RESUMEN
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
Azobenzene photoresponsive elements can be installed on sulfonylureas, yielding optical control over pancreatic beta cell function and insulin release. An obstacle to such photopharmacological approaches remains the use of ultraviolet-blue illumination. Herein, we synthesize and test a novel yellow light-activated sulfonylurea based on a heterocyclic azobenzene bearing a push-pull system.
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Compuestos Azo/química , Células Secretoras de Insulina/efectos de la radiación , Insulina/agonistas , Islotes Pancreáticos/efectos de la radiación , Compuestos de Sulfonilurea/química , Animales , Calcio/metabolismo , Células Cultivadas , Diazóxido/farmacología , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HEK293 , Humanos , Hipoglucemiantes/farmacología , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Luz , Ratones , Procesos Fotoquímicos , Potasio/metabolismo , Transducción de Señal , Compuestos de Sulfonilurea/farmacología , Receptores de Sulfonilureas/genética , Receptores de Sulfonilureas/metabolismoRESUMEN
Diets rich in saturated fats may contribute to the loss of pancreatic ß-cells in type 2 diabetes. JunB, a member of the activating protein 1 (AP-1) transcription factor family, promotes ß-cell survival and mediates part of the beneficial effects of GLP-1 agonists. In this study we interrogated the molecular mechanisms involved in JunB-mediated ß-cell protection from lipotoxicity. The saturated fatty acid palmitate decreased JunB expression, and this loss may contribute to ß-cell apoptosis, as overexpression of JunB protected cells from lipotoxicity. Array analysis of JunB-deficient ß-cells identified a gene expression signature of a downregulated endoplasmic reticulum (ER) stress response and inhibited AKT signaling. JunB stimulates XBP1 expression via the transcription factor c/EBPδ during ER stress, and forced expression of XBP1s rescued the viability of JunB-deficient cells, constituting an important antiapoptotic mechanism. JunB silencing inhibited AKT activation and activated the proapoptotic Bcl-2 protein BAD via its dephosphorylation. BAD knockdown reversed lipotoxic ß-cell death potentiated by JunB siRNA. Interestingly, XBP1s links JunB and AKT signaling as XBP1 knockdown also reduced AKT phosphorylation. GLP-1 agonists induced cAMP-dependent AKT phosphorylation leading to ß-cell protection against palmitate-induced apoptosis. JunB and XBP1 knockdown or IRE1 inhibition decreased AKT activation by cAMP, leading to ß-cell apoptosis. In conclusion, JunB modulates the ß-cell ER stress response and AKT signaling via the induction of XBP1s. The activation of the JunB gene network and the crosstalk between the ER stress and AKT pathway constitute a crucial defense mechanism by which GLP-1 agonists protect against lipotoxic ß-cell death. These findings elucidate novel ß-cell-protective signal transduction in type 2 diabetes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Secretoras de Insulina/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Humanos , Células Secretoras de Insulina/enzimología , Masculino , Persona de Mediana Edad , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Factores de Transcripción del Factor Regulador X , Transducción de Señal , Factores de Transcripción/genética , Proteína 1 de Unión a la X-BoxRESUMEN
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.
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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
Protection against insulitis and diabetes by active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in nonobese diabetic mice has until now mainly been attributed to its immunomodulatory effects, but also protective effects of this hormone on inflammation-induced ß-cell death have been reported. The aim of this study was to clarify the molecular mechanisms by which 1,25(OH)2D3 contributes to ß-cell protection against cytokine-induced ß-cell dysfunction and death. Human and mouse islets were exposed to IL-1ß and interferon-γ in the presence or absence of 1,25(OH)2D3. Effects on insulin secretion and ß-cell survival were analyzed by glucose-stimulated insulin release and electron microscopy or Hoechst/propidium iodide staining, respectively. Gene expression profiles were assessed by Affymetrix microarrays. Nuclear factor-κB activity was tested, whereas effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in ß-cell apoptosis, which was almost completely prevented by 1,25(OH)2D3. In addition, 1,25(OH)2D3 restored insulin secretion from cytokine-exposed islets. Microarray analysis of murine islets revealed that the expression of approximately 4000 genes was affected by cytokines after 6 and 24 hours (n = 4; >1.3-fold; P < .02), of which nearly 250 genes were modified by 1,25(OH)2D3. These genes belong to functional groups involved in immune response, chemotaxis, cell death, and pancreatic ß-cell function/phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D3 against inflammation-induced ß-cell dysfunction and death in human and murine islets, with, in particular, alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH)2D3 against the induction of autoimmune diabetes.
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Calcitriol/metabolismo , Citocinas/metabolismo , Regulación de la Expresión Génica , Inflamación , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Anciano , Animales , Muerte Celular , Línea Celular , Células Cultivadas , Quimiotaxis , Ensayo de Inmunoadsorción Enzimática , Glucosa/metabolismo , Humanos , Interferón gamma/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Ratas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND & AIMS: We investigated whether improving 25-hydroxyvitamin D status in young type 1A diabetic patients reduces reactivity of peripheral blood mononuclear cells against islet autoantigens and associates with beta-cell functional changes. METHODS: Eight patients with 25-hydroxyvitamin D deficiency (<20 ng/ml), out of 15 consecutive young type 1A diabetic subjects received 25-hydroxyvitamin D3 to achieve and maintain levels above 50 ng/ml for up to one year. Peripheral blood mononuclear cell reactivity (Interferon-γ spots) against beta-cell autoantigens (glutamic acid decarboxylase 65-kD isoform, proinsulin and tyrosine phosphatase-like protein IA-2) and C-peptide during mixed meal were assessed before and after 25-hydroxyvitamin D3 replenishment. RESULTS: Target 25-hydroxyvitamin D blood levels were safely reached and maintained. Peripheral blood mononuclear cell reactivity against glutamic acid decarboxylase 65-kD isoform (3.8 ± 4.0 vs. 45 ± 16) and proinsulin (3.5 ± 3.2 vs. 75 ± 51) decreased significantly (p < 0.001 and p < 0.02) upon 25-hydroxyvitamin D3 replenishment, which was correlated with 25-hydroxyvitamin D concentrations. C-peptide values remained stable after one year of treatment. CONCLUSIONS: Safely restored and maintained 25-hydroxyvitamin D levels associated with reduced peripheral blood mononuclear cell reactivity against beta-cell autoantigens with no significant decrease of beta-cell function in this cohort of patients.
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Autoantígenos/metabolismo , Calcifediol/administración & dosificación , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Deficiencia de Vitamina D/tratamiento farmacológico , Adolescente , Péptido C/metabolismo , Calcifediol/sangre , Niño , Diabetes Mellitus Tipo 1/sangre , Femenino , Glutamato Descarboxilasa/metabolismo , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Interferón gamma/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Proinsulina/metabolismo , Deficiencia de Vitamina D/sangreRESUMEN
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.
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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: Sirtuin (SIRT)3 is a mitochondrial protein deacetylase that regulates reactive oxygen species (ROS) production and exerts anti-inflammatory effects. As chronic inflammation and mitochondrial dysfunction are key factors mediating pancreatic beta cell impairment in type 2 diabetes, we investigated the role of SIRT3 in the maintenance of beta cell function and mass in type 2 diabetes. METHODS: We analysed changes in SIRT3 expression in experimental models of type 2 diabetes and in human islets isolated from type 2 diabetic patients. We also determined the effects of SIRT3 knockdown on beta cell function and mass in INS1 cells. RESULTS: SIRT3 expression was markedly decreased in islets isolated from type 2 diabetes patients, as well as in mouse islets or INS1 cells incubated with IL1ß and TNFα. SIRT3 knockdown in INS1 cells resulted in lowered insulin secretion, increased beta cell apoptosis and reduced expression of key beta cell genes. SIRT3 knockdown also blocked the protective effects of nicotinamide mononucleotide on pro-inflammatory cytokines in beta cells. The deleterious effects of SIRT3 knockdown were mediated by increased levels of cellular ROS and IL1ß. CONCLUSIONS/INTERPRETATION: Decreased beta cell SIRT3 levels could be a key step in the onset of beta cell dysfunction, occurring via abnormal elevation of ROS levels and amplification of beta cell IL1ß synthesis. Strategies to increase the activity or levels of SIRT3 could generate attractive therapies for type 2 diabetes.
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Diabetes Mellitus Tipo 2/metabolismo , Regulación hacia Abajo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Sirtuina 3/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Línea Celular , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/inmunología , Diabetes Mellitus Tipo 2/patología , Regulación de la Expresión Génica , Humanos , Secreción de Insulina , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Interferencia de ARN , Ratas , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 3/antagonistas & inhibidores , Sirtuina 3/genética , Técnicas de Cultivo de TejidosRESUMEN
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.
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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.
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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 , RatasRESUMEN
BACKGROUND AND AIMS: GPR40 is a membrane-bound receptor paired with medium and long-chain fatty acids (FFA) as endogenous ligands. Its acute activation potentiates insulin secretion from beta cells, whereas prolonged binding might contribute to the deleterious effects of chronic exposure to FFA. Little information is available on the expression of GPR40 and its regulation in human islets (HI). MATERIAL AND METHODS: HI were prepared by enzymatic digestion and gradient separation from the pancreas of 20 non-diabetic (Ctrl) and 13 type 2 diabetic (T2DM) multiorgan donors, and functional and molecular studies were then performed. RESULTS: By qualitative and quantitative PCR experiments, mRNA expression was shown in HI. Both in T2DM islets and in Ctrl islets pre-exposed for 24 h to 1.0 mmol/l FFA (palmitate:oleate, 2:1), GPR40 mRNA expression was significantly reduced (p<0.01) in the T2DM cells as compared to Ctrl cells. A significant positive correlation was found between glucose-stimulated insulin secretion and GPR40 expression. CONCLUSIONS: These results show the expression of GPR40 in human pancreatic islets which are regulated by FFA. The finding that T2DM islets have a lower GPR40 expression, and the correlation of these genes with insulin secretion, raises the possibility of an involvement of GPR40 in human diabetes beta-cell dysfunction.
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Diabetes Mellitus Tipo 2/metabolismo , Ácidos Grasos no Esterificados/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Anciano , Diabetes Mellitus Tipo 2/fisiopatología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Glucosa/farmacología , Humanos , Insulina/genética , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/fisiopatología , Ligandos , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
AIM: To evaluate the effects of exposure to high glucose (HG) levels and sulphonylurea on isolated human islet-cell function, and to investigate some of the mechanisms that might be involved. METHODS: Islet cells were isolated, using collagenase digestion and gradient purification, from 13 pancreata from non-diabetic multiorgan donors (age: 61.2+/-11.5 years; gender: 7 men/6 women; body mass index: 25.1+/-2.8kg/m(2)). The cells were then cultured for 5 days with normal glucose (NG) concentrations (5.5mmol/L), or NG and HG (16.7mmol/L) levels (alternating every 24h), with or without the addition of therapeutic concentrations of gliclazide (10micromol/L) or glibenclamide (1.0micromol/L). At the end of incubation, functional (glucose-stimulated insulin secretion), morphological (electron microscopy) and molecular (gene and protein expression) studies were performed. RESULTS: Insulin secretion differed significantly between study groups, with marked decreases in the presence of HG plus glibenclamide. Compared with NG, insulin expression decreased significantly with HG, and increased similarly with gliclazide as with glibenclamide. However, exposure to gliclazide, but not glibenclamide, significantly induced expression (at both gene and protein levels) of PDX-1, a fundamental beta-cell differentiation transcription factor, and Ki67, a marker of proliferation. However, gliclazide and glibenclamide did not differ in terms of effects on gene expression of the antiapoptotic molecule Bcl2 (increased significantly with both) and the proapoptotic molecule Bax (decreased significantly with both). CONCLUSION: Gliclazide and glibenclamide have different effects on the changes induced by prolonged exposure of human islet cells to high levels of glucose.
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Gliclazida/farmacología , Glucosa/administración & dosificación , Gliburida/farmacología , Hipoglucemiantes/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , Anciano , Análisis de Varianza , Células Cultivadas , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Hiperglucemia/fisiopatología , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Persona de Mediana Edad , Páncreas/efectos de los fármacos , Páncreas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Donantes de Tejidos , Transactivadores/genética , Transactivadores/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.
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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
Exendin-4 is a dipeptidyl peptidase IV (DPP-IV)-resistant glucagon-like peptide 1 (GLP-1) mimetic and its synthetic counterpart, exenatide, is being used in the therapy of type 2 diabetes (T2DM). No information, however, is currently available as for the direct action of exendin-4 on human T2DM islets. In the present study, we exposed pancreatic islets prepared from non-diabetic and T2DM subjects to exendin-4 for 48 h and found that the compound had several, direct beneficial actions on insulin secretion and the expression of genes involved in beta-cell function and differentiation.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/farmacología , Islotes Pancreáticos/efectos de los fármacos , Péptidos/farmacología , Ponzoñas/farmacología , Anciano , Estudios de Casos y Controles , Exenatida , Femenino , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Persona de Mediana EdadRESUMEN
AIMS/HYPOTHESIS: Pancreatic beta cells have highly developed endoplasmic reticulum (ER) due to their role in insulin secretion. Since ER stress has been associated with beta cell dysfunction, we studied several features of beta cell ER in human type 2 diabetes. METHODS: Pancreatic samples and/or isolated islets from non-diabetic controls (ND) and type 2 diabetes patients were evaluated for insulin secretion, apoptosis (electron microscopy and ELISA), morphometric ER assessment (electron microscopy), and expression of ER stress markers in beta cell prepared by laser capture microdissection and in isolated islets. RESULTS: Insulin release was lower and beta cell apoptosis higher in type 2 diabetes than ND islets. ER density volume was significantly increased in type 2 diabetes beta cells. Expression of alpha-mannosidase (also known as mannosidase, alpha, class 1A, member 1) and UDP-glucose glycoprotein glucosyl transferase like 2 (UGCGL2), assessed by microarray and/or real-time reverse transcriptase polymerase chain reaction (RT-PCR), differed between ND and type 2 diabetes beta cells. Expression of immunoglobulin heavy chain binding protein (BiP, also known as heat shock 70 kDa protein 5 [glucose-regulated protein, 78 kDa] [HSPA5]), X-box binding protein 1 (XBP-1, also known as XBP1) and C/EBP homologous protein (CHOP, also known as damage-inducible transcript 3 [DDIT3]) was not higher in type 2 diabetes beta cell or isolated islets cultured at 5.5 mmol/l glucose (microarray and real-time RT-PCR) than in ND samples. When islets were cultured for 24 h at 11.1 mmol/l glucose, there was induction of BiP and XBP-1 in type 2 diabetes islets but not in ND islets. CONCLUSIONS/INTERPRETATION: Beta cell in type 2 diabetes showed modest signs of ER stress when studied in pancreatic samples or isolated islets maintained at physiological glucose concentration. However, exposure to increased glucose levels induced ER stress markers in type 2 diabetes islet cells, which therefore may be more susceptible to ER stress induced by metabolic perturbations.
Asunto(s)
Diabetes Mellitus Tipo 2/patología , Diabetes Mellitus Tipo 2/fisiopatología , Retículo Endoplásmico/patología , Retículo Endoplásmico/fisiología , Células Secretoras de Insulina/ultraestructura , Anciano , Apoptosis/fisiología , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Diabetes Mellitus Tipo 2/genética , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Chaperón BiP del Retículo Endoplásmico , Femenino , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Glucosa/farmacología , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Células Secretoras de Insulina/fisiología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Persona de Mediana Edad , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Transcripción del Factor Regulador X , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Factores de Transcripción , Proteína 1 de Unión a la X-BoxRESUMEN
BACKGROUND: Decreased beta-cell mass, mainly due to apoptosis, is crucial for the development and progression of type 2 diabetes. Chronic exposure to high glucose levels is a probable underlying mechanism, whereas the role of oral anti-diabetic agents (sulphonylureas in particular) is still unsettled. METHODS: To directly investigate more on such issues, we prepared isolated human islets, which were then cultured for 5 days in continuous normal glucose concentration (NG, 5.5 mmol/L) or normal and high (HG, 16.7 mmol/L) glucose levels (alternating every 24 h), with or without the addition of therapeutical concentration (10 micromol L) of gliclazide or glibenclamide. RESULTS: Intermittent high glucose caused a significant decrease of glucose-stimulated insulin secretion, which was not further affected by either sulphonylurea. Apoptosis, as assessed by electron microscopy, was also significantly increased by alternating high glucose exposure, which was accompanied by altered mitochondria morphology and density volume, and increased concentrations of nitrotyrosine, a marker of oxidative stress. Gliclazide, but not glibenclamide, was able to significantly reduce high glucose induced apoptosis, mitochondrial alterations, and nitrotyrosine concentration increase. CONCLUSION: Therefore, gliclazide protected human beta-cells from apoptosis induced by intermittent high glucose, and this effect was likely to be due, at least in part, to the anti-oxidant properties of the molecule.
Asunto(s)
Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Gliclazida/farmacología , Glucosa/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Cultivadas , Femenino , Glucosa/administración & dosificación , Gliburida/farmacología , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
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.
Asunto(s)
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
Peroxisome proliferator-activated receptors (PPARs) are a subgroup of the superfamily of nuclear receptors, with three distinct main types: alpha, beta and gamma (subdivided into gamma(1) and gamma(2)). Recently, the presence of PPARgamma has been reported in human islets. Whether other PPAR types can be found in human islets, how islet PPARgamma mRNA expression is regulated by the metabolic milieu, their role in insulin secretion, and the effects of a PPARgamma agonist are not known. In this study, human pancreatic islets were prepared by collagenase digestion and density gradient purification from nonobese adult donors. The presence of PPAR mRNAs was assessed by RT-PCR, and the effect was evaluated of exposure for up to 24 h to either 22.2 mmol/l glucose and/or 0.25, 0.5, or 1.0 mmol/l long-chain fatty acid mixture (oleate to palmitate, 2:1). PPARbeta and, to a greater extent, total PPARgamma and PPARgamma(2) mRNAs were expressed in human islets, whereas PPARalpha mRNA was not detected. Compared with human adipose tissue, PPARgamma mRNA was expressed at lower levels in the islets, and PPARbeta at similar levels. The expression of PPARgamma(2) mRNA was not affected by exposure to 22.2 mmol/l glucose, whereas it decreased markedly and time-dependently after exposure to progressively higher free fatty acids (FFA). This latter effect was not affected by the concomitant presence of high glucose. Exposure to FFA caused inhibition of insulin mRNA expression, glucose-stimulated insulin release, and reduction of islet insulin content. The PPARgamma agonists rosiglitazone and 15-deoxy-Delta-(12,14)prostaglandin J(2) prevented the cytostatic effect of FFA as well as the FFA-induced changes of PPAR and insulin mRNA expression. In conclusion, this study shows that PPARgamma mRNA is expressed in human pancreatic islets, with predominance of PPARgamma(2); exposure to FFA downregulates PPARgamma(2) and insulin mRNA expression and inhibits glucose-stimulated insulin secretion; exposure to PPARgamma agonists can prevent these effects.