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Purpose Recent studies point to adipose-derived stem cells (ASCs) as a link between obesity and cancer. We aimed to determine whether survivin, which is highly secreted by ASCs from subjects with obesity, might drive a pro-tumoral phenotype in macrophages. Methods The effect of ASC conditioned medium on the macrophage phenotype was assessed by expression studies. Survivin intracellular localization and internalization were examined by subcellular fractionation and immunofluorescence, respectively. Loss- and gain-of-function studies were performed using adenoviral vectors, and gene expression patterns, migration and invasion capacities of cancer cells were examined. Heterotypic cultures of ASCs, macrophages and cancer cells were established to mimic the tumor microenvironment. Survivin-blocking experiments were used to determine the impact of survivin on both macrophages and cancer cells. Immunohistochemical analysis of survivin was performed in macrophages from ascitic fluids of cancer patients and healthy controls. Results We found that obese-derived ASCs induced a phenotypic switch in macrophages characterized by the expression of both pro- and anti-inflammatory markers. Macrophages were found to internalize extracellular survivin, generating hybrid macrophages with a tumor-associated phenotype that included secretion of survivin. Exogenous expression of survivin in macrophages generated a similar phenotype and enhanced the malignant characteristics of cancer cells by a mechanism dependent on survivin phosphorylation at threonine 34. Survivin secreted by both ASCs from subjects with obesity and tumor-associated macrophages synergistically boosted the malignancy of cancer cells. Importantly, survivin was mainly detected in ascites-associated macrophages from patients with a malignant diagnosis. Conclusion Our data indicate that survivin may serve as a molecular link between obesity and cancer and as a novel marker for tumor-associated macrophages.

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.

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., This study was supported by grants from the Spanish Ministry of Science, Innovation and Universities (PI14/00228 and PI17/01503 to J.V., SAF2015-65019-R to S.F.-V., SAF2014-56819-R and SAF2015-71878-REDT to A.C., BFU2016-78951-R to G.M.-M., PI15/00143 to C.S. and PI15/01562 to A.M., BFU2015-70454-REDT and BFU2017-90578-REDT to S.F.-V. and G.M.-M.) co-financed by the European Regional Development Fund (ERDF). The Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders (CIBERDEM) (CB07708/0012) is an initiative of the Instituto de Salud Carlos III. N.K. is recipient of a predoctoral fellowship from MINECO, Spain (FPI, BES-2016-077745). C.S. acknowledges support from the ‘Ramón y Cajal’ program from MINECO (RYC2013-13186) and S.F.-V. the Miguel Servet tenure-track program (CP10/00438 and CPII16/00008) from the Fondo de Investigación Sanitaria, co-financed by the ERDF.

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.

Crohn&rsquo, s disease (CD) is characterized by compromised immune tolerance to the intestinal commensal microbiota, intestinal barrier inflammation, and hyperplasia of creeping fat (CF) and mesenteric adipose tissue (AT), which seems to be directly related to disease activity. Gut microbiota dysbiosis might be a determining factor in CD etiology, manifesting as a low microbial diversity and a high abundance of potentially pathogenic bacteria. We tested the hypothesis that CF is a reservoir of bacteria through 16S-rRNA sequencing of several AT depots of patients with active and inactive disease and controls. We found a microbiome signature within CF and mesenteric AT from patients, but not in subcutaneous fat. We failed to detect bacterial DNA in any fat depot of controls. Proteobacteria was the most abundant phylum in both CF and mesenteric AT, and positively correlated with fecal calprotectin/C-reactive protein. Notably, the clinical status of patients seemed to be related to the microbiome signature, as those with the inactive disease showed a reduction in the abundance of pathogenic bacteria. Predictive functional profiling revealed many metabolic pathways including lipopolysaccharide biosynthesis and sulfur metabolism overrepresented in active CD relative to that in inactive CD. Our findings demonstrate that microbiota dysbiosis associated with CD pathophysiology is reflected in AT and might contribute to disease severity.

DOI: 10.1016/j.trsl.2016.06.012 URL: http://www.sciencedirect.com/science/article/pii/S1931524416301050 Filiació URV: SI Inclòs a la memòria: SI Angiopoietin-like protein 8 (ANGPTL8), a protein implicated in lipid and glucose homeostasis, is present only in mammals, suggesting that it is involved in processes unique to these vertebrates such as pregnancy and homeothermy. We explored the role of ANGPTL8 in maternal-fetal crosstalk and its relationship with newborn adiposity. In a longitudinal analysis of healthy pregnant women, ANGPTL8 levels decreased progressively during pregnancy although remained higher than levels in the postpartum period. In a cross-sectional observational study of women with or without gestational diabetes mellitus (GDM), and their offspring, ANGPTL8 levels were higher in venous cord blood than those in maternal blood and were significantly lower in GDM patients than those in healthy women. Infants small for gestational age and with low-fat mass had the highest ANGPTL8 cord blood levels. Studies in vitro revealed that ANGPTL8 was secreted by brown adipocytes and its expression was increased in experimental models of white-to-brown fat conversion. In addition, ANGPTL8 induced the expression of markers of brown adipocytes. The high levels of ANGPTL8 found in fetal life together with its relationship with newborn adiposity and brown adipose tissue point to ANGPTL8 as a potential new player in the modulation of the thermogenic machinery during the fetal-neonatal transition.

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-β1 secretion, than lean-derived ASCs. Treatment of lean hASCs with interleukin (IL)-1β mimicked the dysfunctional immune behavior of obese and T2D hASCs. Conversely, combined treatment with IL1RA and TGF-β1 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. Video Highlight: https://youtu.be/ceWOIIZd7Jo

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.

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.

To study whether FGF21 was present in cord blood and explore its relationship with maternal variables and postnatal growth. The study included 157 pregnant women at the beginning of the third trimester; 79 with gestational diabetes (GDM), 78 with normal glucose tolerance (NGT), and their offspring. Glucose metabolism was assessed by oral glucose tolerance test. Insulin resistance was assessed by homeostasis model assessment index–insulin resistance (HOMA-IR). FGF21 was determined in maternal plasma drawn at recruitment and in cord blood obtained at delivery. Offspring weight and height was assessed at birth and at 12, 24 and 48 months. Maternal FGF21 was higher in gestational diabetes than in the normal glucose-tolerant group, whereas similar cord blood FGF21 levels were observed in both groups. Lower cord blood FGF21 was strongly positively correlated with maternal circulating levels. This relationship was independent of mother’s prepregnancy body mass index (BMI), glucose levels and HOMA-IR. Although maternal FGF21 levels were correlated with prepregnancy BMI and HOMA-IR index, no relationship was observed between FGF21 and birth weight. However, cord blood FGF21 levels were correlated with BMI Zeta Score at 12 and 24 months, and this relationship became stronger when only the NGT group was analyzed. FGF21 is present in human cord blood, and its levels are closely correlated with maternal levels. The association observed between cord blood FGF21 and postnatal BMI may suggest a potential role during intrauterine life that may influence future metabolic imbalance.

Adipose tissue (AT) has a central role in obesity-related metabolic imbalance through the dysregulated production of cytokines and adipokines. In addition to its known risk for cardiovascular disease and diabetes, obesity is also a major risk for cancer. We investigated the impact of obesity for the expression of survivin, an antiapoptotic protein upregulated by adipokines and a diagnostic biomarker of tumor onset and recurrence. In a cross-sectional study of 111 subjects classified by body mass index, circulating levels of survivin and gene expression in subcutaneous AT were significantly higher in obese patients and positively correlated with leptin. Within AT, survivin was primarily detected in human adipocyte-derived stem cells (hASCs), the adipocyte precursors that determine AT expansion. Remarkably, survivin expression was significantly higher in hASCs isolated from obese patients that from lean controls and was increased by proinflammatory M1 macrophage soluble factors including IL-1β. Analysis of survivin expression in hASCs revealed a complex regulation including epigenetic modifications and protein stability. Surprisingly, obese hASCs showed survivin promoter hypermethylation that correlated with a significant decrease in its mRNA levels. Nonetheless, a lower level of mir-203, which inhibits survivin protein translation, and higher protein stability, was found in obese hASCs compared with their lean counterparts. We discovered that survivin levels determine the susceptibility of hASCs to apoptotic stimuli (including leptin and hypoxia). Accordingly, hASCs from an obese setting were protected from apoptosis. Collectively, these data shed new light on the molecular mechanisms governing AT expansion in obesity through promotion of hASCs that are resistant to apoptosis, and point to survivin as a potential new molecular player in the communication between AT and tumor cells. Thus, inhibition of apoptosis targeting survivin might represent an effective strategy for both obesity and cancer therapy.

Objective Glycogen metabolism has emerged as a mediator in the control of energy homeostasis and studies in murine models reveal that adipose tissue might contain glycogen stores. Here we investigated the physio(patho)logical role of glycogen in human adipose tissue in the context of obesity and insulin resistance. Methods We studied glucose metabolic flux of hypoxic human adipoctyes by nuclear magnetic resonance and mass spectrometry-based metabolic approaches. Glycogen synthesis and glycogen content in response to hypoxia was analyzed in human adipocytes and macrophages. To explore the metabolic effects of enforced glycogen deposition in adipocytes and macrophages, we overexpressed PTG, the only glycogen-associated regulatory subunit (PP1-GTS) reported in murine adipocytes. Adipose tissue gene expression analysis was performed on wild type and homozygous PTG KO male mice. Finally, glycogen metabolism gene expression and glycogen accumulation was analyzed in adipose tissue, mature adipocytes and resident macrophages from lean and obese subjects with different degrees of insulin resistance in 2 independent cohorts. Results We show that hypoxia modulates glucose metabolic flux in human adipocytes and macrophages and promotes glycogenesis. Enforced glycogen deposition by overexpression of PTG re-orients adipocyte secretion to a pro-inflammatory response linked to insulin resistance and monocyte/lymphocyte migration. Furthermore, glycogen accumulation is associated with inhibition of mTORC1 signaling and increased basal autophagy flux, correlating with greater leptin release in glycogen-loaded adipocytes. PTG-KO mice have reduced expression of key inflammatory genes in adipose tissue and PTG overexpression in M0 macrophages induces a pro-inflammatory and glycolytic M1 phenotype. Increased glycogen synthase expression correlates with glycogen deposition in subcutaneous adipose tissue of obese patients. Glycogen content in subcutaneous mature adipocytes is associated with BMI and leptin expression. Conclusion Our data establish glycogen mishandling in adipose tissue as a potential key feature of inflammatory-related metabolic stress in human obesity., Highlights • Hypoxia redirects extracellular glucose to glycogen synthesis in human adipocytes. • Glycogen modifies the endocrine function of adipocytes and induces insulin resistance. • Glycogen stimulates leptin secretion through an autophagy-dependent mechanism. • Enforced glycogen accumulation in macrophages promotes M1 polarization. • Obesity is associated with higher GS expression and glycogen stores in adipose tissue.

Objective Evidence from mouse models suggests that zinc-α2-glycoprotein (ZAG) is a novel anti-obesity adipokine. In humans, however, data are controversial and its physiological role in adipose tissue (AT) remains unknown. Here we explored the molecular mechanisms by which ZAG regulates carbohydrate metabolism in human adipocytes. Methods ZAG action on glucose uptake and insulin action was analyzed. β1 and β2-adrenoreceptor (AR) antagonists and siRNA targeting PP2A phosphatase were used to examine the mechanisms by which ZAG modulates insulin sensitivity. Plasma levels of ZAG were measured in a lean patient cohort stratified for HOMA-IR. Results ZAG treatment increased basal glucose uptake, correlating with an increase in GLUT expression, but induced insulin resistance in adipocytes. Pretreatment of adipocytes with propranolol and a specific β1-AR antagonist demonstrated that ZAG effects on basal glucose uptake and GLUT4 expression are mediated via β1-AR, whereas inhibition of insulin action is dependent on β2-AR activation. ZAG treatment correlated with an increase in PP2A activity. Silencing of the PP2A catalytic subunit abrogated the negative effect of ZAG on insulin-stimulated AKT phosphorylation and glucose uptake but not on GLUT4 expression and basal glucose uptake. ZAG circulating levels were unchanged in a lean patient cohort stratified for HOMA-IR. Neither glucose nor insulin was associated with plasma ZAG. Conclusions ZAG inhibits insulin-induced glucose uptake in human adipocytes by impairing insulin signaling at the level of AKT in a β2-AR- and PP2A-dependent manner.

This work aimed to explore the link between angiopoietin-like protein 8 (ANGPTL8) and weight loss after metabolic surgery. In the cross-sectional study (n = 100), circulating ANGPTL8 concentrations were significantly lower in morbidly obese than in lean subjects, and strikingly lower in morbidly obese patients with type 2 diabetes mellitus (T2DM). Conversely, ANGPTL8 expression in subcutaneous adipose tissue (SAT) was higher in morbidly obese patients, particularly in those with T2DM, whereas its expression in visceral adipose tissue was unchanged. The main predictors for circulating levels of ANGPTL8 were BMI and T2DM, whereas ANGPTL8 expression in SAT was determined by the presence of T2DM. The prospective cohort studies before and 1 year after bariatric surgery in morbidly obese patients with (n = 45) and without (n = 30) T2DM, revealed a significant increase of circulating ANGPTL8 levels 1 year after the bariatric surgery. Intriguingly, this increment, which was predicted by basal ANGPTL8 concentrations, appeared as a determinant of T2DM remission. In conclusion, circulating ANGPTL8 levels have an inverse relationship with SAT expression. Low basal levels of ANGPTL8 rebound after bariatric surgery. The increment in ANGPTL8 concentrations at 1 month of follow-up after weight loss emerged as a significant predictor of the T2DM remission at 1 year of follow-up.