Christine Rouault

CCL5 Promotes Macrophage Recruitment and Survival in Human Adipose Tissue

Objectives—To examine the role of adipose-produced chemokine, chemokine ligand (CCL) 5, on the recruitment and survival of macrophages in human white adipose tissue (WAT). Methods and Results—CCL5 levels measured by enzyme immunoassay in serum and by real-time polymerase chain reaction in WAT were higher in obese compared to lean subjects. CCL5, but not CCL2, secretion was higher in visceral compared to subcutaneous WAT. CCL5 mRNA expression was positively correlated with the inflammatory macrophage markers as CD11b, tumor necrosis factor-&agr;, and IL-6 in visceral WAT (n=24 obese subjects), and was higher in macrophages than other WAT cells. We found that CCL5 triggered adhesion and transmigration of blood monocytes to/through endothelial cells of human WAT. Whereas in obese WAT apoptotic macrophages were located around necrotic adipocytes, we demonstrated that CCL5, but not CCL2, protected macrophages from free cholesterol-induced apoptosis via activation of the Akt/Erk pathways. Conclusions—CCL5 could participate in the inflammation of obese WAT by recruiting blood monocytes and exerting antiapoptotic properties on WAT macrophages. This specific role of CCL5 on macrophage survival with maintenance of their lipid scavenging function should be taken into account for future therapeutic strategies in obesity-related diseases.

Inhibition of Insulin Secretion by Leptin in Normal Rodent Islets of Langerhans

The recently discovered adipose cell-specific hormone called leptin decreases food intake and increases energy expenditure in rodents through a pathway involving hypothalamic leptin receptors, OB-R. In addition, leptin decreases insulin circulating levels independent of the reduction in food intake. Whether or not the hormone has a direct effect on pancreatic beta-cells is not clear, because previous in vitro studies have led to controversial results depending on the animal model used. The present study was designed to investigate the effects of leptin in islets of Langerhans isolated from normal rodents. Three isoforms of the leptin receptor, OB-Ra, b, and f, were detected by RT-PCR analysis of total RNA from rat islets. In static incubations, leptin (10 ng/ml) did not alter basal insulin secretion nor insulin secretion stimulated by glucose alone, potassium chloride, or ketoisocaproic acid. In contrast, insulin secretion stimulated by glucose + 3-isobutyl 1-methylxanthine (IBMX) was inhibited by 34 +/- 15% (n = 4, P < 0.05). This was further substantiated in perifusion experiments, in which leptin decreased by 31 +/- 3% (n = 5, P < 0.01) glucose + IBMX-stimulated insulin release. Similarly, in mouse islets a significant inhibitory effect of leptin (-31 +/- 4%, n = 6, P < 0.05) was observed only on glucose + IBMX-stimulated insulin secretion, with no effect of the hormone on basal nor glucose-stimulated secretion. Finally, leptin was totally inefficient in islets isolated from obese fa/fa rats, which bear a mutation in OB-R. These results suggest that, in normal rodent islets, leptin specifically inhibits IBMX-potentiated glucose-induced insulin secretion, through a direct effect involving at least one of the three isoforms of OB-R expressed in islets.

Variations in circulating inflammatory factors are related to changes in calorie and carbohydrate intakes early in the course of surgery-induced weight reduction.

BACKGROUNDnObesity is considered a low-grade inflammatory state that improves with weight loss. In addition to acute-phase proteins, other cytokines might contribute to systemic inflammation.nnnOBJECTIVEnOur objective was to compare serum concentrations of a large panel of inflammation-related factors in obese and normal-weight subjects and to determine kinetic changes induced by caloric restriction.nnnDESIGNnThe cohort comprised 14 normal-weight women and 51 obese women who were followed over 2 y after Roux-en-Y gastric bypass. Multiplexed proteomics were used to simultaneously assay 27 cytokines and growth factors in serum.nnnRESULTSnConcentrations of interleukin (IL)-9, IL-1-receptor antagonist, IL-10, interferon-γ-inducible protein 10, macrophage inflammatory protein 1β, monocyte chemoattractant protein 1, IL-8, RANTES (regulated upon activation, normal T cell expressed and secreted), monokine induced by interferon-γ, and vascular endothelial growth factor were found to be elevated in obesity. IL-10 was further elevated in diabetic obese patients, whereas eotaxin was found to be higher only in diabetic subjects. After surgery, many factors showed a biphasic pattern of variation, decreasing sharply at month 3 before rising back to presurgical values at month 6; these changes closely tracked similar kinetic changes in calorie and carbohydrate intake. After 1 y, an overall reduction in cytokines accompanied the reduction in body mass index and an amelioration in metabolic status.nnnCONCLUSIONSnObesity is associated with elevated circulating concentrations of a large panel of cytokines. Coordinated kinetic changes during weight loss suggest an early influence of calorie and carbohydrate intakes, whereas a longer-term reduction in corpulence might prevail in regulating circulating cytokine concentrations. This trial is registered at clincaltrials.gov as NCT00476658.

SMRT-GPS2 corepressor pathway dysregulation coincides with obesity-linked adipocyte inflammation

Low-grade chronic inflammation is a major characteristic of obesity and results from deregulated white adipose tissue function. Consequently, there is interest in identifying the underlying regulatory mechanisms and components that drive adipocyte inflammation. Here, we report that expression of the transcriptional corepressor complex subunits GPS2 and SMRT was significantly reduced in obese adipose tissue, inversely correlated to inflammatory status, and was restored upon gastric bypass surgery-induced weight loss in morbid obesity. These alterations correlated with reduced occupancy of the corepressor complex at inflammatory promoters, providing a mechanistic explanation for elevated inflammatory transcription. In support of these correlations, RNAi-mediated depletion of GPS2 and SMRT from cultured human adipocytes promoted derepression of inflammatory transcription and elevation of obesity-associated inflammatory markers, such as IL-6 and MCP-1. Furthermore, we identified a regulatory cascade containing PPARγ and TWIST1 that controlled the expression of GPS2 and SMRT in human adipocytes. These findings were clinically relevant, because treatment of diabetic obese patients with pioglitazone, an antidiabetic and antiinflammatory PPARγ agonist, restored expression of TWIST1, GPS2, and SMRT in adipose tissue. Collectively, our findings identify alterations in a regulatory transcriptional network in adipocytes involving the dysregulation of a specific corepressor complex as among the initiating events promoting adipose tissue inflammation in human obesity.

Human adipocyte function is impacted by mechanical cues

Fibrosis is a hallmark of human white adipose tissue (WAT) during obesity‐induced chronic inflammation. The functional impact of increased interstitial fibrosis (peri‐adipocyte fibrosis) on adjacent adipocytes remains unknown. Here we developed a novel in vitro 3D culture system in which human adipocytes and decellularized material of adipose tissue (dMAT) from obese subjects are embedded in a peptide hydrogel. When cultured with dMAT, adipocytes showed decreased lipolysis and adipokine secretion and increased expression/production of cytokines (IL‐6, G‐CSF) and fibrotic mediators (LOXL2 and the matricellular proteins THSB2 and CTGF). Moreover, some alterations including lipolytic activity and fibro‐inflammation also occurred when the adipocyte/hydrogel culture was mechanically compressed. Notably, CTGF expression levels correlated with the amount of peri‐adipocyte fibrosis in WAT from obese individuals. Moreover, dMAT‐dependent CTGF promoter activity, which depends on β1‐integrin/cytoskeleton pathways, was enhanced in the presence of YAP, a mechanosensitive co‐activator of TEAD transcription factors. Mutation of TEAD binding sites abolished the dMAT‐induced promoter activity. In conclusion, fibrosis may negatively affect human adipocyte function via mechanosensitive molecules, in part stimulated by cell deformation. Published by John Wiley & Sons, Ltd

Differential effects of macronutrient content in 2 energy-restricted diets on cardiovascular risk factors and adipose tissue cell size in moderately obese individuals: a randomized controlled trial

BACKGROUNDnThe most effective and safe dietary approach for weight loss and its impact on the metabolic functions and morphology of adipose tissue remain unclear.nnnOBJECTIVESnWe evaluated whether an energy-restricted high-protein diet with a low glycemic index and soluble fiber (LC-P-LGI) would be more effective than a low-calorie conventional diet (LC-CONV) on weight loss and related metabolic risk factors. We further determined factors that may influence adipocyte size during energy restriction.nnnDESIGNnThirteen obese participants were randomly assigned in a crossover design to 2 periods of a 4-wk hypocaloric diet as either LC-P-LGI or LC-CONV, separated by 8-wk washout intervals.nnnRESULTSnIn comparison with the LC-CONV diet, the main effect of the LC-P-LGI diet was a greater decrease in adipocyte diameter (P = 0.048), plasma plasminogen activator inhibitor protein-1 (P = 0.019), vascular endothelial growth factor (P = 0.032), and interferon-γ inducible protein 10 (P = 0.010). Whereas fasting plasma glucose and high-sensitivity C-reactive protein decreased only after the LC-P-LGI diet, with no differences between diets, fasting plasma insulin and insulin resistance were lower after the LC-CONV diet. The diet results did not differ for body composition and lipid variables. Kinetic modifications in adipocyte diameter were associated with metabolic variables and genes implicated in adipocyte proliferation, apoptosis, and angiogenesis.nnnCONCLUSIONSnIn comparison with the LC-CONV diet, the LC-P-LGI diet was associated with improvement in some cardiometabolic risk factors and greater reduction in adipocyte size. Profiles of genes involved in inhibiting adipogenesis and angiogenesis, but increasing apoptosis, were correlated with decreased adipocyte size. This study provides insight into the adipose tissue-remodeling changes that induce regulation of adipocyte size during dietary weight loss. This trial was registered at clinicaltrials.gov as NCT01312740.

A monoclonal antibody (B724) to von Willebrand factor recognizing an epitope within the Al disulphide loop (Cys509-Cys695) discriminates between type 2A and type 2B von Willebrand disease

Monoclonal antibody (MoAb) B724 to von Willebrand factor (vWF) completely inhibits its interaction with heparin, sulphatides and botrocetin and consequently botrocetin‐induced binding of vWF to platelets. MoAb B724 has no effect on the binding of vWF to collagen or to ristocetin‐treated platelets nor on vWF‐dependent platelet aggregation induced with ristocetin and asialo‐vWF‐mediated platelet aggregation. MoAb B724 preferentially recognizes a conformation of native vWF, in solution, or immobilized through a coated antibody. It exhibits a markedly lower affinity for vWF immobilized onto collagen or plastic surfaces. Using proteolytic fragments of vWF, B724 epitope was localized within the 512–673 sequence of the Al disulphide loop of vWF, MoAb B724 was used as second antibody in a two‐site ELISA to test a series of patients with type 1, 2 A, 2B and 2N vWD or haemophilia A and recombinant wild type or mutated vWFs. Results were compared with those obtained by control ELISAs performed using polyclonal antibodies. Using MoAb B724, strikingly lower levels of vWFAg were observed in plasma from most patients with type 2B vWD, and in seven out of the eight rvWF mutated close to or within the Al disulphide loop. Therefore MoAb B724, which interferes with this loop involved in the function of vWF, appears to be a useful tool for rapid screening of conformational changes in this region.

Roles of Chemokine Ligand-2 (CXCL2) and Neutrophils in Influencing Endothelial Cell Function and Inflammation of Human Adipose Tissue

The hypertrophied white adipose tissue (WAT) during human obesity produces inflammatory mediators, including cytokines (IL-6 and TNFα) and chemokines ([C-C motif] chemokine ligand 2 and IL-8). These inflammatory factors are preferentially produced by the nonadipose cells, particularly the adipose tissue infiltrating macrophages. We identified the chemokine (C-X-C motif) ligand 2 (CXCL2) by a transcriptomic approach. Because CXCL2 could represent a WAT-produced chemokine, we explored its role in obesity-associated inflammation. CXCL2 levels in serum and mRNA in WAT were higher in obese subjects compared with lean ones. CXCL2 secretions were higher in sc and visceral (vis) WAT from obese compared with lean subjects. In vis WAT, CXCL2 mRNA expression was higher in macrophages compared with other WAT cells and positively correlated with the inflammatory macrophage markers TNFα and IL-6. CXCL2 triggered the in vitro adhesion of the neutrophils, its selective cell targets, to endothelial cells (ECs) of vis WAT (vis WAT-ECs). Immunohistological analysis indicated that activated neutrophils were adherent to the endothelium of vis WAT from human obese subjects. Blood neutrophils from obese subjects released high levels of proinflammatory mediators (IL-8, chemokine motif ligand 2 [CCL2], matrix metalloproteinase [MMP] 9, and myeloperoxidase [MPO]). Visceral WAT-ECs, treated by neutrophil-conditioned media prepared from obese subjects, displayed an increase of the expression of inflammatory molecules associated with senescence and angiogenic capacities. To conclude, CXCL2, a WAT-produced chemokine being up-regulated in obesity, stimulates neutrophil adhesion to vis WAT-ECs. Activated neutrophils in obesity may influence vis WAT-ECs functions and contribute to WAT inflammation.

Long-term exposure of isolated rat islets of Langerhans to supraphysiologic glucose concentrations decreases insulin mRNA levels.

Chronic hyperglycemia has been postulated to contribute to beta-cell dysfunction in type 2 diabetic patients. A deleterious effect of prolonged exposure to high glucose concentrations on insulin gene expression has been demonstrated in insulin-secreting cell lines. This study was designed to investigate in isolated rat islets the effects of long-term exposure to supraphysiologic glucose concentrations on insulin, GLUT2, and glucokinase gene expression. The acute effects of glucose on gene expression were investigated by culturing rat islets in 2.8 or 16.7 mmol/L glucose for 24 hours. Insulin, GLUT2, and glucokinase mRNA levels were assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). As expected, glucose acutely increased relative insulin and GLUT2 mRNA levels by 2.8- +/- 0.5-fold (n = 5, P < .005) and 1.8- +/- 0.3-fold (n = 5, P < .05), respectively, but had no effect on glucokinase gene expression (1.1- +/- 0.1-fold increase, n = 4, NS). These results validate the use of semiquantitative RT-PCR to detect changes in gene expression in rat islets. Islets were then cultured in 5.6 or 16.7 mmol/L glucose for 2, 4, or 6 weeks. Relative insulin mRNA levels were higher in islets cultured in high glucose after 2 weeks (1.8+/-0.1 v 1.0+/-0.1, n = 4, P < .05), identical after 4 weeks (0.9+/-0.1 v 1.00+/-0.2, n = 4, NS), and significantly lower after 6 weeks (0.6+/-0.1 v 1.0+/-0.2, n = 6, P < .05). Relative GLUT2 mRNA levels were higher in islets cultured in high glucose after 2 weeks (1.7+/-0.2 v 1.0+/-0.2, n = 3, P < .05) and then identical in both groups after 4 weeks (1.0+/-0.1 v 1.0+/-0.1, n = 3, NS) and 6 weeks (1.0+/-0.2 v 1.0+/-0.1, n = 6, NS). Relative glucokinase mRNA levels were identical under both culture conditions at 2 (1.4+/-0.4 v 1.0+/-0.2, n = 3, NS), 4 (0.8+/-0.5 v 1.0+/-0.3, n = 3, NS), and 6 (0.9+/-0.2 v 1.0+/-0.1, n = 6, NS) weeks. These results indicate that a 6-week exposure of rat islets to supraphysiologic glucose concentrations decreases insulin mRNA levels without affecting GLUT2 and glucokinase gene expression. We conclude that the phenomenon of glucose toxicity decreasing insulin gene expression is not restricted to transformed cells, and might provide insight into the mechanisms by which chronic hyperglycemia adversely affects beta-cell function.

Endothelial Cells From Visceral Adipose Tissue Disrupt Adipocyte Functions in a Three-Dimensional Setting: Partial Rescue by Angiopoietin-1

During obesity, chronic inflammation of human white adipose tissue (WAT) is associated with metabolic and vascular alterations. Endothelial cells from visceral WAT (VAT-ECs) exhibit a proinflammatory and senescent phenotype and could alter adipocyte functions. We aimed to determine the contribution of VAT-ECs to adipocyte dysfunction related to inflammation and to rescue these alterations by anti-inflammatory strategies. We developed an original three-dimensional setting allowing maintenance of unilocular adipocyte functions. Coculture experiments demonstrated that VAT-ECs provoked a decrease in the lipolytic activity, adipokine secretion, and insulin sensitivity of adipocytes from obese subjects, as well as an increased production of several inflammatory molecules. Interleukin (IL)-6 and IL-1β were identified as potential actors in these adipocyte alterations. The inflammatory burst was not observed in cocultured cells from lean subjects. Interestingly, pericytes, in functional interactions with ECs, exhibited a proinflammatory phenotype with diminished angiopoietin-1 (Ang-1) secretion in WAT from obese subjects. Using the anti-inflammatory Ang-1, we corrected some deleterious effects of WAT-ECs on adipocytes, improving lipolytic activity and insulin sensitivity and reducing the secretion of proinflammatory molecules. In conclusion, we identified a negative impact of VAT-ECs on adipocyte functions during human obesity. Therapeutic options targeting EC inflammation could prevent adipocyte alterations that contribute to obesity comorbidities.