SUCNR1 controls an anti-inflammatory program in macrophages to regulate the metabolic response to obesity.

Succinate is a signaling metabolite sensed extracellularly by succinate receptor 1 (SUNCR1). The accumulation of succinate in macrophages is known to activate a pro-inflammatory program; however, the contribution of SUCNR1 to macrophage phenotype and function has remained unclear. Here we found that activation of SUCNR1 had a critical role in the anti-inflammatory responses in macrophages. Myeloid-specific deficiency in SUCNR1 promoted a local pro-inflammatory phenotype, disrupted glucose homeostasis in mice fed a normal chow diet, exacerbated the metabolic consequences of diet-induced obesity and impaired adipose-tissue browning in response to cold exposure. Activation of SUCNR1 promoted an anti-inflammatory phenotype in macrophages and boosted the response of these cells to type 2 cytokines, including interleukin-4. Succinate decreased the expression of inflammatory markers in adipose tissue from lean human subjects but not that from obese subjects, who had lower expression of SUCNR1 in adipose-tissue-resident macrophages. Our findings highlight the importance of succinate-SUCNR1 signaling in determining macrophage polarization and assign a role to succinate in limiting inflammation.

Crohn's Disease Increases the Mesothelial Properties of Adipocyte Progenitors in the Creeping Fat.

Our understanding of the interplay between human adipose tissue and the immune system is limited. The mesothelium, an immunologically active structure, emerged as a source of visceral adipose tissue. After investigating the mesothelial properties of human visceral and subcutaneous adipose tissue and their progenitors, we explored whether the dysfunctional obese and Crohn's disease environments influence the mesothelial/mesenchymal properties of their adipocyte precursors, as well as their ability to mount an immune response. Using a tandem transcriptomic/proteomic approach, we evaluated the mesothelial and mesenchymal expression profiles in adipose tissue, both in subjects covering a wide range of body-mass indexes and in Crohn's disease patients. We also isolated adipose tissue precursors (adipose-derived stem cells, ASCs) to assess their mesothelial/mesenchymal properties, as well as their antigen-presenting features. Human visceral tissue presented a mesothelial phenotype not detected in the subcutaneous fat. Only ASCs from mesenteric adipose tissue, named creeping fat, had a significantly higher expression of the hallmark mesothelial genes mesothelin (MSLN) and Wilms' tumor suppressor gene 1 (WT1), supporting a mesothelial nature of these cells. Both lean and Crohn's disease visceral ASCs expressed equivalent surface percentages of the antigen-presenting molecules human leucocyte antigen-DR isotype (HLA-DR) and CD86. However, lean-derived ASCs were predominantly HLA-DR dim, whereas in Crohn's disease, the HLA-DR bright subpopulation was increased 3.2-fold. Importantly, the mesothelial-enriched Crohn's disease precursors activated CD4+ T-lymphocytes. Our study evidences a mesothelial signature in the creeping fat of Crohn's disease patients and its progenitor cells, the latter being able to present antigens and orchestrate an immune response.

Adipose tissue mitochondrial dysfunction in human obesity is linked to a specific DNA methylation signature in adipose-derived stem cells.

BACKGROUND: A functional population of adipocyte precursors, termed adipose-derived stromal/stem cells (ASCs), is crucial for proper adipose tissue (AT) expansion, lipid handling, and prevention of lipotoxicity in response to chronic positive energy balance. We previously showed that obese human subjects contain a dysfunctional pool of ASCs. Elucidation of the mechanisms underlying abnormal ASC function might lead to therapeutic interventions for prevention of lipotoxicity by improving the adipogenic capacity of ASCs. METHODS: Using epigenome-wide association studies, we explored the impact of obesity on the methylation signature of human ASCs and their differentiated counterparts. Mitochondrial phenotyping of lean and obese ASCs was performed. TBX15 loss- and gain-of-function experiments were carried out and western blotting and electron microscopy studies of mitochondria were performed in white AT biopsies from lean and obese individuals. RESULTS: We found that DNA methylation in adipocyte precursors is significantly modified by the obese environment, and adipogenesis, inflammation, and immunosuppression were the most affected pathways. Also, we identified TBX15 as one of the most differentially hypomethylated genes in obese ASCs, and genetic experiments revealed that TBX15 is a regulator of mitochondrial mass in obese adipocytes. Accordingly, morphological analysis of AT from obese subjects showed an alteration of the mitochondrial network, with changes in mitochondrial shape and number. CONCLUSIONS: We identified a DNA methylation signature in adipocyte precursors associated with obesity, which has a significant impact on the metabolic phenotype of mature adipocytes.

Obesity and Type 2 Diabetes Alters the Immune Properties of Human Adipose Derived Stem Cells.

Adipose tissue-derived stem cells (ASCs) are proposed as an alternative stem cell source to bone marrow-derived cells for immune cell therapy. However, microenvironmental factors may impact the functionality of this population in human adipose tissue (AT). We hypothesized that the fat depot in addition to the donor phenotype controls the immunomodulatory capacity of ASCs. Focusing on obesity and type 2 diabetes (T2D) as metabolic disorders that might affect the immune response of ASCs, we compared the inflammatory response of ASCs from subcutaneous and visceral AT of age-matched donors (lean n = 4, body mass index [BMI] 21.98 ± 1.9; obese n = 4 BMI 33.1 ± 2.1 and T2D n = 4 BMI 35.3 ± 1.5). Obese and particularly T2D-derived ASCs showed increased expression of inflammatory markers, activation of NLRP3 inflammasome and higher migration, invasion and phagocytosis capacities than those derived from lean donors. Remarkably, ASCs derived from obese and T2D subjects exhibited a reduction in typical immunosuppressive activities attributed to stem cells. Accordingly, obese and T2D-ASCs were less effective in suppressing lymphocyte proliferation, activating the M2 macrophage phenotype, and in increasing TGF-ß1 secretion, than lean-derived ASCs. Treatment of lean hASCs with interleukin (IL)-1ß mimicked the dysfunctional immune behavior of obese and T2D hASCs. Conversely, combined treatment with IL1RA and TGF-ß1 reverted the phenotype of obese- and T2D-ASCs. These data indicate that the donor metabolic phenotype compromises the immunomodulatory properties of ASCs. These results are relevant not only for understanding the physiology of ASCs in terms of cell-based therapies but also for their role as key regulators of the immune response. Stem Cells 2016;34:2559-2573.

Neurogenin3 cooperates with Foxa2 to autoactivate its own expression.

The transcription factor Neurogenin3 functions as a master regulator of endocrine pancreas formation, and its deficiency leads to the development of diabetes in humans and mice. In the embryonic pancreas, Neurogenin3 is transiently expressed at high levels for a narrow time window to initiate endocrine differentiation in scattered progenitor cells. The mechanisms controlling these rapid and robust changes in Neurogenin3 expression are poorly understood. In this study, we characterize a Neurogenin3 positive autoregulatory loop whereby this factor may rapidly induce its own levels. We show that Neurogenin3 binds to a conserved upstream fragment of its own gene, inducing deposition of active chromatin marks and the activation of Neurog3 transcription. Additionally, we show that the broadly expressed endodermal forkhead factors Foxa1 and Foxa2 can cooperate synergistically to amplify Neurogenin3 autoregulation in vitro. However, only Foxa2 colocalizes with Neurogenin3 in pancreatic progenitors, thus indicating a primary role for this factor in regulating Neurogenin3 expression in vivo. Furthermore, in addition to decreasing Neurog3 autoregulation, inhibition of Foxa2 by RNA interference attenuates Neurogenin3-dependent activation of the endocrine developmental program in cultured duct mPAC cells. Hence, these data uncover the potential functional cooperation between the endocrine lineage-determining factor Neurogenin3 and the widespread endoderm progenitor factor Foxa2 in the implementation of the endocrine developmental program in the pancreas.

Characterization of the transcriptional activity of the basic helix-loop-helix (bHLH) transcription factor Atoh8.

The atonal-related Neurogenin/NeuroD family of basic helix-loop-helix (bHLH) transcription factors comprises potent inducers of neuronal and endocrine differentiation programs in the nervous and digestive system. Atonal homolog 8 (Atoh8) displays high similarity in the bHLH domain with NeuroD proteins. Yet, available evidences indicate that Atoh8 has distinctive features including a ubiquitous expression pattern in embryonic tissues and the ability to inhibit differentiation. To gain insights into Atoh8 function, we aimed at identifying Atoh8 targets and investigated the effects of Atoh8 on global gene expression patterns in pancreatic mPAC cells, a model of bHLH-dependent endocrine differentiation. Our data reveal that Atoh8 is a weak transcriptional activator and does not exhibit proendocrine activity. Conversely, it blocks the induction of a reduced group of gene targets of the atonal-related proendocrine factor Neurogenin3. We show that Atoh8 lacks a transactivation domain and possesses intrinsic repressor activity that depends on a conserved Proline-rich domain. Atoh8 binds the ubiquitous E protein E47 and its ability to repress transcription may partly result from its ability to inhibit E47/E47 and Neurogenin3/E47 dimer activities. These results reveal distinctive transcriptional properties of Atoh8 within the atonal-related bHLH family that may be associated with the acquisition of new biological functions.

Genome-wide DNA methylome and transcriptome profiling reveals key genes involved in the dysregulation of adipose-stem cells in Crohn's disease.

BACKGROUND AND AIMS: Crohn's disease (CD) is characterised by the expansion of mesenteric adipose tissue (MAT), named creeping fat (CF), which seems to be directly related to disease activity. Adipose-stem cells (ASCs) isolated from the CF of patients with CD are extremely pro-inflammatory, which persists during disease remission. We hypothesised that the dysfunctional ASCs in CD accumulate epigenetic modifications triggered by the inflammatory environment that could serve as molecular markers. METHODS: Genome-wide DNA methylome and transcriptome profiling were performed in ASCs isolated from MAT adipose-tissue biopsies of patients with active and inactive disease and from non-Crohn's disease patients (non-CD). A validation cohort was used to test the main candidate genes via qPCR in other fat depots and immune cells. RESULTS: We found differences in DNA-methylation and gene expression between ASCs isolated from patients with CD and from non-CD subjects, but we found no differences related to disease activity. Pathway enrichment analysis revealed that oxidative stress and immune response were significantly enriched in active CD and integration analysis identified MAB21L2, a cell fate-determining gene, as the most affected gene in CD. Validation analysis confirmed the elevated gene expression of MAB21L2 in MAT and in adipose tissue macrophages in active CD. We also found a strong association between expression of the calcium channel subunit gene CACNA1H and disease remission, as CACNA1H expression was higher in ASCs and MAT from patients with inactive CD, and correlates negatively with C-reactive protein in peripheral blood mononuclear cells. CONCLUSION: We identified a potential gene signature of CD in ASCs obtained from MAT. Integration analysis highlighted two novel genes demonstrating a negative correlation between promoter DNA methylation and transcription: one linked to ASCs in CD (MAB21L2) and the other (CACNA1H) related to disease remission.

SUCNR1 regulates insulin secretion and glucose elevates the succinate response in people with prediabetes.

Pancreatic ß-cell dysfunction is a key feature of type 2 diabetes, and novel regulators of insulin secretion are desirable. Here we report that the succinate receptor (SUCNR1) is expressed in ß-cells and is up-regulated in hyperglycemic states in mice and humans. We found that succinate acts as a hormone-like metabolite and stimulates insulin secretion via a SUCNR1-Gq-PKC-dependent mechanism in human ß-cells. Mice with ß-cell-specific Sucnr1 deficiency exhibit impaired glucose tolerance and insulin secretion on a high-fat diet, indicating that SUCNR1 is essential for preserving insulin secretion in diet-induced insulin resistance. Patients with impaired glucose tolerance show an enhanced nutritional-related succinate response, which correlates with the potentiation of insulin secretion during intravenous glucose administration. These data demonstrate that the succinate/SUCNR1 axis is activated by high glucose and identify a GPCR-mediated amplifying pathway for insulin secretion relevant to the hyperinsulinemia of prediabetic states.

Gut Microbiota Remodeling and Intestinal Adaptation to Lipid Malabsorption After Enteroendocrine Cell Loss in Adult Mice.

BACKGROUND & AIMS: Enteroendocrine cells (EECs) and their hormones are essential regulators of whole-body energy homeostasis. EECs sense luminal nutrients and microbial metabolites and subsequently secrete various hormones acting locally or at a distance. Impaired development of EECs during embryogenesis is life-threatening in newborn mice and humans due to compromised nutrient absorption. However, the physiological importance of the EEC system in adult mice has yet to be directedly studied. Herein, we aimed to determine the long-term consequences of a total loss of EECs in healthy adults on energy metabolism, intestinal transcriptome, and microbiota. METHODS: We depleted intestinal EECs by tamoxifen treatment of adult Neurog3fl/fl; Villin-CreERT2 male mice. We studied intestinal cell differentiation, food efficiency, lipid absorption, microbiota composition, fecal metabolites, and transcriptomic responses in the proximal and distal small intestines of mice lacking EECs. We also determined the high-fat diet-induced transcriptomic changes in sorted Neurog3eYFP/+ EECs. RESULTS: Induction of EEC deficiency in adults is not life-threatening unless fed with a high-fat diet. Under a standard chow diet, mice lose 10% of weight due to impaired food efficiency. Blood concentrations of cholesterol, triglycerides, and free fatty acids are reduced, and lipid absorption is impaired and delayed in the distal small intestine. Genes controlling lipogenesis, carbohydrate metabolism, and neoglucogenesis are upregulated. Microbiota composition is rapidly altered after EECs depletion and is characterized by decreased α-diversity. Bacteroides and Lactobacillus were progressively enriched, whereas Lachnospiraceae declined without impacting fecal short-chain fatty acid concentrations. CONCLUSIONS: EECs are dispensable for survival in adult male mice under a standard chow diet. The absence of EECs impairs intestinal lipid absorption, leading to transcriptomic and metabolic adaptations and remodeling of the gut microbiota.

EndoC-ßH5 cells are storable and ready-to-use human pancreatic beta cells with physiological insulin secretion.

OBJECTIVES: Readily accessible human pancreatic beta cells that are functionally close to primary adult beta cells are a crucial model to better understand human beta cell physiology and develop new treatments for diabetes. We here report the characterization of EndoC-ßH5 cells, the latest in the EndoC-ßH cell family. METHODS: EndoC-ßH5 cells were generated by integrative gene transfer of immortalizing transgenes hTERT and SV40 large T along with Herpes Simplex Virus-1 thymidine kinase into human fetal pancreas. Immortalizing transgenes were removed after amplification using CRE activation and remaining non-excized cells eliminated using ganciclovir. Resulting cells were distributed as ready to use EndoC-ßH5 cells. We performed transcriptome, immunological and extensive functional assays. RESULTS: Ready to use EndoC-ßH5 cells display highly efficient glucose dependent insulin secretion. A robust 10-fold insulin secretion index was observed and reproduced in four independent laboratories across Europe. EndoC-ßH5 cells secrete insulin in a dynamic manner in response to glucose and secretion is further potentiated by GIP and GLP-1 analogs. RNA-seq confirmed abundant expression of beta cell transcription factors and functional markers, including incretin receptors. Cytokines induce a gene expression signature of inflammatory pathways and antigen processing and presentation. Finally, modified HLA-A2 expressing EndoC-ßH5 cells elicit specific A2-alloreactive CD8 T cell activation. CONCLUSIONS: EndoC-ßH5 cells represent a unique storable and ready to use human pancreatic beta cell model with highly robust and reproducible features. Such cells are thus relevant for the study of beta cell function, screening and validation of new drugs, and development of disease models.

Sequence and epigenetic determinants in the regulation of the Math6 gene by Neurogenin3.

The bHLH factor Neurogenin3 initiates the differentiation program that leads to formation of pancreatic endocrine cells. Math6 is a closely related bHLH factor transiently activated downstream of Neurogenin3 in endocrine progenitors. Here we characterize the Math6 promoter and locate the Neurogenin3 binding site, thus confirming that Math6 is a genuine Neurogenin3 target. We also show that Math6 activation rates are largely controlled by epigenetic mechanisms involving the balance between activating H3K4 and repressive H3K27 methylation marks. High Math6 expression in the embryonic pancreas associates with an H3K4me3-only state, whereas low Math6 expression in differentiated endocrine cells correlates with chromatin dually marked with H3K4me3/H3K27me3, a feature originally associated with developmental genes that are repressed but poised for activation in ES cells. Importantly, we show that Neurogenin3 can trigger the conversion of Math6 from a poorly transcribed bivalent to an active monovalent state in vitro, hence providing a mechanism whereby Neurogenin3 may activate Math6 in endocrine progenitors. Finally, because Neurogenin3-induced changes in histone methylation are observed at other endocrine gene promoters, we propose that this mechanism may contribute to the determination of endocrine cell fate by Neurogenin3 in the pancreas.

DNA Methylation in Gestational Diabetes and its Predictive Value for Postpartum Glucose Disturbances.

CONTEXT: DNA methylation in the diagnosis of gestational diabetes. OBJECTIVE: To assess the value of DNA methylation in the diagnosis of gestational diabetes (GDM) and in the prediction of maternal postpartum glucose disturbances. METHODS: Two-stage observational study performed between July 2006 and December 2010, at University Hospital. Forty-eight randomly selected pregnant women formed the discovery cohort (24 with GDM and 24 controls) and 252 pregnant women (94 with GDM and 158 controls) formed the replication cohort. GDM women were re-evaluated 4 years postpartum. The main outcome measures were GDM, type 2 diabetes or prediabetes at 4 years postpartum. RESULTS: We identified 3 CpG sites related to LINC00917, TRAPPC9, and LEF1 that were differentially methylated in women with GDM and abnormal glucose tolerance; and sites associated with LINC00917 and TRAPPC9 were independently associated with an abnormal glucose tolerance status 4 years postpartum after controlling for clinical variables. Moreover, the site associated with LINC00917 and the combination of the 3 sites had the highest predictive values. CONCLUSION: Our results suggest that some of these sites may be implicated in the development of GDM and postpartum abnormal glucose tolerance.

Glycogen accumulation in adipocyte precursors from elderly and obese subjects triggers inflammation via SIRT1/6 signaling.

Dysfunctional adipocyte precursors have emerged as key determinants for obesity- and aging-related inflammation, but the mechanistic basis remains poorly understood. Here, we explored the dysfunctional adipose tissue of elderly and obese individuals focusing on the metabolic and inflammatory state of human adipose-derived mesenchymal stromal cells (hASCs), and on sirtuins, which link metabolism and inflammation. Both obesity and aging impaired the differentiation potential of hASCs but had a different impact on their proliferative capacity. hASCs from elderly individuals (≥65 years) showed an upregulation of glycolysis-related genes, which was accompanied by increased lactate secretion and glycogen storage, a phenotype that was exaggerated by obesity. Multiplex protein profiling revealed that the metabolic switch to glycogenesis was associated with a pro-inflammatory secretome concomitant with a decrease in the protein expression of SIRT1 and SIRT6. siRNA-mediated knockdown of SIRT1 and SIRT6 in hASCs from lean adults increased the expression of pro-inflammatory and glycolysis-related markers, and enforced glycogen deposition by overexpression of protein targeting to glycogen (PTG) led to a downregulation of SIRT1/6 protein levels, mimicking the inflammatory state of hASCs from elderly subjects. Overall, our data point to a glycogen-SIRT1/6 signaling axis as a driver of age-related inflammation in adipocyte precursors.

Potentiation of morphine analgesia by adjuvant activation of 5-HT7 receptors.

Spinal blockade of 5-HT7 receptors has been reported to inhibit the antinociceptive effect of opioids. In this study, we found that subcutaneous administration of the selective 5-HT7 receptor agonist E-55888 (10 mg/kg) or the antagonist SB-258719 (5 mg/kg) exerted no effect on the tail-flick test in mice. However, E-55888, but not SB-258719, increased (2.6-fold) the analgesic potency of oral morphine. The potentiating effect exerted by E-55888 was prevented by SB-258719. A pharmacokinetic interaction was discarded as morphine plasma and brain concentrations were not significantly modified when co-administered with E-55888. These results reinforce the involvement of 5-HT7 receptors in opioid analgesia and point to a potential use of 5-HT7 receptor agonists as adjuvants of opioid analgesia.

Adipose tissue is a key organ for the beneficial effects of GLP-2 metabolic function.

BACKGROUND AND PURPOSE: Glucagon-like peptide-2 (GLP-2) is a gastrointestinal hormone released in response to nutritional intake that exerts a wide range of effects by activating GLP-2 receptors. In addition to its intestinotrophic effects, GLP-2 also positively influences glucose metabolism under conditions of obesity, but the mechanisms behind this remain unclear. Here, we have investigated the molecular role of the GLP-2/GLP-2 receptor axis in energetic metabolism, focusing on its potential modulatory effects on adipose tissue. EXPERIMENTAL APPROACH: Physiological measurements (body weight, food intake, locomotor activity, and energy expenditure) and metabolic studies (glucose and insulin tolerance tests) were performed on lean and obese mice treated with the protease-resistant GLP-2 analogue teduglutide. KEY RESULTS: Acute but not chronic centrally administered teduglutide decreased food intake and weight-gain. By contrast, chronic activation of peripheral GLP-2 receptors increased body weight-independent glucose tolerance and had anti-inflammatory effects on visceral adipose tissue. Using a gene silencing approach, we found that adipose tissue is necessary for these beneficial effects of teduglutide. Finally, teduglutide regulates the inflammatory state and acts as an anabolic signal in human adipocytes. CONCLUSION AND IMPLICATIONS: Overall, our data identify adipose tissue as a new, clinically relevant, site of action for GLP-2 activity in obesity. LINKED ARTICLES: This article is part of a themed issue on Cellular metabolism and diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.10/issuetoc.

Effects of stem cells from inducible brown adipose tissue on diet-induced obesity in mice.

Adipose-derived mesenchymal stem cells (ASCs) are a promising option for the treatment of obesity and its metabolic co-morbidities. Despite the recent identification of brown adipose tissue (BAT) as a potential target in the management of obesity, the use of ASCs isolated from BAT as a therapy for patients with obesity has not yet been explored. Metabolic activation of BAT has been shown to have not only thermogenic effects, but it also triggers the secretion of factors that confer protection against obesity. Herein, we isolated and characterized ASCs from the visceral adipose tissue surrounding a pheochromocytoma (IB-hASCs), a model of inducible BAT in humans. We then compared the anti-obesity properties of IB-hASCs and human ASCs isolated from visceral white adipose tissue (W-hASCs) in a murine model of diet-induced obesity. We found that both ASC therapies mitigated the metabolic abnormalities of obesity to a similar extent, including reducing weight gain and improving glucose tolerance. However, infusion of IB-hASCs was superior to W-hASCs in suppressing lipogenic and inflammatory markers, as well as preserving insulin secretion. Our findings provide evidence for the metabolic benefits of visceral ASC infusion and support further studies on IB-hASCs as a therapeutic option for obesity-related comorbidities.

Gestational diabetes impacts fetal precursor cell responses with potential consequences for offspring.

Fetal programming has been proposed as a key mechanism underlying the association between intrauterine exposure to maternal diabetes and negative health outcomes in offspring. To determine whether gestational diabetes mellitus (GDM) might leave an imprint in fetal precursors of the amniotic membrane and whether it might be related to adverse outcomes in offspring, a prospective case-control study was conducted, in which amniotic mesenchymal stem cells (AMSCs) and resident macrophages were isolated from pregnant patients, with either GDM or normal glucose tolerance, scheduled for cesarean section. After characterization, functional characteristics of AMSCs were analyzed and correlated with anthropometrical and clinical variables from both mother and offspring. GDM-derived AMSCs displayed an impaired proliferation and osteogenic potential when compared with control cells, accompanied by superior invasive and chemotactic capacity. The expression of genes involved in the inflammatory response (TNFα, MCP-1, CD40, and CTSS) was upregulated in GDM-derived AMSCs, whereas anti-inflammatory IL-33 was downregulated. Macrophages isolated from the amniotic membrane of GDM mothers consistently showed higher expression of MCP-1 as well. In vitro studies in which AMSCs from healthy control women were exposed to hyperglycemia, hyperinsulinemia, and palmitic acid confirmed these results. Finally, genes involved in the inflammatory response were associated with maternal insulin sensitivity and prepregnancy body mass index, as well as with fetal metabolic parameters. These results suggest that the GDM environment could program stem cells and subsequently favor metabolic dysfunction later in life. Fetal adaptive programming in the setting of GDM might have a direct negative impact on insulin resistance of offspring.

Adipose stem cells from patients with Crohn's disease show a distinctive DNA methylation pattern.

BACKGROUND: Crohn's disease (CD) is characterized by persistent inflammation and ulceration of the small or large bowel, and expansion of mesenteric adipose tissue, termed creeping fat (CF). We previously demonstrated that human adipose-derived stem cells (hASCs) from CF of patients with CD exhibit dysfunctional phenotypes, including a pro-inflammatory profile, high phagocytic capacity, and weak immunosuppressive properties. Importantly, these phenotypes persist in patients in remission and are found in all adipose depots explored including subcutaneous fat. We hypothesized that changes in hASCs are a consequence of epigenetic modifications. METHODS: We applied epigenome-wide profiling with a methylation array (Illumina EPIC/850k array) and gene expression analysis to explore the impact of CD on the methylation signature of hASCs isolated from the subcutaneous fat of patients with CD and healthy controls (n = 7 and 5, respectively; cohort I). Differentially methylated positions (p value cutoff < 1 × 10-4 and ten or more DMPs per gene) and regions (inclusion threshold 0.2, p value cutoff < 1 × 10-2 and more than 2 DMRs per gene) were identified using dmpfinder and Bumphunter (minfi), respectively. Changes in the expression of differentially methylated genes in hASCs were validated in a second cohort (n = 10/10 inactive and active CD and 10 controls; including patients from cohort I) and also in peripheral blood mononuclear cells (PBMCs) of patients with active/inactive CD and of healthy controls (cohort III; n = 30 independent subjects). RESULTS: We found a distinct DNA methylation landscape in hASCs from patients with CD, leading to changes in the expression of differentially methylated genes involved in immune response, metabolic, cell differentiation, and development processes. Notably, the expression of several of these genes in hASCs and PBMCs such as tumor necrosis factor alpha (TNFA) and PR domain zinc finger protein 16 (PRDM16) were not restored to normal (healthy) levels after disease remission. CONCLUSIONS: hASCs of patients with CD exhibit a unique DNA methylation and gene expression profile, but the expression of several genes are only partially restored in patients with inactive CD, both in hASCs and PBMCs. Understanding how CD shapes the functionality of hASCs is critical for investigating the complex pathophysiology of this disease, as well as for the success of cell-based therapies. Human adipose-stem cells isolated from subcutaneous fat of patients with Crohn's disease exhibit an altered DNA methylation pattern and gene expression profile compared with those isolated from healthy individuals, with immune system, cell differentiation, metabolic and development processes identified as the main pathways affected. Interestingly, the gene expression of several genes involved in these pathways is only partially restored to control levels in patients with inactive Crohn's disease, both in human adipose-stem cells and peripheral blood mononuclear cells. Understanding how Crohn's disease shapes the functionality of human adipose-stem cells is critical for investigating the complex pathophysiology of this disease, as well as for the success of cell-based therapies.

Role of adipose tissue GLP-1R expression in metabolic improvement after bariatric surgery in patients with type 2 diabetes.

We aimed to explore the relationship between GLP-1 receptor (GLP-1R) expression in adipose tissue (AT) and incretin secretion, glucose homeostasis and weight loss, in patients with morbid obesity and type 2 diabetes undergoing bariatric surgery. RNA was extracted from subcutaneous (SAT) and visceral (VAT) AT biopsies from 40 patients randomized to metabolic gastric bypass, sleeve gastrectomy or greater curvature plication. Biochemical parameters, fasting plasma insulin, glucagon and area under the curve (AUC) of GLP-1 following a standard meal test were determined before and 1 year after bariatric surgery. GLP-1R expression was higher in VAT than in SAT. GLP-1R expression in VAT correlated with weight (r = -0.453, p = 0.008), waist circumference (r = -0.494, p = 0.004), plasma insulin (r = -0.466, p = 0.007), and systolic blood pressure (BP) (r = -0.410, p = 0.018). At 1 year, GLP-1R expression in VAT was negatively associated with diastolic BP (r = -0.361, p = 0.039) and, following metabolic gastric bypass, with the increase of GLP-1 AUC, (R2 = 0.46, p = 0.038). Finally, GLP-1R in AT was similar independently of diabetes outcomes and was not associated with weight loss after surgery. Thus, GLP-1R expression in AT is of limited value to predict incretin response and does not play a role in metabolic outcomes after bariatric surgery.