RESUMEN
Glutaraldehyde preservation is the gold standard for cardiovascular biological prosthesis. However, secondary calcifications and the absence of tissue growth remain major limitations. Our study assessed in vitro and in vivo the biocompatibility of human (fascia lata, pericardium) and porcine tissues (pericardium, peritoneum) treated with a physicochemical procedure for decellularization and non-conventional pathogens inactivation. Biopsies were performed before and after treatment to assess decellularization (HE/Dapi staining/DNA quantification/MHC I/alpha gal immunostaining) and mechanical integrity. Forty-five rats received an abdominal aortic patch of native cryopreserved tissues (n = 20), treated tissues (n = 20) or glutaraldehyde-preserved bovine pericardium (GBP, control, n = 5). Grafts were explanted at 4 weeks and processed for HE/von Kossa staining and immunohistochemistries for lymphocytes (CD3)/macrophages (CD68) histomorphometry. 95% of decellularization was obtained for all tissues except for fascia lata (75%). Mechanical properties were slightly altered. In the in vivo model, a significant increase of CD3 and CD68 infiltrations was found in native and control implants in comparison with decellularized tissues (p < 0.05). Calcifications were found in 3 controls. Decellularized tissues were recolonized. GBP showed the most inflammatory response. This physicochemical treatment improves the biocompatibility of selected xeno/allogeneic tissues in comparison with their respective native cryopreserved tissues and with GBP. Incomplete decellularization is associated with a significantly higher inflammatory response. Our treatment is a promising tool in the field of tissue decellularization and tissue banking.
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Prótesis Vascular , Glutaral/química , Pericardio/química , Andamios del Tejido/química , Animales , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Fenómenos Biomecánicos , Prótesis Vascular/efectos adversos , Complejo CD3/análisis , Bovinos , ADN/análisis , Fascia Lata/química , Fascia Lata/citología , Femenino , Glutaral/efectos adversos , Humanos , Inflamación/etiología , Masculino , Ensayo de Materiales , Pericardio/citología , Peritoneo/química , Peritoneo/citología , Ratas , Ratas Wistar , PorcinosRESUMEN
BACKGROUND: Reconstruction of muscle defects remains a challenge. Our work assessed the potential of an engineered construct made of a human acellular collagen matrix (HACM) seeded with porcine mesenchymal stem cells (MSCs) to reconstruct abdominal wall muscle defects in a rodent model. METHODS: This study compared 2 sources of MSCs (bone-marrow, BMSCs, and adipose, ASCs) in vitro and in vivo for parietal defect reconstruction. Cellular viability and growth factor release (VEGF, FGF-Beta, HGF, IGF-1, TGF-Beta) were investigated under normoxic/hypoxic culture conditions. Processed and recellularized HACMs were mechanically assessed. The construct was tested in vivo in full thickness abdominal wall defect treated with HACM alone vs. HACM+ASCs or BMSCs (n=14). Tissue remodeling was studied at day 30 for neo-angiogenesis and muscular reconstruction. RESULTS: A significantly lower secretion of IGF was observed with ASCs vs. BMSCs under hypoxic conditions (-97.6%, p<0.005) whereas significantly higher VEGF/FGF secretions were found with ASCs (+92%, p<0.001 and +72%, p<0.05, respectively). Processing and recellularization did not impair the mechanical properties of the HACM. In vivo, angiogenesis and muscle healing were significantly improved by the HACM+ASCs in comparison to BMSCs (p<0.05) at day 30. CONCLUSION: A composite graft made of an HACM seeded with ASCs can improve muscle repair by specific growth factor release in hypoxic conditions and by in vivo remodeling (neo-angiogenesis/graft integration) while maintaining mechanical properties.
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Pared Abdominal/cirugía , Abdominoplastia/métodos , Tejido Adiposo/trasplante , Células de la Médula Ósea/citología , Colágeno/farmacología , Hernia Abdominal/cirugía , Células Madre Mesenquimatosas/citología , Tejido Adiposo/citología , Animales , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Hernia Abdominal/patología , Humanos , PorcinosRESUMEN
Aneurysmal bone cystic (ABC) lesions can be primary or secondary (to a trauma or a pre-existing benign or malignant tumour). Specific translocations of the USP6 gene are reported in about 70% of primary but never in secondary ABC lesions. We report two cases of ABC lesions in which imbalanced genomic aberrations were detected at initial presentation and showed complex clonal evolution. These demonstrative observations strengthen the guidelines regarding the diagnostic approach when an ABC is suggested by imaging. Biopsy is mandatory including genomic analysis. When a primary ABC is not clearly proven by the initial biopsy, an extensive curettage should be performed, with pathological examination of all removed tissue in order to exclude a secondary ABC. It also illustrates the added value of genomic analyses in the setting of an ABC lesion: complex clonal aberrations argues for a lesion secondary to a malignant proliferation whereas USP6 rearrangement allows the diagnosis of primary ABC.
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Quistes Óseos Aneurismáticos/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas/genética , Ubiquitina Tiolesterasa/genética , Adolescente , Quistes Óseos Aneurismáticos/diagnóstico por imagen , Quistes Óseos Aneurismáticos/patología , Quistes Óseos Aneurismáticos/cirugía , Femenino , Pruebas Genéticas , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Imagen por Resonancia Magnética , Masculino , RadiografíaRESUMEN
ß-Cell plasticity governs the adjustment of ß-cell mass and function to ensure normoglycemia. The study of how ß-cell mass is controlled and the identification of alternative sources of ß-cells are active fields of research. ß-Cell plasticity has been implicated in numerous physiological and pathological conditions. We developed a mice model in which we induced major ß-cell mass atrophy by implanting insulin pellets (IPI) for 7 or 10 days. The implants were then removed (IPR) to observe the timing and characteristics of ß-cell regeneration in parallel to changes in glycemia. Following IPR, the endocrine mass was reduced by 60% at day 7 and by 75% at day 10, and transient hyperglycemia was observed, which resolved within 1 wk. Five days after IPR, enhanced ß-cell proliferation and an increased frequency of small islets were observed in 7-day IPI mice. ß-Cell mass was fully restored after an additional 2 days. For the 10-day IPI group, ß-cell and endocrine mass were no longer significantly different from those of the control group at 2 wk post-IPR. Furthermore, real-time quantitative PCR analysis of endocrine structures isolated by laser capture microdissection indicated sequentially enhanced expression of the pancreatic transcription factors ß(2)/NeuroD and Pdx-1 post-IPR. Thus, our data suggest this mouse model of ß-cell plasticity not only relies on replication but also involves enhanced cell differentiation plasticity.
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Hipoglucemia/inducido químicamente , Hipoglucemiantes/farmacología , Células Secretoras de Insulina , Insulina/farmacología , Islotes Pancreáticos , ARN Mensajero/análisis , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Aumento de la Célula/efectos de los fármacos , Proliferación Celular , Hiperinsulinismo Congénito , Femenino , Glucosa/metabolismo , Transportador de Glucosa de Tipo 2/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/patología , Células Secretoras de Insulina/fisiología , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/fisiología , Captura por Microdisección con Láser , Ratones , Nesidioblastosis , Reacción en Cadena de la Polimerasa , Regeneración/efectos de los fármacos , Regeneración/fisiología , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Morphological studies of the pancreas in persistent hyperinsulinemic hypoglycemia of infancy (PHHI) have focused on the diagnosis of focal vs. diffuse forms, a distinction that determines the optimal surgical management. ABCC8 or KCNJ11 genomic mutations are present in most of them. AIM: Our aim was to report a new form of PHHI with peculiar morphological and clinical characteristics. RESEARCH DESIGN AND METHODS: Histopathological review of 217 pancreatic PHHI specimens revealed 16 cases morphologically different from diffuse and focal forms. They were analyzed by conventional microscopy, quantitative morphometry, immunohistochemistry, and in situ hybridization. RESULTS: Their morphological peculiarity was the coexistence of two types of islet: large islets with cytoplasm-rich ß-cells and occasional enlarged nuclei and shrunken islets with ß-cells exhibiting little cytoplasm and small nuclei. In small islets, ß-cells had abundant insulin content but limited amount of Golgi proinsulin. Large islets had low insulin storage and high proinsulin production and were mostly confined to a few lobules. No evidence for K(ATP) channels involvement or 11p15 deletion was found. Genomic mutations for ABCC8, KCNJ11, and GCK were absent. Patients had normal birth weight and late hypoglycemia onset and improved with diazoxide. Ten were cured by limited pancreatectomy. Six recurred after surgery and were medically controlled. CONCLUSION: This new form of PHHI is characterized by a morphological mosaicism. Pathologists should recognize this mosaicism on intraoperative frozen sections because it is often curable by partial pancreatectomy. The currently unknown genetic background does not involve the classical genomic mutations responsible for diffuse and focal PHHI.
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Hiperinsulinismo Congénito/patología , Islotes Pancreáticos/patología , Hiperinsulinismo Congénito/genética , Hiperinsulinismo Congénito/cirugía , Femenino , Humanos , Lactante , Recién Nacido , Islotes Pancreáticos/cirugía , Masculino , Repeticiones de Microsatélite , Mosaicismo , Mutación , Pancreatectomía , Resultado del TratamientoRESUMEN
The present study aimed to validate ThermoFisher's (Thermo Fisher Scientific, Runcorn, Cheshire, UK) Papspin (PS) for human papillomavirus (HPV) testing by in-house PCR and by the Hybrid Capture II (HC2) assay and to compare the results with those obtained using Specimen Transport Medium (STM) (Digene Diagnostics, Gaithersburg, MD, USA). Forty-five patients underwent conization for known lesions ranging from atypical squamous cells of undetermined significance (ASC-US) with high-risk HPV (hr-HPV) to high-grade squamous intraepithelial lesion (H-SIL/CIN2+) or adenocarcinoma. Two negative controls were included: one patient with post-menopausal bleeding and another from whom an inflammatory cervical sample was taken without conization. Prior to conization, a gynaecologist collected two cervical samples, fixed in PS or STM, from each patient. All but four cases were tested for panHPV (GP5+/GP6+) and specific hr-HPV subtypes (HPV16, 18, 31,33) by PCR using both media and all were processed for HC2. This study demonstrates that both HPV detection techniques work with PS, showing a specificity of 78.3% for HC2 and 92.8% for PCR compared to 83.8% for HC2 and 92% for PCR using STM. The efficacy of detecting HPV in PS-preserved H-SIL/CIN2+ was very high (96% for PCR using PS and 86% for HC2 using PS), which was in the same range as for PCR using STM, and which was only slightly lower than for HC2 using STM (96% and 89%, respectively). The differences were not statistically significant. It is concluded that ThermoFisher's PS is a valid liquid-based cytology medium for cervical samples, convenient for HPV testing by PCR with GP5+/GP6+ primers and by the HC2 assay.
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Cuello del Útero/virología , Papillomaviridae/clasificación , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Virología/métodos , Adulto , Anciano , Cartilla de ADN/genética , Femenino , Humanos , Persona de Mediana Edad , Hibridación de Ácido Nucleico/métodos , Papillomaviridae/genética , Infecciones por Papillomavirus/virología , Sensibilidad y Especificidad , Manejo de Especímenes/métodosRESUMEN
Pancreatic beta-cells exposed to high glucose concentrations display altered gene expression, function, survival and growth that may contribute to the slow deterioration of the functional beta-cell mass in type 2 diabetes. These glucotoxic alterations may result from various types of stress imposed by the hyperglycaemic environment, including oxidative stress, endoplasmic reticulum stress, cytokine-induced apoptosis and hypoxia. The glucose regulation of oxidative stress-response and integrated stress-response genes in cultured rat islets follows an asymmetric V-shaped profile parallel to that of beta-cell apoptosis, with a large increase at low glucose and a moderate increase at high vs. intermediate glucose concentrations. These observations suggest that both types of stress could play a role in the alteration of the functional beta-cell mass under states of prolonged hypoglycaemia and hyperglycaemia. In addition, beta-cell demise under glucotoxic conditions may also result from beta-cell hypoxia and, in vivo, from their exposure to inflammatory cytokines released locally by non-endocrine islet cells. A better understanding of the relative contribution of each type of stress to beta-cell glucotoxicity and of their pathophysiological cause in vivo may lead to new therapeutic strategies to prevent the slow deterioration of the functional beta-cell mass in glucose intolerant and type 2 diabetic patients.
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Diabetes Mellitus Tipo 2/fisiopatología , Retículo Endoplásmico/fisiología , Glucosa/metabolismo , Células Secretoras de Insulina/fisiología , Estrés Oxidativo/fisiología , Animales , Apoptosis/fisiología , Hipoxia de la Célula/fisiología , Progresión de la Enfermedad , Expresión Génica , Humanos , Hiperglucemia/fisiopatología , Hipoglucemia/fisiopatología , Células Secretoras de Insulina/efectos de los fármacos , RatasRESUMEN
BACKGROUND AND AIMS: Obese and diabetic mice display enhanced intestinal permeability and metabolic endotoxaemia that participate in the occurrence of metabolic disorders. Our recent data support the idea that a selective increase of Bifidobacterium spp. reduces the impact of high-fat diet-induced metabolic endotoxaemia and inflammatory disorders. Here, we hypothesised that prebiotic modulation of gut microbiota lowers intestinal permeability, by a mechanism involving glucagon-like peptide-2 (GLP-2) thereby improving inflammation and metabolic disorders during obesity and diabetes. METHODS: Study 1: ob/ob mice (Ob-CT) were treated with either prebiotic (Ob-Pre) or non-prebiotic carbohydrates as control (Ob-Cell). Study 2: Ob-CT and Ob-Pre mice were treated with GLP-2 antagonist or saline. Study 3: Ob-CT mice were treated with a GLP-2 agonist or saline. We assessed changes in the gut microbiota, intestinal permeability, gut peptides, intestinal epithelial tight-junction proteins ZO-1 and occludin (qPCR and immunohistochemistry), hepatic and systemic inflammation. RESULTS: Prebiotic-treated mice exhibited a lower plasma lipopolysaccharide (LPS) and cytokines, and a decreased hepatic expression of inflammatory and oxidative stress markers. This decreased inflammatory tone was associated with a lower intestinal permeability and improved tight-junction integrity compared to controls. Prebiotic increased the endogenous intestinotrophic proglucagon-derived peptide (GLP-2) production whereas the GLP-2 antagonist abolished most of the prebiotic effects. Finally, pharmacological GLP-2 treatment decreased gut permeability, systemic and hepatic inflammatory phenotype associated with obesity to a similar extent as that observed following prebiotic-induced changes in gut microbiota. CONCLUSION: We found that a selective gut microbiota change controls and increases endogenous GLP-2 production, and consequently improves gut barrier functions by a GLP-2-dependent mechanism, contributing to the improvement of gut barrier functions during obesity and diabetes.
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Ciego/microbiología , Péptido 2 Similar al Glucagón/fisiología , Inflamación/prevención & control , Obesidad/complicaciones , Probióticos/uso terapéutico , Adiposidad/efectos de los fármacos , Adiposidad/fisiología , Animales , Bacterias/aislamiento & purificación , Ciego/fisiopatología , Endotoxemia/etiología , Endotoxemia/prevención & control , Péptido 2 Similar al Glucagón/agonistas , Péptido 2 Similar al Glucagón/antagonistas & inhibidores , Hepatitis/etiología , Hepatitis/prevención & control , Inflamación/etiología , Inflamación/microbiología , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/fisiología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Obesos , Obesidad/microbiología , Obesidad/fisiopatología , Ocludina , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Permeabilidad , Fosfoproteínas/metabolismo , Proglucagón/genética , ARN Mensajero/genética , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
Decreases in both beta-cell function and number can contribute to insulin deficiency in type 2 diabetes. Here, we quantified the beta-cell mass in pancreas obtained at autopsy of 57 type 2 diabetic (T2D) and 52 non-diabetic subjects of European origin. Sections from the body and tail were immunostained for insulin. The beta-cell mass was calculated from the volume density of beta-cells (measured by point-counting methods) and the weight of the pancreas. The pancreatic insulin concentration was measured in some of the subjects. beta-cell mass increased only slightly with body mass index (BMI). After matching for BMI, the beta-cell mass was 41% (BMI < 25) and 38% (BMI 26-40) lower in T2D compared with non-diabetic subjects, and neither gender nor type of treatment influenced these differences. beta-cell mass did not correlate with age at diagnosis but decreased with duration of clinical diabetes (24 and 54% lower than controls in subjects with <5 and >15 years of overt diabetes respectively). Pancreatic insulin concentration was 30% lower in patients. In conclusion, the average beta-cell mass is about 39% lower in T2D subjects compared with matched controls. Its decrease with duration of the disease could be a consequence of diabetes that, with further impairment of insulin secretion, contributes to the progressive deterioration of glucose homeostasis. We do not believe that the small difference in beta-cell mass observed within 5 years of onset could cause diabetes in the absence of beta-cell dysfunction.
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Autopsia/métodos , Diabetes Mellitus Tipo 2/patología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Páncreas/patología , Adulto , Anciano , Anciano de 80 o más Años , Glucemia/metabolismo , Índice de Masa Corporal , Progresión de la Enfermedad , Femenino , Humanos , Resistencia a la Insulina/fisiología , Masculino , Persona de Mediana Edad , Obesidad/complicaciones , Población BlancaRESUMEN
Central-omental obesity plays a causative role in the pathogenesis of the metabolic syndrome. Adipokines are involved in the pathogenesis of this syndrome. However, adipokines secreted by omental adipose tissue (OAT) are still poorly characterized in human obesity. Therefore, we searched for novel adipokines abnormally secreted by OAT in obesity and examined their relationships with some features of metabolic syndrome and the respective contribution of adipocytes vs. stromal-vascular cells. OAT from obese and nonobese men was fractionated into adipocytes and SV cells, which were then cultured. Medium was screened by medium-scale protein arrays and ELISAs. Adipokine mRNA levels were measured by real-time RT-qPCR. We detected 16 cytokines secreted by each cellular fraction of lean and obese subjects. Of the 16 cytokines, six adipokines were newly identified as secretory products of OAT, which were dysregulated in obesity: three chemokines (growth-related oncogen factor, RANTES, macrophage inflammatory protein-1beta), one interleukin (IL-7), one tissue inhibitor of metalloproteinases (TIMP-1), and one growth factor (thrombopoietin). Their secretion and expression were enhanced in obesity, with a relatively similar contribution of the two fractions. The higher proportion of macrophages and endothelial cells in obesity may contribute to this enhanced production as well as changes in intrinsic properties of hypertrophied adipocytes. Accordingly, mRNA concentrations of most of these adipokines increased during adipocyte differentiation. Eventually, expression of the investigated adipokines did correlate with several features of the metabolic syndrome. In conclusion, six adipokines were newly identified as oversecreted by OAT in obesity. These adipokines may link obesity to its cardiovascular or metabolic comorbidities.
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Tejido Adiposo/metabolismo , Citocinas/metabolismo , Síndrome Metabólico/metabolismo , Obesidad/metabolismo , Epiplón/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
AIMS/HYPOTHESIS: Sulfonylurea receptor 1 (SUR1) is the regulatory subunit of ATP-sensitive K channels in beta cells. Morphological methods (immunohistochemistry and sulfonylurea binding) were used to establish the cellular and subcellular location of SUR1 in human and rodent islets. RESULTS: In the human, mouse and rat pancreas, all endocrine cells of the islets were immunolabelled with an anti-SUR1 antibody, whereas tissues containing SUR2 were consistently negative, as were those from Sur1 (also known as Abcc8)(-/-) mice. In beta cells of the three species, the plasma membrane was distinctly stained, but SUR1 was mainly present over the cytoplasm, with an intensity that varied between cells. Electron microscopy showed that SUR1 was immunolocalised in insulin, glucagon and somatostatin granules. In rat beta cells degranulated by in vivo treatment with glibenclamide (known as glyburide in the USA and Canada), the insulin and SUR1 staining intensity was similarly decreased by approximately 45%, whereas SUR1 staining was not changed in non-beta cells. In all islet cells, binding of glibenclamide labelled with fluorescent dipyrromethane boron difluoride (BODIPY-FL) was punctate over the cytoplasm, compatible with the labelling of endocrine granules. A faint labelling persisted in Sur1 (-/-) mice, but it was not different from that obtained with BODIPY-FL alone used as negative control. CONCLUSIONS/INTERPRETATION: Our study immunolocalised SUR1 in alpha, beta and delta cells of human, mouse and rat islets, and for the first time visualised it in the plasma membrane. We also show that SUR1 is abundant in endocrine granules, where its function remains to be established. No specific sulfonylurea-binding sites other than SUR1 are identified in islet cells by the glibenclamide-BODIPY-FL technique.
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Transportadoras de Casetes de Unión a ATP/fisiología , Islotes Pancreáticos/fisiología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Páncreas/fisiología , Canales de Potasio de Rectificación Interna/fisiología , Canales de Potasio/fisiología , Receptores de Droga/fisiología , Transportadoras de Casetes de Unión a ATP/genética , Animales , Cadáver , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/deficiencia , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Ratas , Ratas Wistar , Receptores de Sulfonilureas , Donantes de TejidosRESUMEN
AIMS/HYPOTHESIS: Inadequate chaperone function relative to client protein load in the endoplasmic reticulum triggers an adaptive unfolded protein response (UPR), including the integrated stress response (ISR), the latter being also activated by other types of stresses. It is well established that pancreatic beta cells, which synthesise and secrete insulin upon nutrient stimulation, are markedly affected by pathological disruption or excessive activation of the UPR. However, whether and how physiological nutrient stimulation affects the beta cell UPR has been little investigated. MATERIALS AND METHODS: We compared the effects of increasing glucose concentrations and of endoplasmic reticulum Ca(2+) emptying with thapsigargin on the UPR (X-box binding protein [Xbp1] mRNA splicing and XBP1/activating transcription factor [ATF] 6-target gene expression) and ISR (eukaryotic translation initiation factor 2A phosphorylation, ATF4 protein levels and target gene expression) in isolated rat islets. RESULTS: Thapsigargin strongly increased both UPR and ISR. In comparison, glucose moderately increased the UPR between 5 and 30 mmol/l, but exerted complex effects on the ISR as follows: (1) marked reduction between 2 and 10 mmol/l; (2) moderate increase parallel to the UPR between 10 and 30 mmol/l. These glucose effects occurred within 2 h, were mimicked by other metabolic substrates, but were independent of changes in Ca(2+) influx or insulin secretion. Remarkably, attenuating the glucose stimulation of protein synthesis with a low concentration of cycloheximide prevented UPR activation but not ISR reduction by high glucose. CONCLUSIONS/INTERPRETATION: Nutrient stimulation acutely activates rat islet UPR in a manner dependent on protein synthesis, while exerting complex effects on the ISR. These effects may contribute to nutrient-induced maintenance of the beta cell phenotype.
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Células Secretoras de Insulina/metabolismo , Empalme Alternativo , Animales , Calcio/metabolismo , Medios de Cultivo/metabolismo , Cicloheximida/farmacología , Retículo Endoplásmico/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Secreción de Insulina , Cinética , Chaperonas Moleculares/metabolismo , Fosforilación , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Tapsigargina/farmacologíaRESUMEN
AIMS/HYPOTHESIS: Adiponectin is an adipokine that exhibits insulin-sensitising, fat-burning and anti-inflammatory properties as well as modulatory effects on oxidative stress. We examined whether adiponectin could be induced in a non-adipose tissue, skeletal muscle, in response to metabolic or oxidative aggression both in vivo (in a murine model of type 2 diabetes) and in vitro. METHODS: Obese and diabetic ob/ob mice were used and compared with lean littermates. Some obese mice were treated with the antioxidant probucol for 3 weeks. At the end of the experiment, blood was sampled and tibialis anterior muscles were collected for mRNA measurement and immunohistochemistry. Additional in vitro experiments were performed on C2C12 myotubes cultured for up to 48 h. RESULTS: In spite of hypoadiponectinaemia, Adipoq mRNA levels were markedly increased in the skeletal muscle of ob/ob mice and correlated with systemic oxidative stress. Adipoq upregulation was shown in laser-microdissected myocytes of obese mice. Concomitantly, immunoreactivity for adiponectin was enhanced in obese muscle fibres together with lipid infiltration and local markers of oxidative stress. In cultured C2C12 myotubes, a triglyceride mix and reactive oxygen species producers (H2O2 or a lipoperoxidation end-product) upregulated Adipoq expression and adiponectin production. This effect was reversed by an antioxidant. Finally, treatment of obese mice with probucol also attenuated upregulation in muscle. CONCLUSIONS/INTERPRETATION: The paradoxical upregulation of adiponectin in muscle of obese and diabetic mice may result from lipotoxicity and related oxidative stress. This unexpected finding could be viewed as a local protection to counteract ectopic fat deposition and oxidative damage.
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Adiponectina/biosíntesis , Diabetes Mellitus Tipo 2/fisiopatología , Músculo Esquelético/fisiopatología , Adiponectina/genética , Animales , Antioxidantes/farmacología , Peso Corporal , Células Cultivadas , Cartilla de ADN , Femenino , Regulación de la Expresión Génica , Lípidos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Obesidad , Estrés Oxidativo , Reacción en Cadena de la Polimerasa , Probucol/farmacología , ARN/genética , ARN/aislamiento & purificaciónRESUMEN
We previously showed that the stimulation of heme oxygenase-1 expression by high glucose and hydrogen peroxide (H(2)O(2)) in cultured rat islets is prevented by antioxidants and suggested that this effect of high glucose results from an oxidative stress. However, the role of oxidative stress in high-glucose-induced beta-cell dysfunction is unclear. We therefore compared the preventative effects of N-acetyl-l-cysteine (NAC), a free radical scavenger, and manganese(III)tetrakis (4-benzoic acid)porphyrin (MnTBAP), a superoxide dismutase/catalase mimetic agent, on the alteration of stimulus-secretion coupling induced in rat islets by overnight exposure to hydrogen peroxide (H(2)O(2)-treated islets) or 1-wk culture in 30 vs. 10 mmol/l glucose (High-glucose vs. Control islets). The features of beta-cell dysfunction differed between the two groups: reduced glucose-induced insulin secretion without changes in glucose sensitivity in H(2)O(2)-treated islets; increased sensitivity to glucose with parallel reductions in insulin content and maximal rate of glucose-induced insulin secretion in High-glucose islets. The latter alterations were accompanied by a decrease in preproinsulin without changes in pancreatic and duodenal homeobox gene 1 mRNA levels. The functional alterations induced by H(2)O(2) were significantly prevented by addition of NAC or MnTBAP in the culture medium. In contrast, neither NAC nor MnTBAP affected the functional alterations induced by high glucose. These results suggest that beta-cell dysfunction induced by 1-wk culture in high glucose does not result from an increase in oxidative stress.
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Acetilcisteína/farmacología , Depuradores de Radicales Libres/farmacología , Glucosa/administración & dosificación , Células Secretoras de Insulina/efectos de los fármacos , Metaloporfirinas/farmacología , Animales , Calcio/metabolismo , Glucosa/antagonistas & inhibidores , Glucosa/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Histocitoquímica , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microscopía de Contraste de Fase , Proinsulina/genética , Proinsulina/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
UNLABELLED: Hypervitaminosis A-related liver toxicity may be severe and may even lead to cirrhosis. In the normal liver, vitamin A is stored in hepatic stellate cells (HSC), which are prone to becoming activated and acquiring a myofibroblast-like phenotype, producing large amounts of extracellular matrix. AIMS: In order to assess the relationship between vitamin A intake, HSC activation and fibrosis, we studied nine liver biopsies from patients belonging to a well-characterized series of 41 patients with vitamin A hepatotoxicity. METHODS: Fibrosis was underlined by Sirius-red staining, whereas activated HSC were immunohistochemically identified using an antibody against alpha smooth muscle actin. The volume density (Vv) of sinusoidal and total fibrosis and of sinusoidal and total activated HSC was quantified by the point-counting method. RESULTS: Morphology ranged from HSC hypertrophy and hyperplasia as the sole features to severe architectural distortion. There was a significant positive correlation between Vv of perisinusoidal fibrosis and the daily consumption of vitamin A (P=0.004). CONCLUSION: The close correlation between the severity of perisinusoidal fibrosis and the daily dose of the retinol intake suggests the existence of a dose-effect relationship.
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Hipervitaminosis A/inducido químicamente , Macrófagos del Hígado/efectos de los fármacos , Cirrosis Hepática/inducido químicamente , Vitamina A/efectos adversos , Actinas/metabolismo , Adulto , Anciano , Biomarcadores/metabolismo , Aumento de la Célula/efectos de los fármacos , Femenino , Humanos , Hiperplasia/inducido químicamente , Hiperplasia/patología , Hipervitaminosis A/patología , Inmunohistoquímica , Macrófagos del Hígado/metabolismo , Macrófagos del Hígado/patología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/patología , Masculino , Persona de Mediana EdadRESUMEN
AIM: The aim of this study was to study the Galactosyl alpha(1,3) Gal expression and the vascular tissue distribution prior to and after isolation of porcine pancreatic islets. METHODS: Biopsies of native pancreas were carried out in young (12-15 weeks; n = 4) and adult Landrace pigs (2 years old; n = 7). These pancreases were then digested (Liberase Porcine Islets [PI]) to obtain isolated pancreatic islets from each pancreas. Alpha Gal-specific biotinylated BS-1 isolectin B4 and von Willebrand's Factor (vWF) staining were performed for Galactosyl and vascular structure analysis, respectively. Quantitative Galactosyl expression as well as location of the vascular structure were determined using image analysis in pig islets of different sizes. RESULTS: Vascular structures and Galactosyl expression varied following the islet sizes but not the pig age. In large islets (>100 microm), capillaries were mainly located within the islets, whereas in small islets (<100 microm), 4-fold more vessels were situated at the periphery of the islets. Galactosyl expression followed a comparable distribution than vascular tissue in small and large islets. After isolation, a significant decrease of Gal staining (-49%) was observed, but Galactosyl expression remained positive within both small and large islets. CONCLUSIONS: Galactosyl expression is maintained within pancreatic islets after isolation procedure. Gal knock-out pigs could represent the solution to this hurdle.
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Disacáridos/genética , Islotes Pancreáticos/fisiología , Envejecimiento , Análisis de Varianza , Animales , Biopsia , Regulación de la Expresión Génica , Islotes Pancreáticos/irrigación sanguínea , Páncreas/citología , Páncreas/fisiología , PorcinosRESUMEN
OBJECTIVES: To study the impact of porcine islet size on structural properties and cellular engraftment. METHODS: The endocrine structure and collagen/vascular localization in pig islets were studied before and after enzymatic isolation on the pancreas from 6 young and 6 adult Landrace pigs. Isolated islets from both pig types were transplanted under the kidney capsula of diabetic nude rats to assess cellular engraftment. RESULTS: In comparison with adult pig pancreata, a significantly greater number of small beta cells (<100 microm) were observed before and after isolation (82% vs. 32%, respectively, P < 0.005) from young pig pancreata. Small islets (<100 microm) showed a peripheral vascular structure, whereas large islets showed a more centralized vascular organization, thereby providing protection during the enzymatic digestion procedure. The islet endocrine structure was not affected by the islet size, but a loss of glucagon cells (-7.9%, P < 0.005) was observed in large isolated islets. The purity of islet preparation was better with pancreata from adult than young donors (86% vs. 64%, respectively, P < 0.05). A lack of engraftment was observed for small islets from young pig donors as compared with large islets from adult donors. CONCLUSIONS: Large and well-structured islets, mainly found in adult pig pancreata, probably possess a better potential for cellular engraftment due to centralized vascularization and collagen distribution.
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Supervivencia de Injerto , Células Secretoras de Insulina/citología , Trasplante de Islotes Pancreáticos , Factores de Edad , Animales , Biopsia , Capilares , Separación Celular , Tamaño de la Célula , Colágeno/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/irrigación sanguínea , Islotes Pancreáticos/citología , Ratas , Ratas Endogámicas Lew , Sus scrofa , Trasplante HeterólogoRESUMEN
Somatostatin blocks the release of numerous growth factors and is therefore a potent inhibitor of cell division and/or secretion. It exerts its effects through binding to somatostatin receptors. Five different subtypes of such receptors are identified (SSTR1 to SSTR5), having various tissue expression. The detection of their presence in tumours can be performed on histological sections and has potential therapeutic implications.
Asunto(s)
Neoplasias de las Glándulas Endocrinas/metabolismo , Receptores de Somatostatina/metabolismo , Somatostatina/metabolismo , Humanos , Receptores de Somatostatina/clasificación , Somatostatina/fisiologíaRESUMEN
Chronic hyperglycemia has been shown to induce either a lack of response or an increased sensitivity to glucose in pancreatic beta-cells. We reinvestigated this controversial issue in a single experimental model by culturing rat islets for 1 wk in 10 or 30 mmol/l glucose (G10, Controls; or G30, High-glucose islets) before testing the effect of stepwise glucose stimulation from G0.5 to G20 on key beta-cell stimulus-secretion coupling events. Compared with Controls, the glucose sensitivity of High-glucose islets was markedly increased, leading to maximal stimulation of oxidative metabolism and both triggering and amplifying pathways of insulin secretion in G6 rather than G20, hence to loss of glucose effect above G6. This enhanced glucose sensitivity occurred despite an approximately twofold increase in islet uncoupling protein 2 mRNA expression. Besides this increased glucose sensitivity, the maximal glucose stimulation of insulin secretion in High-glucose islets was reduced by approximately 50%, proportionally to the reduction of insulin content. In High-glucose islets, changes in (45)Ca(2+) influx induced by glucose and diazoxide were qualitatively similar but quantitatively smaller than in Control islets and, paradoxically, did not lead to detectable changes in the intracellular Ca(2+) concentration measured by microspectrofluorimetry (fura PE 3). In conclusion, after 1 wk of culture in G30, the loss of glucose stimulation of insulin secretion in the physiological range of glucose concentrations (G5-G10) results from the combination of an increased sensitivity to glucose of both triggering and amplifying pathways of insulin secretion and an approximately 50% reduction in the maximal glucose stimulation of insulin secretion.