RESUMEN
ß-Cell failure is crucial for the onset and progression of human type 2 diabetes, and a few studies have suggested that inflammation may play a role. Immune cell infiltration has been reported in subpopulations of islets in some cases of human type 2 diabetes, and altered gene expression of a few cytokines and chemokines has been observed in isolated islets and laser captured ß-cells from diabetic subjects. Recent observations on the links between inflammation, apoptosis and autophagy are putting the focus on the possibility that modulating the autophagic processes could protect the ß-cells from cytotoxicity induced by inflammatory mediators.
Asunto(s)
Autofagia/fisiología , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/patología , Inflamación/complicaciones , Células Secretoras de Insulina/patología , Animales , Diabetes Mellitus Tipo 2/inmunología , Expresión Génica , Humanos , Inflamación/patología , Células Secretoras de Insulina/metabolismo , Infiltración Neutrófila/fisiologíaRESUMEN
AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) is a major incretin, mainly produced by the intestinal L cells, with beneficial actions on pancreatic beta cells. However, while in vivo only very small amounts of GLP-1 reach the pancreas in bioactive form, some observations indicate that GLP-1 may also be produced in the islets. We performed comprehensive morphological, functional and molecular studies to evaluate the presence and various features of a local GLP-1 system in human pancreatic islet cells, including those from type 2 diabetic patients. METHODS: The presence of insulin, glucagon, GLP-1, proconvertase (PC) 1/3 and PC2 was determined in human pancreas by immunohistochemistry with confocal microscopy. Islets were isolated from non-diabetic and type 2 diabetic donors. GLP-1 protein abundance was evaluated by immunoblotting and matrix-assisted laser desorption-ionisation-time of flight (MALDI-TOF) mass spectrometry. Single alpha and beta cell suspensions were obtained by enzymatic dissociation and FACS sorting. Glucagon and GLP-1 release were measured in response to nutrients. RESULTS: Confocal microscopy showed the presence of GLP-1-like and PC1/3 immunoreactivity in subsets of alpha cells, whereas GLP-1 was not observed in beta cells. The presence of GLP-1 in isolated islets was confirmed by immunoblotting, followed by mass spectrometry. Isolated islets and alpha (but not beta) cell fractions released GLP-1, which was regulated by glucose and arginine. PC1/3 (also known as PCSK1) gene expression was shown in alpha cells. GLP-1 release was significantly higher from type 2 diabetic than from non-diabetic isolated islets. CONCLUSIONS/INTERPRETATION: We have shown the presence of a functionally competent GLP-1 system in human pancreatic islets, which resides in alpha cells and might be modulated by type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Insulina/metabolismo , Femenino , Humanos , Inmunohistoquímica , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Páncreas/metabolismoRESUMEN
AIMS/HYPOTHESIS: We assessed whether per-arnt-sim (PAS) domain-containing protein kinase (PASK) is involved in the regulation of glucagon secretion. METHODS: mRNA levels were measured in islets by quantitative PCR and in pancreatic beta cells obtained by laser capture microdissection. Glucose tolerance, plasma hormone levels and islet hormone secretion were analysed in C57BL/6 Pask homozygote knockout mice (Pask-/-) and control littermates. Alpha-TC1-9 cells, human islets or cultured E13.5 rat pancreatic epithelia were transduced with anti-Pask or control small interfering RNAs, or with adenoviruses encoding enhanced green fluorescent protein or PASK. RESULTS: PASK expression was significantly lower in islets from human type 2 diabetic than control participants. PASK mRNA was present in alpha and beta cells from mouse islets. In Pask-/- mice, fasted blood glucose and plasma glucagon levels were 25 ± 5% and 50 ± 8% (mean ± SE) higher, respectively, than in control mice. At inhibitory glucose concentrations (10 mmol/l), islets from Pask-/- mice secreted 2.04 ± 0.2-fold (p < 0.01) more glucagon and 2.63 ± 0.3-fold (p < 0.01) less insulin than wild-type islets. Glucose failed to inhibit glucagon secretion from PASK-depleted alpha-TC1-9 cells, whereas PASK overexpression inhibited glucagon secretion from these cells and human islets. Extracellular insulin (20 nmol/l) inhibited glucagon secretion from control and PASK-deficient alpha-TC1-9 cells. PASK-depleted alpha-TC1-9 cells and pancreatic embryonic explants displayed increased expression of the preproglucagon (Gcg) and AMP-activated protein kinase (AMPK)-alpha2 (Prkaa2) genes, implying a possible role for AMPK-alpha2 downstream of PASK in the control of glucagon gene expression and release. CONCLUSIONS/INTERPRETATION: PASK is involved in the regulation of glucagon secretion by glucose and may be a useful target for the treatment of type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/enzimología , Islotes Pancreáticos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Línea Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Glucagón/metabolismo , Células Secretoras de Glucagón/efectos de los fármacos , Células Secretoras de Glucagón/metabolismo , Glucosa/farmacología , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Ratones , Ratones Mutantes , Modelos Biológicos , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/genética , RatasAsunto(s)
Diabetes Mellitus/epidemiología , Hepatopatías/epidemiología , Trasplante de Hígado , Biomarcadores/sangre , Glucemia/metabolismo , Diabetes Mellitus/sangre , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/terapia , Femenino , Prueba de Tolerancia a la Glucosa , Hemoglobina Glucada/metabolismo , Humanos , Hepatopatías/diagnóstico , Hepatopatías/cirugía , Trasplante de Hígado/efectos adversos , Masculino , Persona de Mediana Edad , Estado Prediabético/sangre , Estado Prediabético/diagnóstico , Estado Prediabético/epidemiología , Factores de RiesgoRESUMEN
AIMS/HYPOTHESIS: Type 2 diabetes (T2D) is characterized by a progressive loss of beta-cell function, and the "disappearance" of beta-cells in T2D may also be caused by the process of beta -cell dedifferentiation. Since noradrenergic innervation inhibits insulin secretion and density of noradrenergic fibers is increased in type 2 diabetes mouse models, we aimed to study the relation between islet innervation, dedifferentiation and beta-cell function in humans. METHODS: Using immunohistochemistry and electron microscopy, we analyzed pancreata from organ donors and from patients undergoing pancreatic surgery. In the latter, a pre-surgical detailed metabolic characterization by oral glucose tolerance test (OGTT) and hyperglycemic clamp was performed before surgery, thus obtaining in vivo functional parameters of beta-cell function and insulin secretion. RESULTS: The islets of diabetic subjects were 3 times more innervated than controls (0.91⯱â¯0.21 vs 0.32⯱â¯0.10, n.fibers/islet; pâ¯=â¯0.01), and directly correlated with the dedifferentiation score (râ¯=â¯0.39; pâ¯=â¯0.03). In vivo functional parameters of insulin secretion, assessed by hyperglycemic clamp, negatively correlated with the increase in fibers [beta-cell Glucose Sensitivity (râ¯=â¯-0.84; pâ¯=â¯0.01), incremental second-phase insulin secretion (râ¯=â¯-0.84, pâ¯=â¯0.03) and arginine-stimulated insulin secretion (râ¯=â¯-0.76, pâ¯=â¯0.04)]. Moreover, we observed a progressive increase in fibers, paralleling worsening glucose tolerance (from NGT through IGT to T2D). CONCLUSIONS/INTERPRETATION: Noradrenergic fibers are significantly increased in the islets of diabetic subjects and this positively correlates with beta-cell dedifferentiation score. The correlation between in vivo insulin secretion parameters and the density of pancreatic noradrenergic fibers suggests a significant involvement of these fibers in the pathogenesis of the disease, and indirectly, in the islet dedifferentiation process.
Asunto(s)
Neuronas Adrenérgicas/fisiología , Desdiferenciación Celular/fisiología , Diabetes Mellitus Tipo 2/metabolismo , Gliburida/metabolismo , Secreción de Insulina/fisiología , Células Secretoras de Insulina/metabolismo , Fibras Nerviosas/fisiología , Anciano , Glucemia/metabolismo , Femenino , Intolerancia a la Glucosa/metabolismo , Humanos , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
AIMS/HYPOTHESIS: Beta cell failure is a crucial component in the pathogenesis of type 2 diabetes. One of the proposed mechanisms of beta cell failure is local inflammation, but the presence of pancreatic islet inflammation in type 2 diabetes and the mechanisms involved remain under debate. METHODS: Chemokine and cytokine expression was studied by microarray analysis of laser-capture microdissected islets from pancreases obtained from ten non-diabetic and ten type 2 diabetic donors, and by real-time PCR of human islets exposed to oleate or palmitate at 6 or 28 mmol/l glucose. The cellular source of the chemokines was analysed by immunofluorescence of pancreatic sections from individuals without diabetes and with type 2 diabetes. RESULTS: Microarray analysis of laser-capture microdissected beta cells showed increased chemokine and cytokine expression in type 2 diabetes compared with non-diabetic controls. The inflammatory response in type 2 diabetes was mimicked by exposure of non-diabetic human islets to palmitate, but not to oleate or high glucose, leading to the induction of IL-1beta, TNF-alpha, IL-6, IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2). Interference with IL-1beta signalling abolished palmitate-induced cytokine and chemokine expression but failed to prevent lipotoxic human islet cell death. Palmitate activated nuclear factor kappaB (NF-kappaB) in human pancreatic beta and non-beta cells, and chemically induced endoplasmic reticulum stress caused cytokine expression and NF-kappaB activation similar to that occurring with palmitate. CONCLUSIONS/INTERPRETATION: Saturated-fatty-acid-induced NF-kappaB activation and endoplasmic reticulum stress may contribute to IL-1beta production and mild islet inflammation in type 2 diabetes. This inflammatory process does not contribute to lipotoxicity ex vivo, but may lead to local chemokine release.
Asunto(s)
Quimiocina CCL2/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Palmitatos/farmacología , Anciano , Línea Celular , Quimiocina CXCL1 , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas In Vitro , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , Persona de Mediana Edad , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Radioinmunoensayo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
AIMS/HYPOTHESIS: Beta cell loss contributes to type 2 diabetes, with increased apoptosis representing an underlying mechanism. Autophagy, i.e. the physiological degradation of damaged organelles and proteins, may, if altered, be associated with a distinct form of cell death. We studied several features of autophagy in beta cells from type 2 diabetic patients and assessed the role of metabolic perturbation and pharmacological intervention. METHODS: Pancreatic samples were obtained from organ donors and isolated islets prepared both by collagenase digestion and density gradient centrifugation. Beta cell morphology and morphometry were studied by electron microscopy. Gene expression studies were performed by quantitative RT-PCR. RESULTS: Using electron microscopy, we observed more dead beta cells in diabetic (2.24 +/- 0.53%) than control (0.66 +/- 0.52%) samples (p < 0.01). Massive vacuole overload (suggesting altered autophagy) was associated with 1.18 +/- 0.54% dead beta cells in type 2 diabetic samples and with 0.36 +/- 0.26% in control samples (p < 0.05). Density volume of autophagic vacuoles and autophagosomes was significantly higher in diabetic beta cells. Unchanged gene expression of beclin-1 and ATG1 (also known as ULK1), and reduced transcription of LAMP2 and cathepsin B and D was observed in type 2 diabetic islets. Exposure of non-diabetic islets to increased NEFA concentration led to a marked increase of vacuole accumulation, together with enhanced beta cell death, which was associated with decreased LAMP2 expression. Metformin ameliorated autophagy alterations in diabetic beta cells and beta cells exposed to NEFA, a process associated with normalisation of LAMP2 expression. CONCLUSIONS/INTERPRETATION: Beta cells in human type 2 diabetes have signs of altered autophagy, which may contribute to loss of beta cell mass. To preserve beta cell mass in diabetic patients, it may be necessary to target multiple cell-death pathways.
Asunto(s)
Autofagia/fisiología , Diabetes Mellitus Tipo 2/patología , Células Secretoras de Insulina/patología , Células Secretoras de Insulina/ultraestructura , Anciano , Proteínas Reguladoras de la Apoptosis/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia , Beclina-1 , Catepsina B/genética , Catepsina D/genética , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Femenino , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína 2 de la Membrana Asociada a los Lisosomas , Proteínas de Membrana de los Lisosomas/genética , Masculino , Proteínas de la Membrana/genética , Metformina/farmacología , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Type 2 diabetes (T2D) is characterized by reduction of beta-cell mass and dysfunctional insulin secretion. Understanding beta-cell phenotype changes as T2D progresses should help explain these abnormalities. The normal phenotype should differ from the state of overwork when beta-cells compensate for insulin resistance to keep glucose levels normal. When only mild hyperglycaemia develops, beta-cells are subjected to glucotoxicity. As hyperglycaemia becomes more severe, so does glucotoxicity. beta-Cells in all four of these situations should have separate phenotypes. When assessing phenotype with gene expression, isolated islets have artefacts resulting from the trauma of isolation and hypoxia of islet cores. An advantage comes from laser capture microdissection (LCM), which obtains beta-cell-rich tissue from pancreatic frozen sections. Valuable data can be obtained from animal models, but the real goal is human beta-cells. Our experience with LCM and gene arrays on frozen pancreatic sections from cadaver donors with T2D and controls is described. Although valuable data was obtained, we predict that the approach of taking fresh samples at the time of surgery is an even greater opportunity to markedly advance our understanding of how beta-cell phenotype evolves as T2D develops and progresses.
Asunto(s)
Diabetes Mellitus Tipo 2/patología , Hiperglucemia/patología , Resistencia a la Insulina/fisiología , Células Secretoras de Insulina/fisiología , Estrés Oxidativo/fisiología , Páncreas/patología , Autofagia , Cadáver , Diabetes Mellitus Tipo 2/genética , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Hiperglucemia/fisiopatología , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/patología , Microdisección , Estrés Oxidativo/genéticaRESUMEN
Autophagy is the major mechanism involved in degradation and recycling of intracellular components, and its alterations have been proposed to cause beta cell dysfunction. In this study, we explored the effects of autophagy modulation in human islets under conditions associated to endoplasmic reticulum (ER) stress. Human pancreatic islets were isolated by enzymatic digestion and density gradient purification from pancreatic samples of non-diabetic (ND; n = 17; age 65 ± 21 years; gender: 5 M/12 F; BMI 23.4 ± 3.3 kg/m2) and T2D (n = 9; age 76 ± 6 years; 4 M/5 F; gender: BMI 25.4 ± 3.7 kg/m2) organ donors. Nine ND organ donors were treated for hypertension and 1 for both hypertension and hypercholesterolemia. T2D organ donors were treated with metformin (1), oral hypoglycemic agents (2), diet + oral hypoglycemic agents (3), insulin (3) or insulin plus metformin (3) as for antidiabetic therapy and, of these, 3 were treated also for hypertension and 6 for both hypertension and hypercholesterolemia. Two days after isolation, they were cultured for 1-5 days with 10 ng/ml rapamycin (autophagy inducer), 5 mM 3-methyladenine or 1.0 nM concanamycin-A (autophagy blockers), either in the presence or not of metabolic (0.5 mM palmitate) or chemical (0.1 ng/ml brefeldin A) ER stressors. In ND islets palmitate exposure induced a 4 to 5-fold increase of beta cell apoptosis, which was significantly prevented by rapamycin and exacerbated by 3-MA. Similar results were observed with brefeldin treatment. Glucose-stimulated insulin secretion from ND islets was reduced by palmitate (-40 to 50%) and brefeldin (-60 to 70%), and rapamycin counteracted palmitate, but not brefeldin, cytotoxic actions. Both palmitate and brefeldin induced PERK, CHOP and BiP gene expression, which was partially, but significantly prevented by rapamycin. With T2D islets, rapamycin alone reduced the amount of p62, an autophagy receptor that accumulates in cells when macroautophagy is inhibited. Compared to untreated T2D cells, rapamycin-exposed diabetic islets showed improved insulin secretion, reduced proportion of beta cells showing signs of apoptosis and better preserved insulin granules, mitochondria and ER ultrastructure; this was associated with significant reduction of PERK, CHOP and BiP gene expression. This study emphasizes the importance of autophagy modulation in human beta cell function and survival, particularly in situations of ER stress. Tuning autophagy could be a tool for beta cell protection.
RESUMEN
Liver transplantation with very old donors is safe, but is associated with an increased incidence of ischemic-type biliary lesions and delayed graft function. Normothermic machine perfusion (NMP) is a novel technique for preservation of liver grafts and has the potential to reduce ischemia-reperfusion injury. A case is reported here of a liver transplantation (LT) with a graft from an 83-year-old brain-dead donor. Procurement was with dual perfusion and en bloc, modified fast technique. Donor kidneys were not transplanted due to severe atherosclerosis and poor perfusion. The liver was shipped to the transplantation center and underwent NMP with a blood-based perfusate. During machine perfusion lactates decreased, vascular flow was stable, and bile production restored, and the graft was considered suitable for transplantation. The postoperative course was uneventful and 4 months after surgery the patient is in good clinical condition with normal liver function. To date, few LTs have been performed with NMP in humans, but its preliminary results are promising. NMP allows functional evaluation of the graft and possibly reduction of post-transplantation complications when extended-criteria donor grafts are used.
Asunto(s)
Trasplante de Hígado/métodos , Donantes de Tejidos/provisión & distribución , Anciano de 80 o más Años , Humanos , Preservación de Órganos/métodos , Obtención de Tejidos y Órganos/métodosRESUMEN
Studies in rodents have suggested that Th2 and Th3 cytokines can be effective in reducing proinflammatory and Th1 cytokine-induced islet damage. Whether this is the case with human islets and might be due to a direct action of Th2 and Th3 cytokines is not known. In the present study, we evaluated whether Th2 (500 U/ml IL-4 plus 100 U/ml IL-10) or Th3 (5 ng/ml TGF-1beta) cytokines may prevent the derangements induced on isolated human islets by prolonged (12 or 72 h) exposure to combined proinflammatory (50 U/ml IL-1beta, 1000 U/ml TNF alpha) and Th1 (1000 U/ml interferon gamma) cytokines. Compared with control islets, cells preincubated for 12 or 72 h with proinflammatory and Th1 cytokines showed a significant decrease of glucose-stimulated insulin secretion and a significant increase of nitrites production. The addition of IL-4 plus IL-10 or TGF-1beta in the medium prevented these cytostatic effects in the 12-h incubation experiments, but not after the 72-h exposure period. IL-1beta, interferon gamma, and TNF alpha caused no major change in either islet cell survival or Bcl-2 and Bax mRNA expression after a 12-h incubation; however, a marked increase in the amount of dead cells, with a major decrease of Bcl-2 mRNA expression, was observed after 72 h. The presence of Th2, but not of Th3, cytokines significantly reduced beta-cell death, without any major effect on Bcl-2 and Bax mRNA expression. These results suggest that Th2 and (at lower extent) Th3 cytokines may have a partial, direct protective effect on isolated human islets exposed to the cytostatic and cytotoxic action of proinflammatory and Th1 cytokines.
Asunto(s)
Interleucina-10/farmacología , Interleucina-4/farmacología , Islotes Pancreáticos/efectos de los fármacos , Células Th2/fisiología , Glucosa/farmacología , Humanos , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Interferón gamma/toxicidad , Interleucina-1/toxicidad , Islotes Pancreáticos/fisiología , Islotes Pancreáticos/ultraestructura , Óxido Nítrico/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
BACKGROUND: The potential benefits of islet xenografting in type 1 diabetes include the intriguing, but still unanswered, possibility that the grafted xenoislets may be less subjected to human autoimmune attack. Cytokines may play a major role in the pathogenesis of autoimmune diabetes by causing impairment of insulin release and pancreatic islet cell toxicity. METHODS: We compared insulin secretion, islet cell death and survival, inducible nitric oxide synthase (iNOS) mRNA expression, nitrite production, and Bcl-2 and Bax mRNA expression in isolated human and large mammal (bovine) islets exposed to 50 U/ml recombinant human interleukin-1, 1,000 U/ml recombinant human tumor necrosis factor-alpha and 1,000 U/ml recombinant human interferon-gamma. RESULTS: After 24-hr exposure, a marked decrease of glucose-stimulated insulin secretion was observed with human, but not with bovine islets. After 48-hr exposure, human, but not bovine, pancreatic islets showed a significantly higher percentage of apoptotic cells compared to controls. Treatment of human islets with human cytokines induced up-regulation of iNOS mRNA, increased levels of nitrites, and down-regulation of Bcl-2 mRNA, with unchanged levels of Bax mRNA. These parameters were not affected by cytokines in bovine islets. CONCLUSIONS: Bovine islets are less susceptible than human islets to the effects of human cytokines, which may be a potential advantage of xenotransplantation.
Asunto(s)
Citocinas/farmacología , Islotes Pancreáticos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2 , Animales , Apoptosis/efectos de los fármacos , Bovinos , Supervivencia Celular/efectos de los fármacos , Expresión Génica , Genes bcl-2/genética , Humanos , Etiquetado Corte-Fin in Situ , Insulina/metabolismo , Secreción de Insulina , Interleucina-1/farmacología , Islotes Pancreáticos/citología , Necrosis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Proteínas Proto-Oncogénicas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/farmacología , Proteína X Asociada a bcl-2RESUMEN
This study retrospectively evaluated two groups of pregnant women. Group A women (n=1,338) were universally screened for gestational diabetes mellitus (GDM) and GDM patients were intensively treated. In Group B (n=4,035), screening was performed only in women at high risk for GDM and treatment was conventional. This study confirms the validity of a cost-effective screening program for the diagnosis of GDM and that selective screening may be an option only in a situation where healthcare resources are very scarce and/or universal screening of any kind is not feasible. Once the diagnosis of GDM has been made, metabolic management with an intensive approach is important to reduce maternal and fetal morbidity. Diagnosis of GDM and intensive treatment represent a cost for the public health system, but permit a significant monetary savings in terms of costs linked to maternal and neonatal morbidity.
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Diabetes Gestacional/diagnóstico , Diabetes Gestacional/tratamiento farmacológico , Costos de la Atención en Salud , Tamizaje Masivo/economía , Adulto , Análisis Costo-Beneficio , Diabetes Gestacional/metabolismo , Femenino , Humanos , Italia , Embarazo , Estudios RetrospectivosRESUMEN
We describe a method to consistently prepare human islets for transplantation. By combining a simple collagenase digestion method and a density gradient purification system, we were able to obtain successful isolations (>/=200,000 islet equivalents, >/=50% purity) in 69% of processed glands. No reagent of animal source was used. Isolated islets were morphologically well maintained and functionally competent, with sterility confirmed in 97% of cases. Two patients were transplanted with islets prepared by this method; graft function was demonstrated for a few months. Improved simplicity and consistency, together with adequate quality of the preparations, are the main features of this isolation method.
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Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/citología , Adulto , Separación Celular/métodos , Supervivencia de Injerto , Humanos , Trasplante de Islotes Pancreáticos/fisiología , Persona de Mediana Edad , Preservación de Órganos/métodos , Donantes de Tejidos/estadística & datos numéricos , Recolección de Tejidos y Órganos/métodos , Resultado del TratamientoRESUMEN
This retrospective study investigates the clinical characteristics of gestational diabetes mellitus (GDM) (time of diagnosis, different treatment, metabolic parameters, etc.) in relation to prepregnancy body mass index (BMI) and the influence of BMI on neonatal outcome. 93 GDM women and 110 control subjects were divided into three groups in relation to their prepregnancy BMI: normalweight (Nw), overweight (Ow) and obese (Ob). GDM was diagnosed significantly (p < 0.01) earlier in Ow and Ob than in Nw. Preterm deliveries and cesarean sections resulted significantly (p < 0.01) increased in all BMI categories of GDM respect to matched controls. Prevalence of neonatal macrosomia was higher in GDM patients (44.6%) compared with normal controls (15.4%) and correlated (p < 0.01) with prepregnancy BMI in both groups. Nevertheless in each BMI category the prevalence of macrosomia was significantly higher in GDM patients. The body weight increase during pregnancy was not associated with neonatal macrosomia. This study shows that prepregnancy BMI is an important risk factor for GDM and is predictive for macrosomia specially in women suffering from GDM.
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Peso al Nacer/fisiología , Macrosomía Fetal/etiología , Embarazo en Diabéticas/fisiopatología , Adulto , Femenino , Macrosomía Fetal/patología , Humanos , Recién Nacido , Embarazo , Embarazo en Diabéticas/patología , Estudios RetrospectivosRESUMEN
According to the guidelines of the "Third international workshop conference on GDM", we have examined 2000 pregnant women. The glucose challenge test (GCT) was positive in 408 cases (20.4%) and negative in 1592 (79.6%). The OGTT (Carpenter and Coustan's criteria) was performed in 647 pregnant women. GDM and IGGT prevalence was of 6.25% and 5.5% respectively and normal glucose tolerance (NGT) 88.25%. The GCT effectiveness for GDM and IGGT diagnosis is: sensibility 75.1%, specificity 44%, positive predictive value 46.4% and negative predictive value 74%. GDM and IGGT compared with NGT women were significantly older (p < 0.05) and prepregnancy BMI was higher (p < 0.01); the prevalence of previous macrosomia (p < 0.01), previous gestational diabetes (p < 0.01) and family history for diabetes mellitus (p < 0.05) was greater in GDM and IGCT. The prevalence of preterm delivery was higher in both GDM and IGCT (GDM 12.5% and IGGT 15.4% vs NGT 6%; p < 0.01), as well as the prevalence of cesarean sections (GDM 31.6% vs IGGT 23.5% and NGT 20.3%; p < 0.02), and the occurrence of macrosomia (GDM 27.6%, IGGT 16.6% and NGT 16.2%). In addition a higher prevalence (p < 0.01) of hyperbilirubinaemia, hypoglycemia and hypertrophy cardiomyopathy was observed in newborns from GDM women. Our data show that: GCT has a good specificity for GDM diagnosis, prevalence of GDM in our population is about 6%, GDM is still correlated to an elevated maternal and neonatal morbility.
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Embarazo en Diabéticas/epidemiología , Adulto , Femenino , Humanos , Italia , Tamizaje Masivo , EmbarazoRESUMEN
AIM: We have explored whether the insulin secretory defects induced by glucotoxicity in human pancreatic islets could be prevented by metformin and investigated some of the possible mechanisms involved. METHODS: Human pancreatic islets and INS-1E cells were cultured for 24h with or without high glucose (16.7mM) concentration in the presence or absence of therapeutical concentration of metformin and then glucose-stimulated insulin release, adenine nucleotide levels and mitochondrial complex I and II activities were measured. Islet ultrastructure was analyzed by electron microscopy. RESULTS: Compared to control islets, human islets cultured with high glucose showed a reduced glucose-stimulated insulin secretion that was associated with lower ATP levels and a lower ATP/ADP ratio. These functional and biochemical defects were significantly prevented by the presence of metformin in the culture medium, that was also able to significantly inhibit the activity of mitochondrial complex I especially in beta cells exposed to high glucose. Ultrastructural observations showed that mitochondrial volume density was significantly increased in high glucose cultured islets. The critical involvement of mitochondria was further supported by the observation of remarkably swollen organelles with dispersed matrix and fragmented cristae. Metformin was able to efficiently prevent the appearance of all these ultrastructural alterations in human islets exposed to high glucose. CONCLUSIONS: Our results show that the functional, biochemical and ultrastructural abnormalities observed in human islet cells exposed to glucotoxic condition can be significantly prevented by metformin, further highlighting a direct beneficial effect of this drug on the insulin secreting human pancreatic beta cells.
Asunto(s)
Diabetes Mellitus/prevención & control , Glucosa/efectos adversos , Hipoglucemiantes/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , Metformina/farmacología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Células Cultivadas , Diabetes Mellitus/sangre , Diabetes Mellitus/patología , Femenino , Humanos , Secreción de Insulina , Células Secretoras de Insulina/ultraestructura , Masculino , Microscopía Electrónica , Persona de Mediana EdadRESUMEN
The expression of adiponectin receptors has been demonstrated in human and rat pancreatic beta cells, where globular (g) adiponectin rescues rat beta cells from cytokine and fatty acid-induced apoptosis. The aim of our study was to evaluate whether adiponectin has a direct effect on insulin secretion and the metabolic pathways involved. Purified human pancreatic islets and rat beta cells (INS-1E) were exposed (1 h) to g-adiponectin, and glucose-induced insulin secretion was measured. A significant increase in glucose-induced insulin secretion was observed in the presence of g-adiponectin (1 nmol/l) with respect to control cells in both human pancreatic islets (n = 5, p < 0.05) and INS-1E cells (n = 5, p < 0.001). The effect of globular adiponectin on insulin secretion was independent of AMP-dependent protein kinase (AMPK) activation or glucose oxidation. In contrast, g-adiponectin significantly increased oleate oxidation (n = 5, p < 0.05), and the effect of g-adiponectin (p < 0.001) on insulin secretion by INS-1E was significantly reduced in the presence of etomoxir (1 µmol/l), an inhibitor of fatty acid beta oxidation. g-Adiponectin potentiates glucose-induced insulin secretion in both human pancreatic islets and rat beta cells via an AMPK independent pathway. Increased fatty acid oxidation rather than augmented glucose oxidation is the mechanism responsible. Overall, our data indicate that, in addition to its anti-apoptotic action, g-adiponectin has another direct effect on beta cells by potentiating insulin secretion. Adiponectin, therefore, in addition to its well-known effect on insulin sensitivity, has important effects at the pancreatic level.
Asunto(s)
Adiponectina/farmacología , Glucosa/farmacología , Insulina/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Adenilato Quinasa/metabolismo , Animales , Ácidos Grasos/metabolismo , Humanos , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Oxidación-Reducción/efectos de los fármacos , Ratas , Células Tumorales CultivadasRESUMEN
Induction of the C/EBP homologous protein (CHOP) is considered a key event for endoplasmic reticulum (ER) stress-mediated apoptosis. Type 1 diabetes (T1D) is characterized by an autoimmune destruction of the pancreatic ß-cells. Pro-inflammatory cytokines are early mediators of ß-cell death in T1D. Cytokines induce ER stress and CHOP overexpression in ß-cells, but the role for CHOP overexpression in cytokine-induced ß-cell apoptosis remains controversial. We presently observed that CHOP knockdown (KD) prevents cytokine-mediated degradation of the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1), thereby decreasing the cleavage of executioner caspases 9 and 3, and apoptosis. Nuclear factor-κB (NF-κB) is a crucial transcription factor regulating ß-cell apoptosis and inflammation. CHOP KD resulted in reduced cytokine-induced NF-κB activity and expression of key NF-κB target genes involved in apoptosis and inflammation, including iNOS, FAS, IRF-7, IL-15, CCL5 and CXCL10. This was due to decreased IκB degradation and p65 translocation to the nucleus. The present data suggest that CHOP has a dual role in promoting ß-cell death: (1) CHOP directly contributes to cytokine-induced ß-cell apoptosis by promoting cytokine-induced mitochondrial pathways of apoptosis; and (2) by supporting the NF-κB activation and subsequent cytokine/chemokine expression, CHOP may contribute to apoptosis and the chemo attraction of mononuclear cells to the islets during insulitis.