Human Omental Adipose Tissue Research Articles

Depot- and diabetes-specific differences in norepinephrine-mediated adipose tissue angiogenesis, vascular tone, collagen deposition and morphology in obesity

AimsNorepinephrine (NE) is a known regulator of adipose tissue (AT) metabolism, angiogenesis, vasoconstriction and fibrosis. This may be through autocrine/paracrine effects on local resistance vessel function and morphology. The aims of this study were to investigate, in human subcutaneous and omental adipose tissue (SAT and OAT): NE synthesis, angiogenesis, NE-mediated arteriolar vasoconstriction, the induction of collagen gene expression and its deposition in non-diabetic versus diabetic obese subjects. Materials and methodsSAT and OAT from obese patients were used to investigate tissue NE content, tyrosine hydroxylase (TH) density, angiogenesis including capillary density, angiogenic capacity and angiogenic gene expression, NE-mediated arteriolar vasoconstriction and collagen deposition. Key findingsIn the non-diabetic group, NE concentration, TH immunoreactivity, angiogenesis and maximal vasoconstriction were significantly higher in OAT compared to SAT (p < 0.05). However, arterioles from OAT showed lower NE sensitivity compared to SAT (10−8 M to 10–7.5 M, p < 0.05). A depot-specific difference in collagen deposition was also observed, being greater in OAT than SAT. In the diabetic group, no significant depot-specific differences were seen in NE synthesis, angiogenesis, vasoconstriction or collagen deposition. SAT arterioles showed significantly lower sensitivity to NE (10−8 M to 10–7.5 M, p < 0.05) compared to the non-diabetic group. SignificanceSAT depot in non-diabetic obese patients exhibited relatively low NE synthesis, angiogenesis, tissue fibrosis and high vasoreactivity, due to preserved NE sensitivity. The local NE synthesis in OAT and diabetes desensitizes NE-induced vasoconstriction, and may also explain the greater tissue angiogenesis and fibrosis in these depots.

Loss of Human Beta Cell Identity in a Reconstructed Omental Stromal Cell Environment.

In human type 2 diabetes, adipose tissue plays an important role in disturbing glucose homeostasis by secreting factors that affect the function of cells and tissues throughout the body, including insulin-producing pancreatic beta cells. We aimed here at studying the paracrine effect of stromal cells isolated from subcutaneous and omental adipose tissue on human beta cells. We developed an in vitro model wherein the functional human beta cell line EndoC-βH1 was treated with conditioned media from human adipose tissues. By using RNA-sequencing and western blotting, we determined that a conditioned medium derived from omental stromal cells stimulates several pathways, such as STAT, SMAD and RELA, in EndoC-βH1 cells. We also observed that upon treatment, the expression of beta cell markers decreased while dedifferentiation markers increased. Loss-of-function experiments that efficiently blocked specific signaling pathways did not reverse dedifferentiation, suggesting the implication of more than one pathway in this regulatory process. Taken together, we demonstrate that soluble factors derived from stromal cells isolated from human omental adipose tissue signal human beta cells and modulate their identity.

Omentin inhibits the resistin-induced hypertrophy of H9c2 cardiomyoblasts by inhibiting the TLR4/MyD88/NF-κB signaling pathway.

The recently identified adipocytokine omentin was previously found to be expressed mainly in human omental and visceral adipose tissues. As such, reduced plasma concentrations of omentin were revealed to be associated with increased risks of cardiovascular diseases. Omentin has also been previously demonstrated to exert anti-inflammatory effects. By contrast, resistin is a protein that has been associated with obesity and type-2 diabetes mellitus, and the serum concentration of resistin is increased significantly in these populations. Resistin is involved in mediating inflammation development, where they can promote cardiac hypertrophy in humans through toll-like receptor 4 (TLR4)-related signaling. In the present study, the potential effects of omentin on resistin-induced hypertrophy in H9c2 cardiomyoblasts were investigated. In the absence/presence of omentin, H9c2 cardiomyoblasts were treated with resistin. Omentin was found to significantly inhibit resistin-induced increases in the surface area of H9c2 cardiomyoblasts as determined by immunofluorescence. In addition, omentin significantly inhibited resistin-induced increases in the mRNA expression of atrial natriuretic factor, brain natriuretic peptide, β-myosin heavy chain (which is a characteristic feature of cardiac hypertrophy) and TLR4, which was determined using reverse-transcription-quantitative PCR. According to western blotting results, omentin significantly inhibited resistin-induced ERK phosphorylation, which is an important mediator of cardiomyoblast hypertrophy. Furthermore, omentin significantly inhibited resistin-induced protein expression of TLR4, myeloid differentiation factor 88 (MyD88) and NF-κB phosphorylation, both of which are important members of inflammatory signaling. To conclude, data from the present study suggest that omentin can inhibit resistin-induced H9c2 cardiomyoblast hypertrophy through inhibition of the TLR4/MyD88/NF-κB signaling pathway. Therefore, omentin serve as an attractive therapeutic target against resistin-induced cardiac hypertrophy.

Role of the DNA repair genes H2AX and HMGB1 in human fat distribution and lipid profiles

IntroductionRegional fat distribution strongly relates to metabolic comorbidities. We identified the DNA repair genes H2AX and HMGB1 to be differentially expressed between human subcutaneous (SAT) and omental visceral adipose tissue...

High Level of Palmitic Acid Induced Over-Expressed Methyltransferase Inhibits Anti-Inflammation Factor KLF4 Expression in Obese Status.

Our study is based on the establishment of a cohort of human obese omental adipose tissue and the culture of adipocytes in vitro. To observe the effect of high level of free fatty acid (FFA) on the expression of DNA methyltransferases (DNMTs) and the anti-inflammatory factor Kruppel-like factor 4 (KLF4) in adipocytes and evaluate the role of methyltransferases in FFA inhibiting KLF4 expression. A total of 20 normal patients and 20 obese patients were selected for further test. qRT-PCR and western blot were used to detect the mRNA and protein expression levels of DNMT1/DNMT3a/DNMT3b and KLF4 in human adipose tissue and 3T3-L1 adipocytes which stimulated with saturated fatty acid, palmitic acid (PA). Bisulfite sequencing PCR (BSP) detected methylation status of KLF4 gene in human adipose tissue. It was found that the mRNA and protein expression levels of DNMT1 and DNMT3a in the omental tissue of obese individuals were higher than those in normal group, but the expression of KLF4 was decreased. The positive methylation rate of KLF4 promoter region in obese individuals were significantly higher than those in normal individuals, especially at CpG_33 and CpG_34 sites. Meanwhile compared with non-methylated group at CpG_33 and CpG_34 sites of KLF4 promoter region, the DNMT3a mRNA expression in methylated group were significantly increased. A total of 200μM PA significantly promoted DNMT1, DNMT3a, and DNMT3b and inhibited KLF4 protein expression levels in 3T3-L1 adipocytes. Our findings suggest that under obesity status, the lower expression level of KLF4 of visceral adipose tissue may correlate with palmitic acid promoted DNMTs expression in adipocytes.

The effects of ambient particulate matter on human adipose tissue

ABSTRACTThe effects of particulate matter (PM) air pollution on adipose tissue have mainly been studied in animal models. The aim of this study was to examine the potential associations between PM exposure and 25 cellular markers in human omental (OM) and subcutaneous (SC) adipose tissue. The PM exposure assessments for both PM2.5 (PM <2.5 μm in diameter) and PM10 (<10 μm) were based upon a novel hybrid satellite-based spatio-temporally resolved model. We calculated the PM exposure above the background threshold for 1 week (acute phase), 3 and 6 months (intermediate phase), and 1 year (chronic phase) prior to tissue harvesting and tested the associations with adipose cell metabolic effects using multiple linear regressions and heat maps strategy. Chemokine levels were found to increase after acute and intermediate exposure duration to PM10. The levels of stress signaling biomarkers in the SC and OM tissues rose after acute exposure to PM10 and PM2.5. Macrophage and leucocyte counts were associated with severity of PM exposure in all three duration groups. Adipocyte diameter decreased in all exposure periods. Our results provide evidence for significant contribution of air pollutants exposure to adipose tissue inflammation as well as for pathophysiological mechanisms of metabolic dysregulation that may be involved in the observed responses.

Characterization of regulatory T cells in obese omental adipose tissue in humans.

Obesity-associated visceral adipose tissue (AT) inflammation promotes insulin resistance and type 2 diabetes (T2D). In mice, lean visceral AT is populated with anti-inflammatory cells, notably regulatory T cells (Tregs) expressing the IL-33 receptor ST2. Conversely, obese AT contains fewer Tregs and more proinflammatory cells. In humans, however, there is limited evidence for a similar pattern of obesity-associated immunomodulation. We used flow cytometry and mRNA quantification to characterize human omental AT in 29 obese subjects, 18 of whom had T2D. Patients with T2D had increased proportions of inflammatory cells, including M1 macrophages, with positive correlations to body mass index. In contrast, Treg frequencies negatively correlated to body mass index but were comparable between T2D and non-T2D individuals. Compared to human thymic Tregs, omental AT Tregs expressed similar levels of FOXP3, CD25, IKZF2, and CTLA4, but higher levels of PPARG, CCR4, PRDM1, and CXCL2. ST2, however, was not detectable on omental AT Tregs from lean or obese subjects. This is the first comprehensive investigation into how omental AT immunity changes with obesity and T2D in humans, revealing important similarities and differences to paradigms in mice. These data increase our understanding of how pathways of immune regulation could be targeted to ameliorate AT inflammation in humans.

Identification of biomarkers, pathways and potential therapeutic agents for white adipocyte insulin resistance using bioinformatics analysis

ABSTRACTFor the better understanding of insulin resistance (IR), the molecular biomarkers in IR white adipocytes and its potential mechanism, we downloaded two mRNA expression profiles from Gene Expression Omnibus (GEO). The white adipocyte samples in two databases were collected from the human omental adipose tissue of IR obese (IRO) subjects and insulin-sensitive obese (ISO) subjects, respectively. We identified 86 differentially expressed genes (DEGs) between the IRO and ISO subjects using limma package in R software. Gene Set Enrichment Analysis (GSEA) provided evidence that the most gene sets enriched in kidney mesenchyme development in the ISO subjects, as compared with the IRO subjects. The Gene Ontology (GO) analysis indicated that the most significantly enriched in cellular response to interferon-gamma. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the DEGs were most significantly enriched in cytokine-cytokine receptor interaction. Protein–Protein Interaction (PPI) network was performed with the STRING, and the top 10 hub genes were identified with the Cytohubba. CMap analysis found several small molecular compounds to reverse the altered DEGs, including dropropizine, aceclofenac, melatonin, and so on. Our outputs could empower the novel potential targets to treat omental white adipocyte insulin resistance, diabetes, and diabetes-related diseases.

Impaired Glucocorticoid Suppression of TGFβ Signaling in Human Omental Adipose Tissues Limits Adipogenesis and May Promote Fibrosis.

Visceral obesity is associated with insulin resistance and higher risk of type 2 diabetes and metabolic diseases. A limited ability of adipose tissues to remodel through the recruitment and differentiation of adipose stem cells (ASCs) is associated with adipose tissue inflammation and fibrosis and the metabolic syndrome. We show that the lower adipogenesis of omental (Om) compared with abdominal subcutaneous (Abdsc) ASCs was associated with greater secretion of TGFβ ligands that acted in an autocrine/paracrine loop to activate SMAD2 and suppress adipogenesis. Inhibition of TGFβ signaling rescued Om ASC differentiation. In Abdsc ASCs, low concentrations of dexamethasone suppressed TGFβ signaling and enhanced adipogenesis, at least in part by increasing TGFBR3 protein that can sequester TGFβ ligands. Om ASCs were resistant to these dexamethasone effects; recombinant TGFBR3 increased their differentiation. Pericellular fibrosis, a hallmark of dysfunctional adipose tissue, was greater in Om and correlated with higher level of tissue TGFβ signaling activity and lower ASC differentiation. We conclude that glucocorticoids restrain cell-autonomous TGFβ signaling in ASCs to facilitate adipogenesis and healthy remodeling in Abdsc and these processes are impaired in Om. Therapies directed at overcoming glucocorticoid resistance in visceral adipose tissue may improve remodeling and help prevent metabolic complications of visceral obesity.

Interleukin-1β and prostaglandin-synthesizing enzymes as modulators of human omental and subcutaneous adipose tissue function

IL-1β stimulates expression of prostaglandin (PG)-synthesizing enzymes cyclooxygenase (COX)-2 and aldo-keto reductase (AKR)1B1 in human preadipocytes. We aimed to examine the impact of IL-1β, COX-2 and AKR1B1 on markers of human visceral and subcutaneous adipose tissue function, and to assess whether PG synthesis by these enzymes mediates IL-1β effects. Omental and subcutaneous fat samples were obtained from bariatric surgery patients. PG release and expression of inflammatory and adipogenic markers were assessed in explants treated with COX-2 inhibitor NS-398 or AKR1B1 inhibitor Statil, with or without IL-1β. Preadipocyte differentiation experiments were also performed. IL-1β decreased expression of PPARγ in both fat depots compared to control and increased expression of NF-κB1, IL-6, CCL-5, ICAM-1 and VEGFA, especially in visceral fat for IL-6, CCL-5 and VEGFA. Adding Statil or NS-398 to IL-1β blunted PGF2α and PGE2 release, but did not alter IL-1β effects on adipose tissue function markers. IL-1β down-regulated adipocyte differentiation whereas NS-398 alone increased this process. However, NS-398 did not prevent IL-1β inhibition of adipogenesis. We conclude that IL-1β induces a pro-inflammatory response in human adipose tissues, particularly in visceral fat, and acts independently of concomitant PG release. IL-1β and COX-2 appear to be critical determinants of adipose tissue pathophysiologic remodeling in obesity.

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue.

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease. This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) >35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2-3 g of fat. These cells can be expanded in culture over 10-14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5-6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPARγ agonist-rosiglitazone. This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Proteomic Profile of Adipose Tissue-Derived Exosomes and Their Potential Role on Placental Glucose Metabolism in Gestational Diabetes Mellitus

Emerging studies suggest that adipose tissue derived exosomes involves in regulating insulin action and glucose homeostasis. In this study, we hypothesised the protein content of adipose tissue derived exosomes (exo-AT) differs in normal glucose tolerant (NGT) and gestational diabetes mellitus (GDM) pregnancies which lead to changes in placental glucose metabolism. Human omental adipose tissue was obtained from GDM (n=60) and BMI-matched NGT (n=48) pregnancies at the time of term Caesarean section. Exosomes were isolated from tissue-conditioned media by differential centrifugation and characterised using nanoparticle tracking analysis and SWATH mass spectrometry. The effect of exosomes on human placental cells was evaluated using a Human Glucose Metabolism RT² Profiler PCR Array. The total number of exosomes (number of vesicles/mg adipose tissue/24 hour) were significantly higher in GDM compared to NGT. The number of exosomes positively correlated with maternal BMI and fetal weight. We identified 122 proteins upregulated in the exo-AT from GDM compared to NGT. Ingenuity pathway analysis revealed the upregulated proteins are associated with mitochondrial dysfunction and oxidative phosphorylation (OXPHOS). When compared to NGT exo-AT, GDM exo-AT increased ALDOB, PGK2 and GCK in placental cells. The data obtained in this study suggest that exosomes secreted from adipose tissue regulates placental glucose metabolism in GDM. Improving the communication of exo-AT to placental tissues may serve as an effective intervention strategy to prevent the consequences of GDM such as fetal overgrowth. Disclosure N. Jayabalan: None. V. Ormazabal: None. A. Lai: None. M. Lappas: None.

Triglycerides analysis in adipose tissue from insulin sensitive and insulin resistance obese patients

IntroductionObesity triggers changes in adipose tissue composition and functionality that contribute to the progression of obesity‐associated metabolic diseases such as insulin resistance and type 2 diabetes mellitus (T2DM). However, some obese individuals maintain insulin sensitivity and show better adipose tissue functions compared to their equally obese insulin resistance counterparts. Lipid intermediates produced during triaclyglycerols (TAG) synthesis and lipolysis interfere with the intracellular insulin signaling pathway and development of insulin resistance. This study aims to compare TAG species and their fatty acid composition in adipose tissues from insulin sensitive (IS) and insulin resistant (IR) individuals.MethodsHuman subcutaneous and Omental adipose tissue biopsies were obtained from 64 clinically characterized obese individuals during weight reduction surgery. TAG were extracted from the adipose tissues using the Bligh&amp; Dyer method then subjected to non‐aqueous revers phase UHPLC and full scan mass spectrometry acquisition and data dependent MS/MS on the Thermo LTQ dual cell linear ion trap. TAG and their fatty acid contents were compared between IS and IR individuals and their levels were correlated with metabolic traits of participants and the adipogenesis potential of preadipocyte cultures established from their adipose tissues.ResultsData revealed over 120 unique TAG species identified based on their exact mass in adipose tissues. Analysis of TAG levels suggested a number of depot‐specific TAG species irrespective of insulin sensitivity status as well as specific TAG that decreased or increased with disease progression. Significant correlations between TAG species and metabolic traits were observed including age, body mass index, total cholesterol, interleukin‐6, C‐reactive protein, alkaline phosphatase and adipogenic potential.ConclusionPilot data suggest that adipose tissues from obese individuals exhibit depot and insulin response‐specific lipid signatures that may contribute to the increased risk of IR and T2DM. Future experiments will investigate the functional relevance of these specific lipidomic profiles.Support or Funding InformationAnti‐Doping Lab Qatar Result 2 General characteristics of participants Variables IS (N=18) IR (N=35) T2DM (N=11) p value Age (year) 30.9 (8.5) 32.1 (8.7) 42.7 (11.0) 0.002 BMI (kg.m−2) 41.6 (7.6) 42.4 (6.8) 44 (8.1) 0.71 SBP (mmHg) 122 (17.6) 35 (127.08) 132 (5.4) 0.27 DBP (mmHg) 71.9 (12.5) 35 (72.2) 79.2 (6.2) 0.25 MAP 86.4 (8.2) 90.4 (13.6) 97.4 (6.1) 0.06 Cholesterol (mmol/L) 4.4 (0.81) 4.6 (0.69) 4.8 (0.64) 0.25 LDL (mmol/L) 2.6 (0.72) 2.92 (0.71) 2.6 (0.69) 0.33 HDLC (mmol/L) 1.3 (0.3) 1.19 (0.28) 1.15 (0.26) 0.16 Triglyceride (mmol/L) 1.14 (0.74) 1.29 (0.59) 2.35 (1.16) 0.00 Leptin (ng/ml) 68.07 (30.5) 62.09 (25.1) 39.8 (31.5) 0.04 Adiponectin (ng/ml) 5.6 (4.9) 3.7 (2.2) 3.05(1.09) 0.12 IL‐6 (pg/ml) 3.8 (2.04) 4.3 (2.4) 4.4 (2.3) 0.70 FBG (mmol/L) 4.9 (0.7) 5.8 (1.06) 9.5 (5.8) 0.00 Insulin (mIU/L) 7.07 (2.2) 18.3 (9.05) 16.6 (7.1) 0.00 HOMA‐IR 1.4 (0.4) 5.2 (3.2) 6.6(3.9) 0.00 Compared to BMI‐matched IS subjects, IR and T2DM individuals had higher circulating levels of TAG and lower leptin

IRS1 DNA promoter methylation and expression in human adipose tissue are related to fat distribution and metabolic traits

The SNP variant rs2943650 near IRS1 gene locus was previously associated with decreased body fat and IRS1 gene expression as well as an adverse metabolic profile in humans. Here, we hypothesize that these effects may be mediated by an interplay with epigenetic alterations. We measured IRS1 promoter DNA methylation and mRNA expression in paired human subcutaneous and omental visceral adipose tissue samples (SAT and OVAT) from 146 and 41 individuals, respectively. Genotyping of rs2943650 was performed in all individuals (N = 146). We observed a significantly higher IRS1 promoter DNA methylation in OVAT compared to SAT (N = 146, P = 8.0 × 10−6), while expression levels show the opposite effect direction (N = 41, P = 0.011). OVAT and SAT methylation correlated negatively with IRS1 gene expression in obese subjects (N = 16, P = 0.007 and P = 0.010). The major T-allele is related to increased DNA methylation in OVAT (N = 146, P = 0.019). Finally, DNA methylation and gene expression in OVAT correlated with anthropometric traits (waist- circumference waist-to-hip ratio) and parameters of glucose metabolism in obese individuals. Our data suggest that the association between rs2943650 near the IRS1 gene locus with clinically relevant variables may at least be modulated by changes in DNA methylation that translates into altered IRS1 gene expression.

Depot Dependent Effects of Dexamethasone on Gene Expression in Human Omental and Abdominal Subcutaneous Adipose Tissues from Obese Women.

Glucocorticoids promote fat accumulation in visceral compared to subcutaneous depots, but the molecular mechanisms involved remain poorly understood. To identify long-term changes in gene expression that are differentially sensitive or responsive to glucocorticoids in these depots, paired samples of human omental (Om) and abdominal subcutaneous (Abdsc) adipose tissues obtained from obese women during elective surgery were cultured with the glucocorticoid receptor agonist dexamethasone (Dex, 0, 1, 10, 25 and 1000 nM) for 7 days. Dex regulated 32% of the 19,741 genes on the array, while 53% differed by Depot and 2.5% exhibited a Depot*Dex concentration interaction. Gene set enrichment analysis showed Dex regulation of the expected metabolic and inflammatory pathways in both depots. Cluster analysis of the 460 transcripts that exhibited an interaction of Depot and Dex concentration revealed sets of mRNAs for which the responses to Dex differed in magnitude, sensitivity or direction between the two depots as well as mRNAs that responded to Dex only in one depot. These transcripts were also clearly depot different in fresh adipose tissue and are implicated in processes that could affect adipose tissue distribution or functions (e.g. adipogenesis, triacylglycerol synthesis and storage, insulin action). Elucidation of the mechanisms underlying the depot differences in the effect of Dex on the expression of specific genes and pathways that regulate adipose function may offer novel insights into understanding the biology of visceral adipose tissues and their links to metabolic health.

Role of cannabinoid receptor 1 in human adipose tissue for lipolysis regulation and insulin resistance

We recently showed that the peripheral cannabinoid receptor type 1 (CNR1) gene is upregulated by the synthetic glucocorticoid dexamethasone. CNR1 is highly expressed in the central nervous system and has been a drug target for the treatment of obesity. Here we explore the role of peripheral CNR1 in states of insulin resistance in human adipose tissue. Subcutaneous adipose tissue was obtained from well-controlled type 2 diabetes subjects and controls. Subcutaneous adipose tissue gene expression levels of CNR1 and endocannabinoid synthesizing and degrading enzymes were assessed. Furthermore, paired human subcutaneous adipose tissue and omental adipose tissue from non-diabetic volunteers undergoing kidney donation or bariatric surgery, was incubated with or without dexamethasone. Subcutaneous adipose tissue obtained from volunteers through needle biopsy was incubated with or without dexamethasone and in the presence or absence of the CNR1-specific antagonist AM281. CNR1 gene and protein expression, lipolysis and glucose uptake were evaluated. Subcutaneous adipose tissue CNR1 gene expression levels were 2-fold elevated in type 2 diabetes subjects compared with control subjects. Additionally, gene expression levels of CNR1 and endocannabinoid-regulating enzymes from both groups correlated with markers of insulin resistance. Dexamethasone increased CNR1 expression dose-dependently in subcutaneous adipose tissue and omental adipose tissue by up to 25-fold. Dexamethasone pre-treatment of subcutaneous adipose tissue increased lipolysis rate and reduced glucose uptake. Co-incubation with the CNR1 antagonist AM281 prevented the stimulatory effect on lipolysis, but had no effect on glucose uptake. CNR1 is upregulated in states of type 2 diabetes and insulin resistance. Furthermore, CNR1 is involved in glucocorticoid-regulated lipolysis. Peripheral CNR1 could be an interesting drug target in type 2 diabetes and dyslipidemia.

Genome-wide DNA promoter methylation and transcriptome analysis in human adipose tissue unravels novel candidate genes for obesity

Objective/methodsDNA methylation plays an important role in obesity and related metabolic complications. We examined genome-wide DNA promoter methylation along with mRNA profiles in paired samples of human subcutaneous adipose tissue (SAT) and omental visceral adipose tissue (OVAT) from non-obese vs. obese individuals. ResultsWe identified negatively correlated methylation and expression of several obesity-associated genes in our discovery dataset and in silico replicated ETV6 in two independent cohorts. Further, we identified six adipose tissue depot-specific genes (HAND2, HOXC6, PPARG, SORBS2, CD36, and CLDN1). The effects were further supported in additional independent cohorts. Our top hits might play a role in adipogenesis and differentiation, obesity, lipid metabolism, and adipose tissue expandability. Finally, we show that in vitro methylation of SORBS2 directly represses gene expression. ConclusionsTaken together, our data show distinct tissue specific epigenetic alterations which associate with obesity.

Adipocyte progenitor cells initiate monocyte chemoattractant protein-1-mediated macrophage accumulation in visceral adipose tissue.

ObjectiveMacrophages are important producers of obesity-induced MCP-1; however, initial obesity-induced increases in MCP-1 production precede M1 macrophage accumulation in visceral adipose tissue (VAT). The initial cellular source of obesity-induced MCP-1 in vivo is currently unknown. Preliminary reports based on in vitro studies of preadipocyte cell lines and adherent stroma-vascular fraction cells suggest that resident stromal cells express MCP-1. In the past several years, elegant methods of identifying adipocyte progenitor cells (AdPCs) have become available, making it possible to study these cells in vivo. We have previously published that global deletion of transcription factor Inhibitor of Differentiation 3 (Id3) attenuates high fat diet-induced obesity, but it is unclear if Id3 plays a role in diet-induced MCP-1 production. We sought to determine the initial cellular source of MCP-1 and identify molecular regulators mediating MCP-1 production.MethodsId3+/+ and Id3−/− mice were fed either a standard chow or HFD for varying lengths of time. Flow cytometry, semi-quantitative real-time PCR, ELISAs and adoptive transfers were used to assess the importance of AdPCs during diet-induced obesity. Flow cytometry was also performed on a cohort of 14 patients undergoing bariatric surgery.ResultsFlow cytometry identified committed CD45−CD31−Ter119−CD29+CD34+Sca-1+CD24− adipocyte progenitor cells as producers of high levels of MCP-1 in VAT. High-fat diet increased AdPC numbers, an effect dependent on Id3. Loss of Id3 increased p21Cip1 levels and attenuated AdPC proliferation, resulting in reduced MCP-1 and M1 macrophage accumulation in VAT, compared to Id3+/+ littermate controls. AdPC rescue by adoptive transfer of 50,000 Id3+/+ AdPCs into Id3−/− recipient mice increased MCP-1 levels and M1 macrophage number in VAT. Additionally, flow cytometry identified MCP-1-producing CD45−CD31−CD34+CD44+CD90+ AdPCs in human omental and subcutaneous adipose tissue, with a higher percentage in omental adipose. Furthermore, high surface expression of CD44 marked abundant MCP-1 producers, only in visceral adipose tissue.ConclusionsThis study provides the first in vivo evidence, to our knowledge, that committed AdPCs in VAT are the initial source of obesity-induced MCP-1 and identifies the helix-loop-helix transcription factor Id3 as a critical regulator of p21Cip1 expression, AdPC proliferation, MCP-1 expression and M1 macrophage accumulation in VAT. Inhibition of Id3 and AdPC expansion, as well as CD44 expression in human AdPCs, may serve as unique therapeutic targets for the regulation of adipose tissue inflammation.

Abstract 271: Id3 Promotes Obesity-Induced Inflammatory Macrophage Accumulation Through Proliferation of MCP-1-Producing Adipocyte Progenitor Cells

High fat diet (HFD)-induced increases in MCP-1 are a hallmark feature of adipose tissue inflammation during obesity. Macrophages are the main producers of MCP-1 in established obesity; however, early increases in obesity-induced production of MCP-1 precede migration of M1 macrophages to adipose tissue. Global deletion of transcription factor Inhibitor of Differentiation 3 (Id3) attenuates HFD-induced obesity and adipose tissue inflammation. Specifically, Id3-/- mice have less visceral adipose tissue (VAT), fewer M1 adipose tissue macrophages, and attenuated HFD-induced MCP-1 production. The source of initial obesity-induced MCP-1 in vivo, as well as the role that Id3 plays in regulation of MCP-1, is currently unknown. Flow cytometry demonstrated that committed CD45-CD31-Ter119-CD29+CD34+Sca-1+CD24- adipocyte progenitor cells (AdPCs) express high levels of MCP-1. HFD increased the number of AdPCs in an Id3-dependent manner. Loss of Id3 increased levels of p21Cip1 and attenuated HFD-induced proliferation of AdPCs, resulting in lower levels of MCP-1 and reduced M1 macrophage numbers in VAT, compared to Id3+/+ littermate controls. Adoptive transfer of 50,000 Id3+/+ AdPCs into Id3-/- recipient mice restored adipose tissue inflammation via increased MCP-1 levels and M1 macrophage accumulation in VAT. Notably, the M1:M2 macrophage ratio significantly increased due to injection of AdPCs. Additionally, flow cytometry identified MCP-1-producing CD45-CD31-CD34+CD44+CD90+ AdPCs in human omental and subcutaneous adipose tissue. The percentage of AdPCs expressing MCP-1 was higher in omental adipose compared to subcutaneous adipose. Furthermore, high surface expression of CD44 marked abundant MCP-1 producers, only in omental adipose tissue. The present study provides the first in vivo evidence that committed AdPCs are the source of early obesity-induced MCP-1. Id3 is a critical regulator of p21Cip1 expression and proliferation in AdPCs, and in this manner, promotes overall MCP-1 expression and M1 macrophage accumulation in VAT. Inhibition of Id3 and AdPC expansion, as well as CD44 expression in human AdPCs, may serve as unique therapeutic targets for the regulation of adipose tissue inflammation.