enriched diet for 16–20 weeks.ResultsDKO mice showed lower cholesterol and plasma glucose concentrations and improved insulin sensitivity compared with LdlrKO mice. Untargeted proteomic analyses of epididymal white adipose tissue revealed that inflammation- and fibrosis-related pathways were downregulated in DKO mice. In addition, genetic deletion of MMP12 led to alterations in immune cell composition and a reduction in plasma monocyte chemoattractant protein-1 in peripheral blood which indicated decreased low-grade systemic inflammation. Aortic en face analyses and staining of aortic valve sections demonstrated reduced atherosclerotic plaque size and collagen content, which was paralleled by an improved relaxation pattern and endothelial function of the aortic rings and more elastic aortic sections in DKO compared to LdlrKO mice. Shotgun proteomics revealed upregulation of anti-inflammatory and atheroprotective markers in the aortas of DKO mice, further supporting our data. In humans, MMP12 serum concentrations were only weakly associated with clinical and laboratory indicators of CMDs.ConclusionWe conclude that the genetic deletion of MMP12 ameliorates obesity-induced low-grade inflammation, white adipose tissue dysfunction, biomechanical properties of the aorta, and the development of atherosclerosis. Therefore, therapeutic strategies targeting MMP12 may represent a promising approach to combat CMDs.

White Adipose Tissue Dysfunction: Pathophysiology and Emergent Measurements.

White adipose tissue (AT) dysfunction plays an important role in the development of cardiometabolic alterations associated with obesity. AT dysfunction is characterized by the loss of the expansion capacity of the AT, an increment in adipocyte hypertrophy, and changes in the secretion profile of adipose cells, associated with accumulation of macrophages and inflammation. Since not all people with an excess of adiposity develop comorbidities, it is necessary to find simple tools that can evidence AT dysfunction and allow the detection of those people with the potential to develop metabolic alterations. This review focuses on the current pathophysiological mechanisms of white AT dysfunction and emerging measurements to assess its functionality.

The Role of IL-13 and IL-4 in Adipose Tissue Fibrosis

White adipose tissue (WAT) fibrosis, characterized by an excess of extracellular (ECM) matrix components, is strongly associated with WAT inflammation and dysfunction due to obesity. Interleukin (IL)-13 and IL-4 were recently identified as critical mediators in the pathogenesis of fibrotic diseases. However, their role in WAT fibrosis is still ill-defined. We therefore established an ex vivo WAT organotypic culture system and demonstrated an upregulation of fibrosis-related genes and an increase of α-smooth muscle actin (αSMA) and fibronectin abundance upon dose-dependent stimulation with IL-13/IL-4. These fibrotic effects were lost in WAT lacking il4ra, which encodes for the underlying receptor controlling this process. Adipose tissue macrophages were found to play a key role in mediating IL-13/IL-4 effects in WAT fibrosis as their depletion through clodronate dramatically decreased the fibrotic phenotype. IL-4-induced WAT fibrosis was partly confirmed in mice injected intraperitoneally with IL-4. Furthermore, gene correlation analyses of human WAT samples revealed a strong positive correlation of fibrosis markers with IL-13/IL-4 receptors, whereas IL13 and IL4 correlations failed to confirm this association. In conclusion, IL-13 and IL-4 can induce WAT fibrosis ex vivo and partly in vivo, but their role in human WAT remains to be further elucidated.

Impaired white adipose tissue fatty acid metabolism in mice fed a high-fat diet worsened by arsenic exposure, primarily affecting retroperitoneal adipose tissue

Fatty acid (FA) metabolism dysfunction of white adipose tissue (WAT) underlies obesity and insulin resistance in response to high calorie intake and/or endocrine-disrupting chemicals (EDCs), among other factors. Arsenic is an EDC that has been associated with metabolic syndrome and diabetes. However, the combined effect of a high-fat diet (HFD) and arsenic exposure on WAT FA metabolism has been little studied.FA metabolism was evaluated in visceral (epididymal and retroperitoneal) and subcutaneous WAT of C57BL/6 male mice fed control or HFD (12 and 40% kcal fat, respectively) for 16 weeks together with an environmentally relevant chronic arsenic exposure through drinking water (100 μg/L) during the second half of the study.In mice fed HFD, arsenic potentiated the increase of serum markers of selective insulin resistance in WAT and fatty acid re-esterification and the decrease of the lipolysis index. Retroperitoneal was the WAT most affected, where the combination of arsenic and HFD in contrast to HFD, generated higher adipose weight, larger adipocytes, increased triglyceride content, and decreased fasting stimulated lipolysis evidenced by lower phosphorylation of HSL and perilipin. At the transcriptional level, arsenic in mice fed either diet downregulated genes involved in fatty acid uptake (LPL, CD36), oxidation (PPARα, CPT1), lipolysis (ADRß3) and glycerol transport (AQP7 and AQP9). Additionally, arsenic potentiated hyperinsulinemia induced by HFD, despite a slight increase in weight gain and food efficiency.Thus, the second hit of arsenic in sensitized mice by HFD worsens fatty acid metabolism impairment in WAT, mainly retroperitoneal, along with an exacerbated insulin resistance phenotype.

Adipocyte-specific FXR-deficiency protects adipose tissue from oxidative stress and insulin resistance and improves glucose homeostasis

ObjectiveObesity is associated with metabolic dysfunction of white adipose tissue (WAT). Activated adipocytes secrete pro-inflammatory cytokines resulting in the recruitment of pro-inflammatory macrophages, which contribute to WAT insulin resistance. The bile acid (BA)-activated nuclear Farnesoid X Receptor (FXR) controls systemic glucose and lipid metabolism. Here, we studied the role of FXR in adipose tissue function. MethodsWe first investigated the immune phenotype of epididymal WAT (eWAT) from high fat diet (HFD)-fed whole-body FXR-deficient (FXR−/−) mice by flow cytometry and gene expression analysis. We then generated adipocyte-specific FXR-deficient (Ad-FXR−/−) mice and analyzed systemic and eWAT metabolism and immune phenotype upon HFD feeding. Transcriptomic analysis was done on mature eWAT adipocytes from HFD-fed Ad-FXR−/− mice. ResultseWAT from HFD-fed whole-body FXR−/− and Ad-FXR−/− mice displayed decreased pro-inflammatory macrophage infiltration and inflammation. Ad-FXR−/− mice showed lower blood glucose concentrations, improved systemic glucose tolerance and WAT insulin sensitivity and oxidative stress. Transcriptomic analysis identified Gsta4, a modulator of oxidative stress in WAT, as the most upregulated gene in Ad-FXR−/− mouse adipocytes. Finally, chromatin immunoprecipitation analysis showed that FXR binds the Gsta4 gene promoter. ConclusionsThese results indicate a role for the adipocyte FXR-GSTA4 axis in controlling HFD-induced inflammation and systemic glucose homeostasis.

Inhibition of VEGF-B signaling prevents non-alcoholic fatty liver disease development by targeting lipolysis in the white adipose tissue

Hepatic steatosis is a hallmark of non-alcoholic fatty liver disease (NAFLD), a common comorbidity in type 2 diabetes mellitus (T2DM). The pathogenesis of NAFLD is complex and involves the crosstalk between the liver and the white adipose tissue (WAT). Vascular endothelial growth factor B (VEGF-B) has been shown to control tissue lipid accumulation by regulating the transport properties of the vasculature. The role of VEGF-B signaling and the contribution to hepatic steatosis and NAFLD in T2DM is currently not understood. C57BL/6J mice treated with a neutralizing antibody against VEGF-B, or mice with adipocyte-specific overexpression or under-expression of VEGF-B (AdipoqCre+/VEGF-BTG/+ mice and AdipoqCre+/Vegfbfl/+mice) were subjected to a 6-month high-fat diet (HFD), or chow-diet, whereafter NAFLD development was assessed. VEGF-B expression was analysed in WAT biopsies from patients with obesity and NAFLD in a pre-existing clinical cohort (n= 24 patients with NAFLD and n= 24 without NAFLD) and correlated to clinicopathological features. Pharmacological inhibition of VEGF-B signaling in diabetic mice reduced hepatic steatosis and NAFLD by blocking WAT lipolysis. Mechanistically we show, by using HFD-fed AdipoqCre+/VEGF-BTG/+ mice and HFD-fed AdipoqCre+/Vegfbfl/+mice, that inhibition of VEGF-B signaling targets lipolysis in adipocytes. Reducing VEGF-B signaling ameliorated NAFLD by decreasing WAT inflammation, resolving WAT insulin resistance, and lowering the activity of the hormone sensitive lipase. Analyses of human WAT biopsies from individuals with NAFLD provided evidence supporting the contribution of VEGF-B signaling to NAFLD development. VEGF-B expression levels in adipocytes from two WAT depots correlated with development of dysfunctional WAT and NAFLD in humans. Taken together, our data from mouse models and humans suggest that VEGF-B antagonism may represent an approach to combat NAFLD by targeting hepatic steatosis through suppression of lipolysis. Non-alcoholic fatty liver disease (NAFLD) is a common comorbidity in type 2 diabetes mellitus (T2DM) and has a global prevalence of between 25-29%. There are currently no approved drugs for NAFLD, and given the scale of the ongoing diabetes epidemics, there is an urgent need to identify new treatment options. Our work suggests that VEGF-B antagonism may represent an approach to combat NAFLD by targeting hepatic steatosis through suppression of lipolysis. The neutralizing anti-VEGF-B antibody, which was used in this study, has already entered clinical trials for patients with diabetes. Therefore, we believe that our results are of great general interest to a broad audience, including patients and patient organizations, the medical community, academia, the life science industry and the public.

Zinc finger protein 251 deficiency impairs glucose metabolism by inducing adipocyte hypertrophy

Zinc finger protein (ZFP) 251 is a member of the C2H2 ZFP family containing a Krüppel-associated box domain that might mainly act as a transcriptional repressor. However, its cellular function remains largely unknown. Here, we discovered that ZFP251 deficiency caused glucose intolerance in mice. This phenotype was associated with impaired insulin signaling due to hypertrophic changes in white adipose tissue (WAT). Gene ontology analysis revealed that ZFP251 deficiency affected the expression of genes associated with adipocyte differentiation and lipid and fatty acid metabolism. Consistent with in vivo results, hypertrophic changes were observed in Zfp251 knockdown (KD) 3T3-L1 adipocytes. In addition, Zfp251 KD 3T3-L1 preadipocytes exhibited cell cycle arrest in G0/G1 phase, leading to impaired differentiation into mature adipocytes, upon which abnormal mitotic clonal expansion and reduced expression of adipogenic markers were exhibited. These results suggest that ZFP251 deficiency causes impaired adipogenesis and adipocyte hypertrophy, leading to dysfunction of WAT.

RF10 | PMON249 MicroRNA-21 Ablation Exacerbates Androgen-Mediated White Adipose Tissue Dysfunction and Impaired Insulin Signaling in a Mouse Model of Polycystic Ovary Syndrome

Abstract Background Polycystic ovary syndrome (PCOS) is characterized by oligo- or anovulation, polycystic ovaries, and hyperandrogenism. PCOS is associated with white adipose tissue (WAT) expansion and dysfunction, which has been linked to impaired insulin signaling in a number of metabolic diseases. MicroRNAs dysregulation in WAT is involved in the development of obesity and its associated complications. MicroRNA-21 (miR-21), the most abundant circulatory microRNA in women with PCOS, regulates androgen receptor (AR) expression in prostate cancer however, its role in WAT dysfunction in PCOS, and the associated systemic insulin resistance, is unknown. Using a well-established PCOS model of hyperandrogenemia we aim to test the hypothesis that miR-21 regulates AR expression to modulate androgen-mediated WAT dysfunction and the associated local and systemic insulin resistance. Methods Three-week-old miR-21 knockout (miR-21KO) or wild type (WT) female mice were treated with dihydrotestosterone (DHT, 8 mg/Silastic tube) or vehicle for 90 days (n=6/group). Body composition (EchoMRI) and oral glucose tolerance test (OGTT) were assessed. HOMA-IR index was calculated from fasting serum glucose and insulin levels. Retroperitoneal fat (RPF), a WAT depot that is linked to poor plasma glycemic indices and systemic inflammation, was collected to assess adipocyte size and perform molecular determinations. RPF miR-21 levels, as well as gene and protein expression of AR, and insulin signaling markers (IRS-1/2, insulin receptor-β, PI3K, AKT, Glut4, GSK-α, PTEN) were quantified by RT-qPCR and Western blot. Results In WT mice, DHT increased body weight (25.07 ± 0.52 vs 21.79 ± 0.47 g, p<0.05), fat mass (4.60 ± 0.46 vs 1.98 ± 0.12 g, p<0.05), RPF mass (29.68 ± 3.39 vs. 172.64 ± 32.75 g, p<0.05), RPF miR-21 (2.5-fold) and AR expression (2.1-fold). Adipocyte size analysis revealed a hypertrophic response to DHT, which was associated with impaired OGTT (186.10 ± 5.99 vs 250.70 ± 14.76 mg. min/dL, p<0.05) and downregulation of IRS-1/2, insulin receptor-β, and AKT, indicating impaired insulin signaling. MiR-21 ablation had no effect on DHT-mediated total fat or RPF mass increases, but it exacerbated DHT-mediated AR upregulation, RPF hypertrophy, and insulin resistance as measured by the HOMA-IR index. DHT-mediated reduction in GSK-α, which mediates obesity-induced inflammation, was abolished in miR-21KO mice. Glut4 protein was only downregulated in miR-21KO DHT-treated mice, despite DHT downregulating Glut4 mRNA in both strains. Furthermore, DHT only upregulated PTEN, a negative insulin signaling regulator and known target of miR-21 in miR-21KO mice. Conclusion and significance: These findings imply that WAT miR-21 plays a protective role in PCOS by alleviating androgen-mediated adipose tissue structural and molecular derangements, as well as the associated local and systemic insulin resistance via downregulating AR. Modulation of miR-21 levels in adipose tissue could be a novel therapeutic approach for the treatment of PCOS-related metabolic derangements. Presentation: Saturday, June 11, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

CLSTN3 gene variant associates with obesity risk and contributes to dysfunction in white adipose tissue

ObjectiveWhite adipose tissue (WAT) possesses the remarkable remodeling capacity, and maladaptation of this ability contributes to the development of obesity and associated comorbidities. Calsyntenin-3 (CLSTN3) is a transmembrane protein that promotes synapse development in brain. Even though this gene has been reported to be associated with adipose tissue, its role in the regulation of WAT function is unknown yet. We aim to further assess the expression pattern of CLSTN3 gene in human adipose tissue, and investigate its regulatory impact on WAT function. MethodsIn our study, we observed the expression pattern of Clstn3/CLSTN3 gene in mouse and human WAT. Genetic association study and expression quantitative trait loci analysis were combined to identify the phenotypic effect of CLSTN3 gene variant in humans. This was followed by mouse experiments using adeno-associated virus-mediated human CLSTN3 overexpression in inguinal WAT. We investigated the effect of CLSTN3 on WAT function and overall metabolic homeostasis, as well as the possible underlying molecular mechanism. ResultsWe observed that CLSTN3 gene was routinely expressed in human WAT and predominantly enriched in adipocyte fraction. Furthermore, we identified that the variant rs7296261 in the CLSTN3 locus was associated with a high risk of obesity, and its risk allele was linked to an increase in CLSTN3 expression in human WAT. Overexpression of CLSTN3 in inguinal WAT of mice resulted in diet-induced local dysfunctional expansion, liver steatosis, and systemic metabolic deficiency. In vivo and ex vivo lipolysis assays demonstrated that CLSTN3 overexpression attenuated catecholamine-stimulated lipolysis. Mechanistically, CLSTN3 could interact with amyloid precursor protein (APP) in WAT and increase APP accumulation in mitochondria, which in turn impaired adipose mitochondrial function and promoted obesity. ConclusionTaken together, we provide the evidence for a novel role of CLSTN3 in modulating WAT function, thereby reinforcing the fact that targeting CLSTN3 may be a potential approach for the treatment of obesity and associated metabolic diseases.

Positive Effects of a Mediterranean Diet Supplemented with Almonds on Female Adipose Tissue Biology in Severe Obesity.

It has been suggested that weight-loss-independent Mediterranean diet benefits on cardiometabolic health and diabetes prevention may be mediated, at least in part, through the modulation of white adipose tissue (WAT) biology. This study aimed to evaluate the short-term effects of a dietary intervention based on the Mediterranean diet supplemented with almonds (MDSA) on the main features of obesity-associated WAT dysfunction. A total of 38 women with obesity were randomly assigned to a 3-month intervention with MDSA versus continuation of their usual dietary pattern. Subcutaneous (SAT) and visceral adipose tissue (VAT) biopsies were obtained before and after the dietary intervention, and at the end of the study period, respectively. MDSA favored the abundance of small adipocytes in WAT. In SAT, the expression of angiogenesis genes increased after MDSA intervention. In VAT, the expression of genes implicated in adipogenesis, angiogenesis, autophagy and fatty acid usage was upregulated. In addition, a higher immunofluorescence staining for PPARG, CD31+ cells and M2-like macrophages and increased ADRB1 and UCP2 protein contents were found compared to controls. Changes in WAT correlated with a significant reduction in circulating inflammatory markers and LDL-cholesterol levels. These results support a protective effect of a Mediterranean diet supplemented with almonds on obesity-related WAT dysfunction.

77-OR: Native LDL Upregulate the NLRP3 Inflammasome in Human Adipose Tissue: A Mechanism for Higher Risk for Type 2 Diabetes in Subjects with HyperapoB

Background and Hypothesis: Elevated plasma apoB-lipoproteins (hyperapoB, mostly LDL) predicts the incidence of type 2 diabetes (T2D) and its risk factors including systemic activation of the interleukin-1 system (measured as plasma IL-1 receptor antagonist, IL-1Ra) . Moreover, native LDL induce white adipose tissue (WAT) dysfunction. Underlying mechanisms for these findings remain unclear. Activation of WAT NLRP3 inflammasome and secretion of IL-1β promotes WAT dysfunction and related risk factors for T2D. We hypothesized that native LDL upregulate WAT NLRP3 inflammasome/IL-1β pathway. Methods: This data represents baseline analysis of a 3-month clinical trial with omega-3 fatty acid supplementation in nondiabetic men (N=13) and women (N=27) . We assessed insulin secretion and sensitivity by Botnia clamps, postprandial fat metabolism after a high-fat-meal (68% fat) , and priming and/or activation of NLRP3 inflammasome in subject hip WAT biopsies ex vivo using native LDL, lipopolysaccharides (LPS) (priming control) and/or ATP (activation control) . Results: Incubation of WAT with native LDL (1.2 g apoB/L) increased IL-1β secretion in ATP- but not LPS-treated WAT. Subjects with higher than median plasma apoB (1.23±0.15 g/L) had higher WAT IL-1β-secretion than those with lower apoB, using multiple combination of LDL, LPS and/or ATP, independent of adiposity, macronutrient intake or plasma lipids. WAT NLRP3 mRNA expression and IL-1β secretion correlated positively with hyperinsulinemia, insulin resistance, postprandial hypertriglyceridemia and hyperchylomicronemia and plasma IL-1Ra. Adjusting for sex and WAT IL-1β-secretion or sex and plasma IL-1Ra eliminated the association of plasma apoB with these risk factors (except hypertriglyceridemia) . Conclusion: Native LDL are priming signals of WAT NLRP3 inflammasome, which may explain higher risk for T2D in subjects with hyperapoB. ClinicalTrials.gov (NCT04496154) . Disclosure S.Bissonnette: None. V.Lamantia: None. Y.Cyr: None. M.Devaux: None. R.Rabasa-lhoret: Consultant; HLS Therapeutics Inc., Pfizer Inc., Other Relationship; Abbott Diabetes, AstraZeneca, Boehringer Ingelheim International GmbH, Dexcom, Inc., Eli Lilly and Company, Insulet Corporation, Janssen Pharmaceuticals, Inc., Medtronic, Merck & Co., Inc., Novo Nordisk Canada Inc., Sanofi, Vertex Pharmaceuticals Incorporated, Research Support; Canadian Institutes of Health Research, Cystic Fibrosis Canada, Diabetes Canada, Fondation Francophone pour la Recherche en Diabète (FFRD) , JDRF, National Institutes of Health, Société Francophone du Diabète (SFD) , Speaker's Bureau; Canadian Medical & Surgical Knowledge Translation Research Group (CMS) , CPD Network, Tandem Diabetes Care, Inc. M.Chretien: None. M.Saleh: None. M.Faraj: None. Funding Canadian Institutes of Health Research (CIHR) (FRN# 123409)

High‐Fat Diet Exacerbates Androgen‐Mediated Obesity and White Adipose Tissue Hypertrophy in a Mouse Model of Polycystic Ovary Syndrome

BackgroundPolycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in reproductive age women. PCOS is characterized by hyperandrogenemia, oligo‐ or anovulation and polycystic ovaries and is associated with obesity and white adipose tissue (WAT) dysregulation. The deleterious metabolic profile of women with PCOS is exacerbated by obesity. However, the molecular mechanisms that mediate WAT dysfunction in PCOS, as well as its modulation by the diet, are poorly understood. We aim to investigate the effect of an obesogenic diet on androgen‐mediated WAT hypertrophy by assessing the adipocyte size of the different WAT depots [mesenteric fat (MF), subcutaneous fat (SCF) and retroperitoneal fat (RPF)] using a well‐established model of PCOS in mice.MethodsThree‐week old female mice (C57BL/6J) were implanted with Silastic tubes filled with the androgen dihydrotestosterone (DHT, 8.0 mg, S.C.) or vehicle (empty tubes). Animals were fed high‐fat diet (HFD; 60% kcal fat) or low‐fat diet (LFD; 10% kcal fat, sucrose‐matched) for 90 days. Weekly body weights and body composition (EchoMRI) were assessed. Animals were sacrificed and multiple WAT depots (MF, SCF and RPF) were harvested for histological analysis. Formalin‐fixed paraffin‐embedded tissues from the three WAT depots were sectioned (5 µm thick) and stained with hematoxylin and eosin. The stained sections were imaged at 40X magnification. Adipocyte size (cross‐sectional area) was determined using ImageJ software with the Adiposoft plugin [~2,000 adipocytes/section, 3 sections/animal, n = 6/group]. GraphPad Prism 8.4.2 was used for statistical analysis, and P‐values of 0.05 were considered statistically significant.ResultsLFD‐fed DHT‐treated mice showed significant increases in body weight (28.00 ± 0.60 vs. 23.59 ± 0.41 g, p<0.05), lean mass (21.70 ± 0.30 vs. 20.04 ± 0.23 g, p<0.05), and fat mass (6.20 ± 0.50 vs. 3.42 ± 0.33 g, p<0.05) compared to their vehicle‐treated controls. Adipocyte size analysis showed that LFD‐fed DHT‐ treated mice have significantly larger adipocytes when assessed in MF (1512.0 ± 15.55 vs. 833.3 ± 6.50 µm2, p<0.05), SCF (1907.0 ± 22.04 vs. 1395.0 ± 12.43 µm2, p<0.05) and RPF (2284.0 ± 26.20 vs. 1396.0 ± 11.61 µm2, p<0.05), compared to their vehicle‐treated controls. Notably, HFD‐fed DHT‐treated mice showed greater MF and RPF adipocyte size compared to HFD‐fed vehicle‐treated mice, which was not observed in SCF. Interestingly, HFD exacerbated DHT‐mediated increases in body weight (1.54‐fold), lean mass (1.17‐fold), and fat mass (3.03‐fold) compared to LFD‐fed DHT‐treated mice. Additionally, the increase in adipocyte size in all fat depots in response to DHT was greater in mice fed HFD by 1.50‐fold compared to those fed LFD.Conclusion and significanceThe hypertrophic effect of androgens on the WAT is modulated by the diet. Increased adipocyte size is associated with insulin resistance in women with PCOS. Our results may help explain why obesity is associated with a worsened metabolic profile in women with PCOS at the cellular level. Weight loss with a low‐fat diet may be an effective approach to ameliorate WAT dysfunction and the subsequent metabolic dysregulation in PCOS.

Preventing White Adipocyte Browning during Differentiation In Vitro: The Effect of Differentiation Protocols on Metabolic and Mitochondrial Phenotypes.

Mitochondrial dysfunction in white adipose tissue is strongly associated with obesity and its metabolic complications, which are important health challenges worldwide. Human adipose-derived stromal/stem cells (hASCs) are a promising tool to investigate the underlying mechanisms of such mitochondrial dysfunction and to subsequently provide knowledge for the development of treatments for obesity-related pathologies. A substantial obstacle in using hASCs is that the key compounds for adipogenic differentiation in vitro increase mitochondrial uncoupling, biogenesis, and activity, which are the signature features of brown adipocytes, thus altering the white adipocyte phenotype towards brown-like cells. Additionally, commonly used protocols for hASC adipogenic differentiation exhibit high variation in their composition of media, and a systematic comparison of their effect on mitochondria is missing. Here, we compared the five widely used adipogenic differentiation protocols for their effect on metabolic and mitochondrial phenotypes to identify a protocol that enables in vitro differentiation of white adipocytes and can more faithfully recapitulate the white adipocyte phenotype observed in human adipose tissue. We developed a workflow that included functional assays and morphological analysis of mitochondria and lipid droplets. We observed that triiodothyronine- or indomethacin-containing media and commercially available adipogenic media induced browning during in vitro differentiation of white adipocytes. However, the differentiation protocol containing 1 μM of the peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone prevented the browning effect and would be proposed for adipogenic differentiation protocol for hASCs to induce a white adipocyte phenotype. Preserving the white adipocyte phenotype in vitro is a crucial step for the study of obesity and associated metabolic diseases, adipose tissue pathologies, such as lipodystrophies, possible therapeutic compounds, and basic adipose tissue physiology.