Histamine is an important mediator in the development of allergic reactions. Only a small subset of human cell types is able to produce histamine. No previous studies have shown that human neutrophils are among them. The present work was undertaken to analyze whether human neutrophils produce histamine, and to determine what agonists are involved in histamine production by human neutrophils. The expression of histidine decarboxylase in human neutrophils was established by quantitative PCR, Western blotting, and flow cytometry analysis. The activity of the enzyme was determined by ELISA, which measured histamine in the culture supernatant of neutrophils stimulated with a set of classical agonists. Human neutrophils are bona fide histamine-producing cells. Neutrophils store ?0.29 pg/cell and release ?50% of the histamine content in an antigen-dependent manner and on stimulation with other neutrophil agonists. Basal expression of histidine decarboxylase, the rate-limiting enzyme in histamine production, is higher in neutrophils from patients with allergies than from healthy donors. Our results cannot be ascribed to cell contamination for several reasons. LPS failed to induce histamine release by basophils, whereas it induced histamine release by neutrophils; and we did not detect basophils, monocytes, or lymphocytes in our neutrophil preparations. Eosinophils, albeit detected, were only 0.001-0.004% of the final cell population, and they did not store or release histamine on antigen or LPS stimulation. Antigens to which patients with allergies were sensitized stimulated release of histamine from neutrophils. These observations represent a novel view of neutrophils as possible source of histamine in the allergic diseases.
OBJECTIVE: Both glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are gut hormones involved in energy homoeostasis. Obesity, insulin resistance and hyperglycaemia are significant confounders when GLP-1 and PYY secretion is assessed. Thus, we evaluated GLP-1 and PYY response after fat load in morbidly obese patients with different degrees of insulin resistance and glycemic status. DESIGN: We studied 40 morbidly obese subjects (mean age, 40·6 ± 1·3 years; mean BMI, 53·1 ± 1·2 kg/m(2) ) divided into groups according to their glycemic status: normal fasting glucose (NFG) group, impaired fasting glucose (IFG) group and type 2 diabetes mellitus (T2D) group. NFG patients were additionally subclassified, according to the homoeostasis model assessment of insulin resistance (HOMAIR ), into a low insulin-resistance (LIR) group (HOMAIR <3·9) or a high insulin-resistance (HIR) group (HOMAIR ?3·9). MEASUREMENTS: Lipid emulsion was administered orally and measurements made at baseline and 180 min postprandially of levels of GLP-1, PYY, insulin, glucose, free fatty acids, triglycerides and leptin. RESULTS: At the 180-minute postprandial reading, GLP-1 and PYY had increased in LIR-NFG subjects (41·84%, P = 0·01; 35·7%, P = 0·05; respectively), whereas no changes were observed in HIR-NFG, IFG or T2D subjects. CONCLUSIONS: These results suggest that in morbidly obese subjects, both insulin resistance and abnormal glucose metabolism (IFG or T2D) impair the GLP-1 and PYY response to fat load. The implications of this attenuated enteroendocrine response should be elucidated by further studies.
Munc18c is associated with glucose metabolism and could play a relevant role in obesity. However, little is known about the regulation of Munc18c expression. We analyzed Munc18c gene expression in human visceral (VAT) and subcutaneous (SAT) adipose tissue and its relationship with obesity and insulin.
The increase in glucagon-like peptide-1 (GLP-1) activity has emerged as a useful therapeutic tool for the treatment of type 2 diabetes mellitus. The actions of GLP-1 on ?-cells and the nervous and digestive systems are well known. The action of this peptide in adipose tissue (AT), however, is still poorly defined. Furthermore, no relationship has been established between GLP-1 receptor (GLP-1R) in AT and obesity and insulin resistance (IR). We provide evidence for the presence of this receptor in AT and show that its mRNA and protein expressions are increased in visceral adipose depots from morbidly obese patients with a high degree of IR. Experiments with the 3T3-L1 cell line showed the lipolytic and lipogenic dose-dependent effect of GLP-1. Moreover, GLP-1 stimulated lipolysis in 3T3-L1 adipocytes in a receptor-dependent manner involving downstream adenylate cyclase/cAMP signaling. Our data also demonstrate that the expression of the GLP-1R in AT correlated positively with the homeostasis model assessment index in obese IR subjects. Furthermore, prospective studies carried out with patients that underwent biliopancreatic diversion surgery showed that subjects with high levels of GLP-1R expression in AT, which indicates a deficit of GLP-1 in this tissue, were those whose insulin sensitivity improved after surgery, suggesting the potential relationship between AT GLP-1R and insulin sensitivity amelioration in obese subjects. Altogether these results indicate that the GLP-1/GLP-1R system in AT represents another potential candidate for improving insulin sensitivity in obese patients.
Several epidemiological studies have related an increase of lipids in the postprandial state to an individual risk for the development of CVD, possibly due to the increased plasma levels of TAG and fatty acids (FA) through enzymes of FA metabolism. The interaction between nutrition and the human genome determines gene expression and metabolic response. The aim of the present study was to evaluate the influence of a fat overload on the gene mRNA levels of lipogenic regulators in peripheral blood mononuclear cells (PBMC) from patients with the metabolic syndrome. The study included twenty-one patients with criteria for the metabolic syndrome who underwent a fat overload. Measurements were made before and after the fat overload of anthropometric and biochemical variables and also the gene mRNA levels of lipogenic factors. The main results were that the fat overload led to an increased mRNA levels of sterol regulatory element binding protein-1 (SREBP1), retinoid X receptor ? (RXR?) and liver X receptor ? (LXR?) in PBMC, and this increase was associated with the FA synthase (FASN) mRNA levels. We also found that TAG levels correlated with FASN mRNA levels. In addition, there was a positive correlation of SREBP1 with RXR? and of LXR? with the plasma lipoperoxide concentration. The fat overload led to an increase in regulators of lipogenesis in PBMC from patients with the metabolic syndrome.
A paradoxical but common finding in the obesity clinic is the identification of individuals who can be considered inappropriately healthy for their degree of obesity. We think that studying these obese but metabolically healthy individuals and comparing them with equally obese but insulin-resistant individuals could provide important insights into the mechanistic link between adipose tissue expansion and associated metabolic alterations. In the present study, we investigated whether there are differences in inflammatory and insulin signalling pathways in VAT (visceral adipose tissue) that could account for the metabolic differences exhibited by morbidly obese individuals who are either insulin-resistant (IR-MO) or paradoxically insulin-sensitive (NIR-MO). Our results indicate that there are pathways common to obesity and unrelated to insulin resistance and others that are discriminative for insulin resistance for a similar degree of obesity. For instance, all morbidly obese patients, irrespective of their insulin resistance, showed increased expression of TNFalpha (tumour necrosis factor alpha) and activation of JNK1/2 (c-Jun N-terminal kinase 1/2). However, the IR-MO group showed significantly elevated expression levels of IL (interleukin)-1beta and IL-6 and increased macrophage infiltrates compared with non-obese individuals and NIR-MO. IkappaBalpha [inhibitor of NF-kappaB (nuclear factor kappaB) alpha], the activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) and NF-kappaB were discriminative of the state of insulin resistance and correlated with differential changes in IRS-1 (insulin receptor substrate 1) expression and Akt activation between IR-MO and NIR-MO individuals. Our results support the concept that NIR-MO individuals lack the inflammatory response that characterizes the IR-MO patient and that IL-6, IL-1beta, ERK and NF-kappaB are important effectors that mediate the inflammation effects promoting insulin resistance.
Studies indicate that both alterations in leukocyte and endothelial cell adhesion molecules and the renin angiotensin system are involved in the pathogenesis of atherosclerosis processes in human hypertension. The present work was undertaken to investigate whether angiotensin II (Ang II) regulates the expression of CD62L on human neutrophils. Human neutrophils were stimulated with Ang II in the presence of various AT1-receptor antagonists and protein kinase inhibitors, and CD62L cell surface expression was detected by flow cytometry. We report for the first time that Ang II down-regulated CD62L from the surface of human neutrophils, a process which was independent of neutrophil adhesion to endothelium since neutrophils were still able to adhere to human umbilical vein endothelial cells even under doses that almost completely release CD62L from the cell surface. This process occurred through pathways involving AT1 receptors, extracellular signal-regulated kinases 1 and 2 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase, and calcineurin, ruling out a role for p38 MAPK and small GTPases in the process.
The endogenous proangiogenic properties of adipose tissue are well recognized. Although the adult human thymus has long been known to degenerate into fat tissue, it has never been considered as a potential source of angiogenic factors. We have investigated the expression of diverse angiogenic factors, including vascular endothelial growth factor A and B, angiopoietin 1, and tyrosine-protein kinase receptor-2 (an angiopoietin receptor), and then analyzed their physiological role on endothelial cell migration and proliferation, two relevant events in angiogenesis. The detection of the gene and protein expression of the various proteins has been performed by immunohistochemistry, Western blotting, and quantitative real-time polymerase chain reaction. We show, for the first time, that adult thymus fat produces a variety of angiogenic factors and induces the proliferation and migration of human umbilical cord endothelial cells. Based on these findings, we suggest that this fat has a potential angiogenic function that might affect thymic function and ongoing adipogenesis within the thymus.
Fragile X syndrome is the most common genetic cause of mental disability. The mechanisms underlying the pathogenesis remain unclear and specific treatments are still under development. Previous studies have proposed an abnormal hypothalamic-pituitary-adrenal axis and high cortisol levels are demonstrated in the fragile X patients. Additionally, we have previously described that NADPH-oxidase activation leads to oxidative stress in the brain, representing a pathological mechanism in the fragile X mouse model. Fmr1-knockout mice develop an altered free radical production, abnormal glutathione homeostasis, high lipid and protein oxidation, accompanied by stress-dependent behavioral abnormalities and pathological changes in the first months of postnatal life. Chronic pharmacological treatment with alpha-tocopherol reversed pathophysiological hallmarks including free radical overproduction, oxidative stress, Rac1 and alpha-PKC activation, macroorchidism, and also behavior and learning deficits. The restoration of the oxidative status in the fragile X mouse emerges as a new and promising approach for further therapeutic research in fragile X syndrome.
It is well known that the adult human thymus degenerates into fat tissue; however, it has never been considered as a potential source of angiogenic factors. Recently, we have described that this fat (TAT) produces angiogenic factors and induces human endothelial cell proliferation and migration, indicating its potential angiogenic properties.
Fragile X syndrome is the most common form of inherited mental retardation. It is typically caused by a mutation of the Fragile X mental-retardation 1 (Fmr1) gene. To better understand the role of the Fmr1 gene and its gene product, the fragile X mental-retardation protein in central nervous system functions, an fmr1 knockout mouse that is deficient in the fragile X mental-retardation protein was bred. In the present study, fragile X mental retardation 1-knockout and wild-type mice are used to determine behaviour and oxidative stress alterations, including reduced glutathione, oxidized glutathione and thiobarbituric acid-reactive substances, before and after chronic treatment with melatonin or tianeptine. Reduced glutathione levels were reduced in the brain of fmr1-knockout mice and chronic melatonin treatment normalized the glutathione levels compared with the control group. Lipid peroxidation was elevated in brain and testes of fmr1-knockout mice and chronic melatonin treatment prevents lipid peroxidation in both tissues. Interestingly, chronic treatment with melatonin alleviated the altered parameters in the fmr1-knockout mice, including abnormal context-dependent exploratory and anxiety behaviours and learning abnormalities. Chronic treatment with tianeptine (a serotonin reuptake enhancer) did not normalize the behaviour in fmr1-knockout mice. The prevention of oxidative stress in the fragile X mouse model, by an antioxidant compound such as melatonin, emerges as a new and promising approach for further investigation on treatment trials for the disease.
Obesity is associated with a low-grade chronic inflammation state. As a consequence, adipose tissue expresses pro-inflammatory cytokines that propagate inflammatory responses systemically elsewhere, promoting whole-body insulin resistance and consequential islet ?-cell exhaustation. Thus, insulin resistance is considered the early stage of type 2 diabetes. However, there is evidence of obese individuals that never develop diabetes indicating that the mechanisms governing the association between the increase of inflammatory factors and type 2 diabetes are much more complex and deserve further investigation. We studied for the first time the differences in insulin signalling and inflammatory pathways in blood and visceral adipose tissue (VAT) of 20 lean healthy donors and 40 equal morbidly obese (MO) patients classified in high insulin resistance (high IR) degree and diabetes state. We studied the changes in proinflammatory markers and lipid content from serum; macrophage infiltration, mRNA expression of inflammatory cytokines and transcription factors, activation of kinases involved in inflammation and expression of insulin signalling molecules in VAT. VAT comparison of these experimental groups revealed that type 2 diabetic-MO subjects exhibit the same pro-inflammatory profile than the high IR-MO patients, characterized by elevated levels of IL-1?, IL-6, TNF?, JNK1/2, ERK1/2, STAT3 and NF?B. Our work rules out the assumption that the inflammation should be increased in obese people with type 2 diabetes compared to high IR obese. These findings indicate that some mechanisms, other than systemic and VAT inflammation must be involved in the development of type 2 diabetes in obesity.
Calcineurin (protein phosphatase 2B) (CN) comprises a family of serine/threonine phosphatases that play a pivotal role in signal transduction cascades in a variety of cells, including neutrophils. Angiotensin II (Ang II) increases both activity and de novo synthesis of CN in human neutrophils. This study focuses on the role that intracellular redox status plays in the induction of CN activity by Ang II. Both de novo synthesis of CN and activity increase promoted by Ang II were downregulated when cells were treated with L-buthionine-(S,R)-sulfoximine, an inhibitor of synthesis of the antioxidant glutathione. We have also investigated the effect of pyrrolidine dithiocarbamate and phenazine methosulfate, which are antioxidant and oxidant compounds, respectively, and concluded that the intracellular redox status of neutrophils is highly critical for Ang II-induced increase of CN expression and activity. Results obtained in neutrophils from hypertensive patients were very similar to those obtained in these cells on treatment with Ang II. We have also addressed the possible functional implication of CN activation in the development of hypertension. Present findings indicate that downregulation of hemoxygenase-1 expression in neutrophils from hypertensive subjects is likely mediated by CN, which acts by hindering translocation to the nucleus of the transcription factor NRF2. These data support and extend our previous results and those from other authors on modulation of CN expression and activity levels by the intracellular redox status.
Aging negatively affects angiogenesis which is found to be linked to declined vascular endothelial growth factor (VEGF) production. Adult human thymus degenerates into fat tissue (thymus adipose tissue (TAT)). Recently, we described that TAT from cardiomyopathy ischemic subjects has angiogenic properties. The goal of our study was to analyze whether aging could also impair angiogenic properties in TAT as in other adipose tissue such as subcutaneous (subcutaneous adipose tissue (SAT)). SAT and TAT specimens were obtained from 35 patients undergoing cardiac surgery, making these tissues readily available as a prime source of adipose tissue. Patients were separated into two age-dependent groups; middle-aged (n?=?18) and elderly (n?=?17). Angiogenic, endothelial, and adipogenic expression markers were analyzed in both tissues from each group and correlations were examined between these parameters and also with age. There were no significant differences in subjects from either group in clinical or biological variables. Angiogenic markers VEGF-A, B, C, and D and adipogenic parameters, peroxisome proliferator-activated receptors (PPAR?2), FABP4, and ADRP showed elevated expression levels in TAT from elderly patients compared to the middle-aged group, while in SAT, expression levels of these isoforms were significantly decreased in elderly patients. VEGF-R1, VEGF-R2, VEGF-R3, Thy1, CD31, CD29, and VLA1 showed increased levels in TAT from the elderly compared to the middle-aged, while in SAT these levels displayed a decline with aging. Also, in TAT, angiogenic and endothelial parameters exhibited strong positive correlations with age. TAT appears to be the most appropriate source of angiogenic and endothelial factors in elderly cardiomyopathy subjects compared to SAT.
The expansion of adipose tissue is linked to the development of its vasculature, which appears to have the potential to regulate the onset of obesity. However, at present, there are no studies highlighting the relationship between human adipose tissue angiogenesis and obesity-associated insulin resistance (IR).
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