Human Visceral Adipose Tissue Research Articles

Butyrate and tributyrin reduce LPS-induced inflammatory cytokine production from human visceral fat.

The current obesity crisis has resulted in many people with excess adipose tissue suffering from chronic inflammation. This inflammation is largely due to the release of cytokines and chemokines from visceral fat. The aim of this study was to identify potential anti-inflammatory agents that might alleviate obesity-induced chronic inflammation. To identify agents that might alleviate this obesity-induced chronic inflammation we have developed a simple protocol for incubating intact pieces of human visceral adipose tissue in 35 mm tissue culture plates, in the presence of low-dose lipopolysaccharide (LPS) and co-incubating these samples with potential anti-inflammatory agents. RNA-Seq analysis was performed to identify enriched gene expression signatures among the most significantly differentially expressed genes. From this screen, we have identified the short-chain fatty acid (SCFA) sodium butyrate and its triacylglyceride form, tributyrin, as effective agents, significantly reducing the production of LPS-induced inflammatory cytokines and chemokines from all adipose tissue samples tested. As well, these agents appear to be non-toxic at the concentrations tested. RNA-Seq analysis has revealed that IL36γ is one of the most upregulated genes in response to LPS and one of the most downregulated when sodium butyrate is added to human fat samples stimulated with LPS. IL-36γ ELISAs confirmed this holds true at the protein level as well. These studies suggest that the short-chain fatty acid, sodium butyrate, and its triacylglyceride form, tributyrin, might alleviate the chronic inflammation that is associated with many individuals with obesity.

Abstract 178: Adipose tissue upregulates expression of RANKL in B-cell acute lymphoblastic leukemia through prostaglandin E2 signaling

Abstract Obesity increases the risk of developing cancer and worsens treatment outcome. Our lab has shown that adipose tissue contributes to B-cell acute lymphoblastic leukemia (B-ALL) resistance to chemotherapy both in vitro and in vivo. However, the mechanisms by which adipose tissue causes ALL cell resistance are complex and not completely understood. We therefore implanted obese mice (n=3) with syngeneic BCR/ABL B-ALL cells and performed single-cell RNA sequencing (scRNAseq) of ALL collected from epididymal adipose tissue vs. bone marrow. Our data uncovered a strong adipose microenvironment effect on the ALL transcriptome, identifying 577 differentially expressed genes using a log2 fold threshold of 0.25 and Bonferroni corrected p-values <0.05. Notably, we found that ALL cells in adipose tissue upregulate expression of Tnfsf11, the gene that encodes receptor activator of nuclear factor kappa beta ligand (RANKL). 93% of ALL cells in adipose tissue express RANKL compared to 33% in marrow, along with a log2 fold expression increase of 2.12 (p<1 × 10−100, Wilcoxon test). Because ALL expression of RANKL has been linked to bone resorption and CNS invasion, both of which have increased incidence in obese patients, we elected to explore this further. Ex vivo culture with human visceral adipose tissue validated ALL cell RANKL upregulation, demonstrating a 1.63 and 17.5-fold increase in gene expression after 48 hours in BV173 and RS4;11 B-ALL cell lines, respectively (n=4-6, both p<0.01, paired t test). Soluble RANKL was not detected in media from ALL cells cultured alone, detectable in media conditioned by human adipose tissue (0.018 pM), but increased in media from adipose-ALL cocultures (RS4;11: 0.065 pM, BV173: 0.317 pM, n=1). One potential candidate signal to stimulate ALL cell RANKL expression is the inflammatory mediator prostaglandin E2 (PGE2), which is known to be released by adipose tissue. Physiological concentrations of PGE2 (200 ng/mL) caused a similar increase in ALL cell RANKL gene expression as adipose tissue in both cell lines (n=6). PGE2 treatment also caused a mild resistance of ALL cells to daunorubicin chemotherapy in vitro, increasing the EC50 from 27±4 to 53±12 nM in RS4;11 (n=6, p<0.05), and from 39±11 to 62±19 nM in BV173 (n=6, p=0.06). Adipose tissue induction of RANKL expression was partially blocked by the addition of PGE2 receptor EP2 and EP4 blockers. Taken together, our results suggest that PGE2 produced by the adipose microenvironment activates ALL cell RANKL expression, which could potentially contribute to CNS invasion, bone breakdown, and chemoresistance. Citation Format: Michael Cohen, Jia Tan, Tyler Kuk, Blesscamal Nadeak, In Sook Ahn, Xia Yang, Etan Orgel, Steven D. Mittelman. Adipose tissue upregulates expression of RANKL in B-cell acute lymphoblastic leukemia through prostaglandin E2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 178.

Zinc Transporter ZnT1 mRNA Expression Is Negatively Associated with Leptin Serum Concentrations but Is not Associated with Insulin Resistance or Inflammatory Markers in Visceral Adipose Tissue.

The aim of this study was to evaluate the relationship between biomarkers of chronic inflammation, insulin resistance, and zinc transporter ZnT1 expression in human visceral adipose tissue. Visceral adipose tissue obtained from 47 adults undergoing laparoscopic surgery for cholecystectomy was used to analyze ZnT1 mRNA expression by RT-qPCR. ZnT1 mRNA levels were compared between subjects with normal weight, overweight, and obesity. A significantly lower ZnT1 expression was observed in overweight and obesity compared with normal-weight subjects (p = 0.0016). Moreover, subjects with normal weight had significantly higher serum zinc concentration (97.7 ± 13.1mg/L) than subjects with overweight (87.0 ± 12.8mg/L) and obesity (83.1 ± 6.6mg/L) (p = 0.002). Pearson test showed a positive correlation between serum zinc concentrations and ZnT1 mRNA expression in visceral adipose tissue (r = 0.323; p = 0.031) and a negative correlation with body mass index (r = - 0.358; p = 0.013). A linear regression model was used to analyze the associations between ZnT1 mRNA expression and serum zinc levels, insulin resistance (HOMA2-IR), serum adipokines (leptin and adiponectin), and serum inflammation biomarkers (tumor necrosis factor alpha, interleukin-6, and C-reactive protein). Interestingly, leptin concentrations were negatively associated with ZnT1 mRNA expression (p = 0.012); however, no significant associations were found for the rest of the analyzed variables. Future research is needed to analyze the causality of negative association between ZntT1 expression in visceral adipose tissue and leptin.

Clinical and molecular profiling of human visceral adipose tissue reveals impairment of vascular architecture and remodeling as an early hallmark of dysfunction

Adipose tissue dysfunction is more related to insulin resistance than body mass index itself and an alteration in adipose tissue function is thought to underlie the shift from metabolically healthy to unhealthy obesity. Herein, we performed a clustering analysis that revealed distinct visceral adipose tissue gene expression patterns in patients with obesity at distinct stages of metabolic dysregulation. We have built a cross-sectional cohort that aims at reflecting the evolution of the metabolic sequelae of obesity with the main objective to map the sequential events that play a role in adipose tissue dysfunction from the metabolically healthy (insulin-sensitive) state to several incremental degrees of metabolic dysregulation, encompassing insulin resistance establishment, pre-diabetes, and type 2 diabetes. We found that insulin resistance is mainly marked by the downregulation of adipose tissue vasculature remodeling-associated gene expression, suggesting that processes like angiogenesis and adaptative expansion/retraction ability suffer early dysregulation. Prediabetes was characterized by compensatory growth factor-dependent signaling and increased response to hypoxia, while type 2 diabetes was associated with loss of cellular response to insulin and hypoxia and concomitant upregulation of inflammatory markers. Our findings suggest a putative sequence of dysregulation of biological processes that is not linear and has multiple distinct phases across the metabolic dysregulation process, ultimately culminating in the climax of adipose tissue dysfunction in type 2 diabetes. Several studies have addressed the transcriptomic changes in adipose tissue of patients with obesity. However, to the best of our knowledge, this is the first study unraveling the potential molecular mechanisms associated with the multi-step evolution of adipose tissue dysfunction along the metabolic sequelae of obesity.

Positive correlation of ANGPTL8 expression in human visceral adipose tissue with body mass index

BackgroundAngiopoietin-like protein 8 (ANGPTL8) is an important regulator of lipid metabolism. We aimed to investigate the difference of ANGPTL8 expression in different depots of adipose tissues between individuals with and without obesity, and its correlation with various metabolic parameters. MethodsSubcutaneous (SAT) and visceral adipose tissue (VAT) samples were collected from patients who underwent bariatric or intra-abdominal surgery. Expression levels of ANGPTL8, monoglyceride lipase (MGL), monocyte chemoattractant protein-1 (MCP-1), leptin and adiponectin (APM1) were determined using real-time quantitative polymerase chain reaction. The correlation of ANGPTL8 expression with various metabolic parameters and other gene expression levels was analyzed using Person's correlation analysis. Logistic regression was used to establish a prediction model of obesity. ResultsTotally 330 subjects (obese: 281, non-obese: 49) were recruited. ANGPTL8 expression in VAT was significantly higher in the obesity group than in the non-obesity group (P = 0.0096). ANGPTL8 expression in VAT was positively correlated with body mass index (BMI) (r = 0.1169, P < 0.05) and was independently associated with obesity (O.R., 1.246; 95 % C.I. 1.013–21.533, P = 0.038). We also found the gene expression of ANGPTL8 in SAT and VAT was negatively correlated with APM1 expression in respective SAT and VAT. ConclusionsANGPTL8 expression levels in VAT were higher in subjects with obesity, and positively correlated with BMI. This suggests a role of ANGPTL8 in the pathophysiology of obesity and may pave the way for novel treatment target of obesity.

Adipose tissue macrophage dysfunction is associated with a breach of vascular integrity in NASH

In non-alcoholic fatty liver disease (NAFLD), monocytes infiltrate visceral adipose tissue promoting local and hepatic inflammation. However, it remains unclear what drives inflammation and how the immune landscape in adipose tissue differs across the NAFLD severity spectrum. We aimed to assess adipose tissue macrophage (ATM) heterogeneity in a NAFLD cohort. Visceral adipose tissue macrophages from lean and obese patients, stratified by NAFLD phenotypes, underwent single-cell RNA sequencing. Adipose tissue vascular integrity and breaching was assessed on a protein level via immunohistochemistry and immunofluorescence to determine targets of interest. We discovered multiple ATM populations, including resident vasculature-associated macrophages (ResVAMs) and distinct metabolically active macrophages (MMacs). Using trajectory analysis, we show that ResVAMs and MMacs are replenished by a common transitional macrophage (TransMac) subtype and that, during NASH, MMacs are not effectively replenished by TransMac precursors. We postulate an accessory role for MMacs and ResVAMs in protecting the adipose tissue vascular barrier, since they both interact with endothelial cells and localize around the vasculature. However, across the NAFLD severity spectrum, alterations occur in these subsets that parallel an adipose tissue vasculature breach characterized by albumin extravasation into the perivascular tissue. NAFLD-related macrophage dysfunction coincides with a loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. Our study describes for the first time the myeloid cell landscape in human visceral adipose tissue at single-cell level within a cohort of well-characterized patients with non-alcoholic fatty liver disease. We report unique non-alcoholic steatohepatitis-specific transcriptional changes within metabolically active macrophages (MMacs) and resident vasculature-associated macrophages (ResVAMs) and we demonstrate their spatial location surrounding the vasculature. These dysfunctional transcriptional macrophage states coincided with the loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. Our study provides a theoretical basis for new therapeutic strategies to be directed towards reinstating the endogenous metabolic, homeostatic and cytoprotective functions of ResVAMs and MMacs, including their role in protecting vascular integrity.

Abstract 11591: Mechanism of Finerenone-Induced Vasorelaxation in Arteries From Human Visceral Adipose Tissue

Introduction: Inadequate perfusion has emerged as a main determinant of adipose tissue (AT) dysfunction in obesity, sparking interest about possible ways to improve it. Among several mechanisms, mineralcorticoid receptor (MR) signaling has been shown to play a key role in the cross-talk between AT and the vasculature. Hypothesis: This study, therefore, tested the vasoactive properties of finerenone, a nonsteroidal MR antagonist, in arteries harvested from human AT. Methods: Small arteries (&lt;1 mm in diameter) were isolated from visceral AT and studied ex vivo in a wire myograph. After vessels had been contracted, changes in vascular tone were determined under different experimental conditions. Results: Finerenone elicited a concentration-dependent relaxation in arteries of obese individuals contracted with the thromboxane A2 analogue U46619 (n=9; P&lt;0001 vs. baseline); the steroidal MR antagonist potassium canrenoate, by contrast, did not induce any vasorelaxation (n=3; P&gt;0.05). The relaxing effect of finerenone on U46619-induced constriction was not different in arteries from lean (n=3) and obese subjects (P=0.155). Mechanistically, the vasorelaxing response to finerenone was not influenced by pre-incubation with the nitric oxide (NO) synthase inhibitor L-NAME (n=4; P=0.19 vs. saline), hence ruling out an involvement of NO. Interestingly, finerenone was also able to relax arteries contracted with endothelin-1 (n=7; P&lt;0.001) or with high-K + solution (n=4; P&lt;0001), as well as those contracted with the L-type Ca 2+ -channel agonist BayK8644 (n=5; P=0.007); similarly, the calcium-channel blocker nifedipine induced relaxation of arteries contracted with BayK8644 (n=3; P&lt;0.001). Conclusions: Finerenone induces NO-independent vasorelaxation in arterioles from human visceral AT, likely through antagonism of L-type Ca2 + -channels. This finding identifies a novel action of finerenone to improve AT perfusion, thereby potentially expanding the emerging observations about the cardiovascular benefits of this agent.

Human visceral adipose tissue microvascular endothelial cell isolation and establishment of co-culture with white adipocytes to analyze cell-cell communication

Communication between adipocytes and endothelial cells (EC) is suggested to play an important role in the metabolic function of white adipose tissue. In order to generate tools to investigate in detail the physiology and communication of EC and adipocytes, a method for isolation of adipose microvascular EC from visceral adipose tissue (VAT) biopsies of subjects with obesity was developed. Moreover, mature white adipocytes were isolated from the VAT biopsies by a method adapted from a previously published Membrane aggregate adipocytes culture (MAAC) protocol. The identity and functionality of the cultivated and isolated adipose microvascular EC (AMvEC) was validated by imaging their morphology, analyses of mRNA expression, fluorescence activated cell sorting (FACS), immunostaining, low-density lipoprotein (LDL) uptake, and in vitro angiogenesis assays. Finally, we established a new trans filter co-culture system (membrane aggregate adipocyte and endothelial co-culture, MAAECC) for the analysis of communication between the two cell types. EC-adipocyte communication in this system was validated by omics analyses, revealing several altered proteins belonging to pathways such as metabolism, intracellular transport and signal transduction in adipocytes co-cultured with AMvEC. In reverse experiments, induction of several pathways including endothelial development and functions was found in AMvEC co-cultured with adipocytes. In conclusion, we developed a robust method to isolate EC from small quantities of human VAT. Furthermore, the MAAECC system established during the study enables one to study the communication between primary white adipocytes and EC or vice-versa and could also be employed for drug screening.

Four-and-a-Half LIM-Domain Protein 2 (FHL2) Induces Neuropeptide Y (NPY) in Macrophages in Visceral Adipose Tissue and Promotes Diet-Induced Obesity.

Obesity is characterized by the expansion of the adipose tissue, usually accompanied by inflammation, with a prominent role of macrophages infiltrating the visceral adipose tissue (VAT). This chronic inflammation is a major driver of obesity-associated comorbidities. Four-and-a-half LIM-domain protein 2 (FHL2) is a multifunctional adaptor protein that is involved in the regulation of various biological functions and the maintenance of the homeostasis of different tissues. In this study, we aimed to gain new insights into the expression and functional role of FHL2 in VAT in diet-induced obesity. We found enhanced FHL2 expression in the VAT of mice with Western-type diet (WTD)-induced obesity and obese humans and identified macrophages as the cellular source of enhanced FHL2 expression in VAT. In mice with FHL2 deficiency (FHL2KO), WTD feeding resulted in reduced body weight gain paralleled by enhanced energy expenditure and uncoupling protein 1 (UCP1) expression, indicative of activated thermogenesis. In human VAT, FHL2 was inversely correlated with UCP1 expression. Furthermore, macrophage infiltration and the expression of the chemokine MCP-1, a known promotor of macrophage accumulation, was significantly reduced in WTD-fed FHL2KO mice compared with wild-type (wt) littermates. While FHL2 depletion did not affect the differentiation or lipid metabolism of adipocytes in vitro, FHL2 depletion in macrophages resulted in reduced expressions of MCP-1 and the neuropeptide Y (NPY). Furthermore, WTD-fed FHL2KO mice showed reduced NPY expression in VAT compared with wt littermates, and NPY expression was enhanced in VAT resident macrophages of obese individuals. Stimulation with recombinant NPY induced not only UCP1 expression and lipid accumulation but also MCP-1 expression in adipocytes. Collectively, these findings indicate that FHL2 is a positive regulator of NPY and MCP-1 expression in macrophages and herewith closely linked to the mechanism of obesity-associated lipid accumulation and inflammation in VAT. Thus, FHL2 appears as a potential novel target to interfere with the macrophage-adipocyte crosstalk in VAT for treating obesity and related metabolic disorders.

Mitochondrial sourcing of interferogenic ligands and an autoantigen in human obesity-associated metaflammation.

Visceral adipose tissue (VAT) inflammation contributes to metabolic dysregulation in obesity. VAT recruitment and activation of plasmacytoid dendritic cells (pDCs) through toll-like receptor 9 (TLR9) recognition of self-DNA, leading to induction of type I interferons, arecrucial innate triggers for this VAT inflammation. It was hypothesized that mitochondrial DNA (mtDNA) can contribute to TLR9 activation in VAT-recruited pDCs in obesity, and this study aimed to identify the carrier protein for ligand access to TLR9 and to explorewhether this also provides for a source of autoantigensin this context. VAT samples, used for gene expression studies as well as adipose explant cultures, were collected from patients with obesity (n = 54) and lean patients (n = 10). Supernatants from human pDC cultures, treated with adipose explant culture supernatants, were used for interferon α ELISA. Venous plasma, from patients with (n = 114) and without (n = 45) obesity, was used for an ELISA for autoantibodies. MtDNA from VAT in obesity, in complex with mitochondrial transcription factor A protein (TFAM), acts as interferogenic ligands for pDCs. Humoral autoreactivity against TFAM is also induced in obesity. Interferogenic ligands and an autoantigen can be sourced from dysfunctional mitochondria in VAT of humans with obesity. Further therapeutic and prognostic potential for this immune mechanism in obesity warrants exploration.

Dysregulation of Inflammation, Oxidative Stress, and Glucose Metabolism-Related Genes and miRNAs in Visceral Adipose Tissue of Women with Type 2 Diabetes Mellitus.

BACKGROUND Human visceral adipose tissue (VAT), now identified as an endocrine organ, plays a significant role in impaired fasting glucose and diabetes through the deregulated metabolism and adipogenesis of visceral adipocytes in obesity. Our study focuses on exploring the link between inflammation, oxidative stress, and glucose metabolism-associated genes with corresponding miRNAs in human visceral adipocytes and VAT from individuals with glucose metabolism disorders. MATERIAL AND METHODS We examined the expression of ATM, NFKB1, SOD2, INSR, and TIGAR, along with their related miRNAs using PCR, in two contexts:1 - During the three-stage visceral adipogenesis under normal glucose levels (5.5 millimoles), intermittent, and chronic hyperglycemia (30 millimoles).2 - In visceral adipose tissue from subjects (34 F, 18 M) with normal glucose metabolism, impaired fasting glucose, and type 2 diabetes mellitus. RESULTS Both chronic and intermittent hyperglycemia similarly influenced ATM, NFKB1, TIGAR, SOD2, INSR gene expression in visceral adipocytes, with corresponding changes in a few tested miRNAs (eg, let-7g-5p, miR-145-5p, miR-21-5p). Anthropometric and biochemical parameters led us to focus on female subjects. Our results showed transactivation of NFKB1, TIGAR, miR-10b-5p, miR-132-3p, miR-20a-5p, miR-21-5p, and miR-26a-5p exclusively in type 2 diabetes mellitus. Upregulated molecules (excluding miR-10b-5p and miR-20a-5p) positively correlated with glucose metabolism markers. CONCLUSIONS The genes studied may undergo miRNA interferences and hyperglycemic memory in visceral adipocytes under hyperglycemic conditions. VAT from women with type 2 diabetes mellitus, but not with impaired fasting glucose, showed transactivated miRNAs and a molecular dysregulation of TIGAR and NFKB1, possibly enhancing inflammation, oxidative stress, and disrupted glucose metabolism. These findings highlight the epigenetic and molecular disturbances in VAT related to glucose metabolism abnormalities. However, additional research is necessary to further understand their biological significance.

BMPER is a marker of adipose progenitors and adipocytes and a positive modulator of adipogenesis

Autocrine and paracrine signaling regulating adipogenesis in white adipose tissue remains largely unclear. Here we used single-cell RNA-sequencing (RNA-seq) and single nuclei RNA-sequencing (snRNA-seq) to identify markers of adipose progenitor cells (APCs) and adipogenic modulators in visceral adipose tissue (VAT) of humans and mice. Our study confirmed the presence of major cellular clusters in humans and mice and established important sex and diet-specific dissimilarities in cell proportions. Here we show that bone morphogenetic protein (BMP)-binding endothelial regulator (BMPER) is a conserved marker for APCs and adipocytes in VAT in humans and mice. Further, BMPER is highly enriched in lineage negative stromal vascular cells and its expression is significantly higher in visceral compared to subcutaneous APCs in mice. BMPER expression and release peaked by day four post-differentiation in 3T3-L1 preadipocytes. We reveal that BMPER is required for adipogenesis both in 3T3-L1 preadipocytes and in mouse APCs. Together, this study identified BMPER as a positive modulator of adipogenesis.

ASSESSMENT OF MACROPHAGE INFLAMMATORY ACTIVITY ON VISCERAL ADIPOSE TISSUE IN HIGH-FAT DIET-INDUCED OBESE MICE BY 18F- FDG PET/CT

Objective: Obesity induced inflamed visceral adipose tissue (VAT) secretes pro-inflammatory cytokines thereby promoting systemic inflammation and insulin resistance which further exacerbate obesity-related cardiovascular disease (CVD). Macrophages are the key players in the development of obesity-associated VAT inflammation. Extensive clinical studies have reported that 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) can be used to evaluate the macrophage inflammatory activity on VAT in human beings. However, due to difficulty in human VAT biopsy, pathologic correlations were lacking and there was a few study on preclinical animal models. Here, we investigated whether 18F-FDG PET/CT could reflect the macrophage inflammatory activity on VAT in high-fat diet-induced obese mice. Design and method: Obese animal models were induced by a high-fat diet (60% fat) for 20 weeks using the male C57BL/6 mice. Insulin tolerance test was performed to evaluate the status of insulin resistance and C-reactive protein (CRP) was measured to assess the status of systemic inflammation. All animals underwent 18F-FDG PET/CT before sacrifice. Macrophage inflammatory activity was evaluated using the maximum standardized uptake value (SUVmax). Flow cytometry-, histological-, and molecular analyses were performed on harvested VAT. Results: All obese animals showed insulin resistance which resembled the human metabolic syndrome, a key pathophysiological process that contributes to increase CVD risk. VAT SUVmax was increased in obese mice and significantly correlated with the levels of CRP. Furthermore, VAT from obese mice showed increased macrophage infiltration, compared to normal mice. Conclusions: 18F-FDG PET/CT could visualize and evaluate the macrophage inflammatory activity on obesity-driven inflamed VAT in obese mice model. Our preclinical study strongly supports the clinical application of 18F-FDG PET/CT in the assessment of VAT inflammation to patients who are vulnerable to CVD.

PKN1 Kinase: A Key Player in Adipocyte Differentiation and Glucose Metabolism.

Adipocyte dysfunction is the driver of obesity and correlates with insulin resistance and the onset of type 2 diabetes. Protein kinase N1 (PKN1) is a serine/threonine kinase that has been shown to contribute to Glut4 translocation to the membrane and glucose transport. Here, we evaluated the role of PKN1 in glucose metabolism under insulin-resistant conditions in primary visceral adipose tissue (VAT) from 31 patients with obesity and in murine 3T3-L1 adipocytes. In addition, in vitro studies in human VAT samples and mouse adipocytes were conducted to investigate the role of PKN1 in the adipogenic maturation process and glucose homeostasis control. We show that insulin-resistant adipocytes present a decrease in PKN1 activation levels compared to nondiabetic control counterparts. We further show that PKN1 controls the adipogenesis process and glucose metabolism. PKN1-silenced adipocytes present a decrease in both differentiation process and glucose uptake, with a concomitant decrease in the expression levels of adipogenic markers, such as PPARγ, FABP4, adiponectin and CEBPα. Altogether, these results point to PKN1 as a regulator of key signaling pathways involved in adipocyte differentiation and as an emerging player of adipocyte insulin responsiveness. These findings may provide new therapeutic approaches for the management of insulin resistance in type 2 diabetes.

Investigation of changes in DNA methylation associated with alterations in gene expression resulting in differences between lean and obese adipogenesis

DNA methylation (DNAm) is an important epigenetic regulator controlling various cellular activities, including cell proliferation and differentiation. In the present work, we examined alterations in DNAm associated with obesity using methylome and transcriptome data from 27 purified adipocytes (ACs) and 7 preadipocytes (preACs) in human visceral adipose tissue (VAT) samples. We identified differentially methylated probes (DMPs) using two methods: (i) DMPs that were obtained from a comparison of the DNAm levels between AC and preAC samples (AGDMPs) and (ii) DMPs that were obtained from a comparison of the DNAm levels between obese and lean AC samples (ACDMPs). These two classes of DMPs were obtained to identify a relationship between adipogenesis and obesity. We also investigated how hyper and hypomethylation of the promoter and/or gene body differentially affected changes in gene expression by estimating the odds ratios (ORs) of changes in gene expression without DMPs in the background. Interestingly, the magnitude of DNAm alterations during AC differentiation was greater under lean conditions than under obese conditions. In conclusion, several adipogenesis-related genes were affected by complicated methylation changes and ultimately cause differences in gene expression in ACs under lean and obese conditions.

Exploring Adipose Tissue Functions.

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Oleic acid regulates the circadian rhythm of adipose tissue in obesity

The effect of oleic acid (OA) on the regulation of the circadian rhythm present in human visceral (VAT) and subcutaneous (SAT) adipose tissue from patients with morbid obesity has not been analyzed yet. VAT and SAT explants from patients with morbid obesity were incubated with OA to analyze the circadian regulation of clock and other genes related to lipid metabolism (SREBP-1c, FAS, LPL and CPT1), and their association with baseline variables and the improvement of these patients after bariatric surgery. There were significant differences in amplitude and acrophase in VAT with respect to SAT. In VAT, body weight negatively correlated with BMAL1 and CRY1 amplitude, and REVERBα acrophase; body mass index (BMI) negatively correlated with REVERBα acrophase; and waist circumference negatively correlated with PER3 acrophase. In SAT, BMI negatively correlated with CLOCK amplitude, and CLOCK, REVERBα and CRY2 MESOR; and waist circumference negatively correlated with PER3 amplitude and acrophase. A greater short-term improvement of body weight, BMI and waist circumference in patients with morbid obesity after bariatric surgery was associated with a lower CRY1 and CRY2 amplitude and an earlier PER1 and PER3 acrophase in SAT. OA produced a more relevant circadian rhythm and increased the amplitude of most clock genes and lipid metabolism-related genes. OA regulated the acrophase of most clock genes in VAT and SAT, placing CLOCK/BMAL1 in antiphase with regard to the other genes. OA increased the circadian rhythmicity, although with slight differences between adipose tissues. These differences could determine its different behavior in obesity.

The relevance of EGFR, ErbB receptors and neuregulins in human adipocytes and adipose tissue in obesity

ObjectiveTo investigate the potential role of EGFR, ErbBs receptors and neuregulins in human adipose tissue physiology in obesity. MethodsGene expression analysis in human subcutaneous (SAT) and visceral (VAT) adipose tissue in three independent cohorts [two cross-sectional (N = 150, N = 87) and one longitudinal (n = 25)], and in vitro gene knockdown and overexpression experiments were performed. ResultsWhile both SAT and VAT ERBB2 and ERBB4 mRNA increased in obesity, SAT EGFR mRNA was negatively correlated with insulin resistance, but did not change in obesity. Of note, both SAT and VAT EGFR mRNA were significantly associated with adipogenesis and increased during human adipocyte differentiation. In vitro experiments revealed that EGFR, but not ERBB2 and ERBB4, gene knockdown in preadipocytes and in fully differentiated human adipocytes resulted in decreased expression of adipogenic-related genes. ERBB2 gene knockdown also reduced gene expression of fatty acid synthase in fully differentiated adipocytes. In addition, neuregulin 2 (NRG2) mRNA was associated with expression of adipogenic genes in human adipose tissue and adipocytes, and its overexpression increased expression of EGFR and relevant adipogenic genes. ConclusionsThis study demonstrates the association between adipose tissue ERBB2 and obesity, confirms the relevance of EGFR on human adipogenesis, and suggests a possible adipogenic role of NRG2.

LBSUN87 Human Cidec Transgene Improves Lipid Metabolism And Protects Against High Fat-diet Induced Glucose Intolerance In Mice

Abstract CIDEC expression in adipose tissue positively correlates with insulin sensitivity in obese humans. Further, a single-nucleotide CIDEC variant (E186X) causes lipodystrophy, hypertriglyceridemia, and insulin resistance. To establish the unknown mechanistic link between CIDEC and maintenance of systemic glucose homeostasis, we generated transgenic mouse models expressing CIDEC (Ad-CIDECtg) and CIDEC E186X variant (Ad-CIDECmut) transgene specifically in the adipose tissue. Ad-CIDECtg but not Ad-CIDECmut mice were protected against high-fat diet (HFD)-induced glucose intolerance. Transcriptomics and lipidomics revealed the role of CIDEC in lipid metabolism. Serum triglycerides, cholesterol, and low-density lipoproteins were lower in HFD-fed Ad-CIDECtg mice compared to their littermate controls. Mechanistically, CIDEC regulates the enzymatic activity of ATGL via interacting with its activator, CGI-58, to reduce free fatty acid (FFA) release and lipotoxicity. We confirmed that CIDEC is indeed a vital regulator of lipolysis in adipose tissue of obese humans. Finally, treatment with recombinant CIDEC decreased triglyceride breakdown in visceral human adipose tissue. Our study unravels a central pathway whereby adipocyte-specific CIDEC plays a pivotal role in regulating adipose lipid-metabolism and whole-body glucose homeostasis. In sum, our findings identify human CIDEC as a potential 'drug' or a 'druggable' target to reverse obesity-induced lipotoxicity and glucose intolerance. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Low miR-222 expression in human visceral adipose tissue is associated with insulin resistance and PTEN and p53 mRNA levels.

The aim of this is study was to analyse the expression of miR-193b, miR-378, miR-Let7-d, and miR-222 in human visceral adipose tissue (VAT), as well as their association with obesity, insulin resistance (IR), and their role in the regulation of genes controlling adipose tissue homeostasis, including adipocytokines, the phosphatase and tension homologue (PTEN), and tumour protein 53 (p53). VAT was obtained from normal-weight (NW), overweight, and obese (OW/OB) subjects with and without IR. Stem-loop RT-qPCR was used to evaluate miRNA expression levels. miRTarBase 4.0, miRWalk, and DIANA-TarBase v8 were used for prediction of validated target gene of the miRNA analysed. A qPCR was used to evaluate PTEN, p53, leptin (LEP), and adiponectin (ADIPOQ) mRNA. miR-222 was lower in IR subjects, and miR-222 and miR-378 negatively correlated with HOMA-IR. PTEN and p53 are miR-222 direct targets according to databases. mRNA expression of PTEN and p53 was lower in OW/OB subjects with and without IR, compared to NW group and its levels positively associated with miR-222. Additionally, p53 and PTEN are positively associated with serum leptin levels. On the other hand, miR-193b and miR-378 negatively correlated with serum leptin but not with mRNA levels. Moreover, miR-Let-7d negatively correlated with serum adiponectin but not with adiponectin mRNA levels. Lower miR-222 levels are associated with IR, and PTEN and p53 expression; the implication of these genes in adipose tissue homeostasis needs more research.