RESUMEN
Liver disease is a major cause of premature death. Oxidative stress in the liver represents a key disease driver. Compounds, such as dimethyl fumarate (DMF), can activate the antioxidant response and are used clinically to treat disease. In this study, we tested the protective properties of DMF before or after paracetamol exposure. Following DMF administration, Nrf2 nuclear translocation was tracked at the single-cell level and target gene transactivation confirmed. Next, the protective properties of DMF were examined following paracetamol exposure. Transcriptomic and biochemical analysis revealed that DMF rescue was underpinned by reduced Nf-kB and TGF-ß signaling and cell senescence. Following on from these studies, we employed a Zebrafish model to study paracetamol exposure in vivo. We combined a genetically modified Zebrafish model, expressing green fluorescent protein exclusively in the liver, with automated microscopy. Pre-treatment with DMF, prior to paracetamol exposure, led to reduced liver damage in Zebrafish demonstrating protective properties.
RESUMEN
Reactive oxygen species (ROS) contribute to target-cell damage in inflammatory and iron-overload diseases. Little is known about iron transport regulation during inflammatory attack. Through a combination of in vitro and in vivo studies, we show that the proinflammatory cytokine IL-1ß induces divalent metal transporter 1 (DMT1) expression correlating with increased ß cell iron content and ROS production. Iron chelation and siRNA and genetic knockdown of DMT1 expression reduce cytokine-induced ROS formation and cell death. Glucose-stimulated insulin secretion in the absence of cytokines in Dmt1 knockout islets is defective, highlighting a physiological role of iron and ROS in the regulation of insulin secretion. Dmt1 knockout mice are protected against multiple low-dose streptozotocin and high-fat diet-induced glucose intolerance, models of type 1 and type 2 diabetes, respectively. Thus, ß cells become prone to ROS-mediated inflammatory damage via aberrant cellular iron metabolism, a finding with potential general cellular implications.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de Transporte de Catión/metabolismo , Células Secretoras de Insulina/metabolismo , Interleucina-1beta/farmacología , Hierro/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/genética , Diabetes Mellitus Experimental , Dieta Alta en Grasa , Intolerancia a la Glucosa , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/citología , Ratones , Ratones Noqueados , Modelos Biológicos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
The Reg3 peptides INGAP-PP and human Reg3α/ß (HIP) have been hypothesized to stimulate ß-cell neogenesis in the pancreas. Administration of INGAP-PP has been shown to cause an increase in ß-cell mass in multiple animal models, reverse streptozotocin (STZ) induced diabetes in mice and reduces HbA1c levels in type 2 diabetic humans. In this study, we have examined the ability of the INGAP-PP and HIP peptides to induce ß-cell formation in vivo in normal mice through short-term administration of the peptides. We assessed the peptides ability to induce an increase in extra-islet insulin-positive cell clusters by looking at ß-cell number by point counting morphometry on pancreata that had been randomized using the smooth fractionator principle in non-diabetic NMRI mice after short-term injections of the peptides (5 d). Five day continuous BrdU labeling was used to determine if the new ß-cells were derived from replicating ß-cells. Real time quantitative RT-PCR and immuno-histochemistry was used to analyze changes in pancreatic transcription factor expression. A 1.5- to 2-fold increase in the volume of small extra-islet insulin-positive clusters post 5 d treatment with INGAP-PP and HIP as compared with mice treated with a non-peptide control or scrambled peptide (p<0.05) (n = 7) was found. Five day continuous BrdU infusion during the 5 d period showed little or no incorporation in islets or small insulin clusters. Five days of treatment with INGAP-PP or HIP, showed a tendency toward increased levels of pancreatic progenitor markers such as Ngn3, Nkx6.1, Sox9 and Ins. These are the first studies to compare and indicate that the human Reg3 α/ß (HIP) peptide has similar bioactivity in vivo as INGAP by causing formation of small ß-cell clusters in extra-islet pancreatic tissue after only 5 d of treatment. Upregulation of pancreatic transcription factors may be part of the mechanism of action.
Asunto(s)
Antígenos de Neoplasias/farmacología , Biomarcadores de Tumor/farmacología , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/análisis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Recuento de Células , Femenino , Expresión Génica , Proteínas de Homeodominio/análisis , Proteínas de Homeodominio/genética , Insulina/análisis , Insulina/genética , Células Secretoras de Insulina/química , Lectinas Tipo C , Ratones , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Proteínas Asociadas a Pancreatitis , Factor de Transcripción SOX9/análisis , Factor de Transcripción SOX9/genética , Factores de Tiempo , Factores de Transcripción/análisis , Regulación hacia ArribaRESUMEN
We retrospectively studied whether treatment with esomeprazole improved HbA1(c) levels in type 2 diabetic patients. We selected 21 patients who had been treated with esomeprazole for 11 ± 3 months and 21 controls. HbA1(c) levels decreased in the esomeprazole-treated group. Our data indicate that proton pump inhibitors may improve glycaemic control in type 2 diabetic patients.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Esomeprazol/uso terapéutico , Inhibidores de la Bomba de Protones/uso terapéutico , Anciano , Anciano de 80 o más Años , Glucemia , Femenino , Hemoglobina Glucada/metabolismo , Humanos , Hipoglucemiantes/uso terapéutico , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
BACKGROUND: High doses of anti-inflammatory drugs, such as aspirin and salicylates, improve glucose metabolism in insulin resistant and type 2 diabetic patients. It has also been shown that the glucose lowering effect is related to the unspecific ability of these drugs to inhibit inhibitor kinaseß (IKKß). In this study we have investigated the effect of a selective IKKß-inhibitor on beta cell survival and the prevention of diet induced type 2 diabetes in the gerbil Psammomys obesus (P. obesus). METHODOLOGY/PRINCIPAL FINDINGS: P. obesus were fed a diabetes inducing high energy diet for one month in the absence or presence of the IKKß-inhibitor. Body mass, blood glucose, HbA(1C), insulin production and pancreatic insulin stores were measured. The effects on beta cell survival were also studied in INS-1 cells and primary islets. The cells were exposed to IL-1ß and subsequently reactive oxygen species, insulin release and cell death were measured in the absence or presence of the IKKß-inhibitor. In primary islets and beta cells, IL-1ß induced the production of reactive oxygen species, reduced insulin production and increased beta cell death, which were all reversed by pre-treatment with the IKKß-inhibitor. In P. obesus the IKKß-inhibitor prevented the development of hyperglycaemia and hyperinsulinaemia, and maintained pancreatic insulin stores with no effect on body weight. CONCLUSIONS/SIGNIFICANCE: Inhibition of IKKß activity prevents diet-induced diabetes in P. obesus and inhibits IL-1ß induced reactive oxygen species, loss of insulin production and beta cell death in vitro.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Dieta , Gerbillinae , Islotes Pancreáticos/fisiopatología , FN-kappa B/antagonistas & inhibidores , Animales , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Quinasa I-kappa B/antagonistas & inhibidores , Interleucina-1beta/farmacología , Islotes Pancreáticos/metabolismo , FN-kappa B/metabolismo , RatasRESUMEN
In order to investigate the effect and mechanism of liraglutide and vildagliptin in diabetic Psammomys obesus, we examined proliferation and apoptosis of beta-cells, beta-cell mass (BCM), and pancreatic insulin content after zero, six and fourteen days of treatment compared to control groups. One group of animals was kept on low-energy diet and seven groups were given high-energy diet (HED) that induced diabetes over a four week period. Non-fasting morning blood glucose, body weight, HbA(1C) and pancreatic insulin content were measured and beta cell mass (BCM), proliferation and apoptosis frequencies were determined using stereological point counting. Liraglutide significantly reduced blood glucose and even normalized it in all animals treated for six days and in 11 out of 17 animals treated for fourteen days. HED increased BCM and treatment with liraglutide did not change this. However, compared to the vehicle-treated animals pancreatic insulin content was normalized in animals treated for six and fourteen days with liraglutide. In contrast, vildagliptin, in doses causing full inhibition of plasma DPP-IV activity, neither reduced blood glucose nor altered HED-induced increases in BCM or pancreatic insulin content. These results suggest that liraglutide restores normoglycaemia and improves glycaemic control in P. obesus by increasing their insulin content and improving the function of the beta-cells. In contrast, vildagliptin does not improve glycaemic control in P. obesus nor affect beta-cell insulin content.
Asunto(s)
Adamantano/análogos & derivados , Glucemia/efectos de los fármacos , Diabetes Mellitus Tipo 2 , Péptido 1 Similar al Glucagón/análogos & derivados , Insulina/sangre , Nitrilos/farmacología , Pirrolidinas/farmacología , Adamantano/farmacología , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Gerbillinae , Péptido 1 Similar al Glucagón/farmacología , Péptido 1 Similar al Glucagón/uso terapéutico , Inmunohistoquímica , Liraglutida , Masculino , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Estándares de Referencia , VildagliptinaRESUMEN
The desert gerbil Psammomys obesus, an established model of type 2 diabetes (T2D), has previously been shown to lack pancreatic and duodenal homeobox gene 1 (Pdx-1) expression. Pdx-1 deficiency leads to pancreas agenesis in both mice and humans. We have therefore further examined the pancreas of P. obesus during embryonic development. Using Pdx-1 antisera raised against evolutionary conserved epitopes, we failed to detect Pdx-1 immunoreactivity at any time points. However, at E14.5, Nkx6.1 immunoreactivity marks the nuclei of all epithelial cells of the ventral and dorsal pancreatic buds and the only endocrine cell types found at this time point are glucagon and PYY. At E18.5 the pancreas is well branched and both glucagon- and ghrelin-positive cells are scattered or found in clusters, whereas insulin-positive cells are not found. At E22.5, the acini of the exocrine pancreas are starting to mature, and amylase and carboxypeptidase A immunoreactivity is found scattered and not in all acini. Ghrelin-, glucagon-, PYY-, gastrin-, somatostatin (SS)-, pancreatic polypeptide (PP)-, and insulin-immunoreactive cells are found scattered or in small groups within or lining the developing ductal epithelium as marked by cytokeratin 19. Using degenerate PCR, the P. obesus Neurogenin-3 (Ngn-3) gene was cloned. Nucleotide and amino acid sequences show high homology with known Ngn-3 sequences. Using specific antiserum, we can observe that Ngn-3-immunoreactive cells are rare at E14.5 but readily detectable at E18.5 and E22.5. In conclusion, despite the lack of detection of Pdx-1, the P. obesus pancreas develops similarly to Muridae species, and the Ngn-3 sequence and expression pattern is highly conserved in P. obesus.
Asunto(s)
Páncreas/metabolismo , Hormonas Pancreáticas/biosíntesis , Factores de Transcripción/biosíntesis , Animales , Clonación Molecular , Gerbillinae , Edad Gestacional , Humanos , Inmunohistoquímica , Hibridación in Situ , Ratones , Páncreas/embriología , Hormonas Pancreáticas/genética , Ratas , Especificidad de la Especie , Factores de Transcripción/genéticaRESUMEN
Type 1 diabetes (T1D) is characterized by selective autoimmune destruction of the insulin producing beta-cells in the islets of Langerhans. When the beta-cells are destroyed exogenous administration of insulin is necessary for maintenance of glucose homeostasis. Allogeneic islet transplantation has been used as a means to circumvent the need for insulin administration and has in some cases been able to restore endogenous insulin production for years. However, long life immunosuppression is needed to prevent the graft from being rejected and destroyed. Changes in protein expression pattern during spontaneous diabetes development in the diabetes prone BioBreeding rat (BB-DP) have previously been described. In the present study, we have investigated if any of the changes seen in the protein expression pattern during spontaneous diabetes development are also present during allograft rejection of BB-DP rat islets. Two hundred neonatal islets were syngeneically transplanted under the kidney capsule of 30 day old BB-DP rats and removed prior to and at onset of diabetes. Allogeneically transplanted islets from BB-DP rats were removed before onset of allograft rejection and at maximal islet graft inflammation (rejection). The protein expression profiles of the transplants were visualised by two-dimensional gel (2-DG) electrophoresis, analysed and compared. In total, 2590 protein spots were visualised and of these 310 changed expression (p < 0.01) in syngeneic islet transplants in the BB-DP rats from 7 days after transplantation until onset of diabetes. In BB-DP islets transplanted to WK rats 53 protein spots (p < 0.01) showed changes in expression when comparing islet grafts removed 7 days after transplantation with islet grafts removed 12 days after transplantation where mononuclear cell infiltration is at its maximum. Only four protein spots (1%) were significantly changed in both syngeneic (autoimmune) and allogeneic islet destruction. When comparing protein expression changes in syngeneic BB-DP islet transplants from 37 days after transplantation to onset of diabetes with protein expression changes in allografts from day 7 to 12 after transplantation only three spot were found to commonly change expression in both situations. In conclusion, a large number of protein expression changes were detected in both autoimmune islet destruction and allogeneic islet rejection, only two overlaps were detected, suggesting that autoimmune islet destruction and allogeneic islet rejection may result from different target cell responses to signals induced by the cellular infiltrate. Whether this reflects activation of distinct signalling pathways in islet cells is currently unknown and need to be further investigated.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Rechazo de Injerto/inmunología , Células Secretoras de Insulina/inmunología , Trasplante de Islotes Pancreáticos/métodos , Proteómica/métodos , Animales , Animales Recién Nacidos , Diabetes Mellitus Tipo 1/metabolismo , Electroforesis en Gel Bidimensional , Rechazo de Injerto/metabolismo , Histocitoquímica , Humanos , Procesamiento de Imagen Asistido por Computador , Células Secretoras de Insulina/metabolismo , Masculino , Biosíntesis de Proteínas/inmunología , Ratas , Ratas Endogámicas BB , Ratas Endogámicas WF , Organismos Libres de Patógenos EspecíficosRESUMEN
Pro-apoptotic cytokines are toxic to the pancreatic beta-cells and have been associated with the pathogenesis of Type 1 diabetes (T1D). Proteome analysis of IL-1beta exposed isolated rat islets identified galectin-3 (gal-3) as the most up-regulated protein. Here analysis of human and rat islets and insulinoma cells confirmed IL-1beta regulated gal-3 expression of several gal-3 isoforms and a complex in vivo expression profile during diabetes development in rats. Over-expression of gal-3 protected beta-cells against IL-1beta toxicity, with a complete blockage of JNK phosphorylation, essential for IL-1-mediated apoptosis. Mutation scanning of regulatory and coding regions of the gal-3 gene (LGALS3) identified six polymorphisms. A haplotype comprising three cSNPs showed significantly increased transmission to unaffected offspring in 257 T1D families and replicated in an independent set of 170 T1D families. In summary, combined proteome-transcriptome-genome and functional analyses identify gal-3 as a candidate gene/protein in T1D susceptibility that may prove valuable in future intervention/prevention strategies.
Asunto(s)
Citocinas/toxicidad , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Galectina 3/fisiología , Regulación de la Expresión Génica , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/inmunología , Animales , Apoptosis/genética , Apoptosis/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Galectina 3/genética , Regulación de la Expresión Génica/efectos de los fármacos , Genómica , Haplotipos , Humanos , Células Secretoras de Insulina/química , Interleucina-1/toxicidad , Mutación , Fosfotransferasas/metabolismo , Polimorfismo de Nucleótido Simple , Proteómica , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Ratas EndogámicasRESUMEN
The proinflammatory cytokines interleukin-1beta (IL-1beta), interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) are toxic to pancreatic beta-cells and are implicated in the pathogenesis of type 1 diabetes. We have previously found that GH and prolactin (PRL) stimulate both proliferation and insulin production in pancreatic beta-cells and rat insulin-producing INS-1 cells. Here we report that human (h) GH can prevent the apoptotic effects of IL-1beta, IFN-gamma and TNF-alpha in INS-1 and INS-1E cells. Using adenovirus-mediated gene transfer, we found that the anti-apoptotic effect of hGH is abrogated by expression of a dominant negative signal transducer and activator of transcription (STAT5) mutant in INS-1E cells. hGH and the cytotoxic cytokines was found to additively increase suppressor of cytokine signalling-3 mRNA expression after 4 h of exposure. In order to identify possible targets for the STAT5-mediated protection of INS-1E cells, we studied the effect of hGH on activation of the transcription factors STAT1 and nuclear factor-kappaB (NF-kappaB) by IFN-gamma and IL-1beta+TNF-alpha respectively. Gel retardation experiments showed that hGH affects neither IFN-gamma+TNF-alpha-induced STAT1 DNA binding nor IL-1beta and IFN-gamma+TNF-alpha-induced NFkappaB DNA binding. The lack of influence of hGH on cytokine-mediated activation of STAT1 and NFkappaB is in accordance with the finding that hGH had only a minor effect on cytokine-induced inducible nitric oxide synthase (iNOS) gene expression and in fact augmented the IL-1beta-stimulated nitric oxide production. As the anti-apoptotic Bcl-xL gene has been shown to harbour a STAT5-binding element we measured the expression of Bcl-xL as well as the pro-apoptotic Bax. We found that hGH increased the Bcl-xL/Bax ratio both in the absence and in the presence of cytotoxic cytokines. In conclusion, these results suggested that GH and PRL protect beta-cells against cytotoxic cytokines via STAT5-dependent mechanisms distal to iNOS activation possibly at the level of Bcl-xL.
Asunto(s)
Hormona de Crecimiento Humana/farmacología , Islotes Pancreáticos/metabolismo , Factor de Transcripción STAT5/metabolismo , Animales , Apoptosis , Western Blotting/métodos , Humanos , Etiquetado Corte-Fin in Situ , Insulina/metabolismo , Interferón gamma/metabolismo , Interleucina-1/metabolismo , Islotes Pancreáticos/citología , Ratones , Óxido Nítrico/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The c-jun N-terminal kinase (JNK) signaling pathway mediates IL-1beta-induced apoptosis in insulin-secreting cells, a mechanism relevant to the destruction of pancreatic beta-cells in type 1 and 2 diabetes. However, the mechanisms that contribute to IL-1beta activation of JNK in beta-cells are largely unknown. In this study, we investigated whether Ca(2+) plays a role for IL-1beta-induced JNK activation. In insulin-secreting rat INS-1 cells cultured in the presence of 11 mm glucose, combined pharmacological blockade of L- and T-type Ca(2+) channels suppressed IL-1beta-induced in vitro phosphorylation of the JNK substrate c-jun and reduced IL-1beta-stimulated activation of JNK1/2 as assessed by immunoblotting. Inhibition of IL-1beta-induced in vitro kinase activity toward c-jun after collective L- and T-type Ca(2+) channel blockade was confirmed in primary rat and ob/ob mouse islets and in mouse betaTC3 cells. Ca(2+) influx, specifically via L-type but not T-type channels, contributed to IL-1beta activation of JNK. Activation of p38 and ERK in response to IL-1beta was also dependent on L-type Ca(2+) influx. Membrane depolarization by KCl, exposure to high glucose, treatment with Ca(2+) ionophore A23187, or exposure to thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPase, all caused an amplification of IL-1beta-induced JNK activation in INS-1 cells. Finally, a chelator of intracellular free Ca(2+) [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl], an inhibitor of calmodulin (W7), and inhibitors of Ca(2+)/calmodulin-dependent kinase (KN62 and KN93) partially reduced IL-1beta-stimulated c-jun phosphorylation in INS-1 or betaTC3 cells. Our data suggest that Ca(2+) plays a permissive role in IL-1beta activation of the JNK signaling pathway in insulin-secreting cells.
Asunto(s)
Calcio/fisiología , Insulina/metabolismo , Interleucina-1/farmacología , Islotes Pancreáticos/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Secreción de Insulina , Membranas Intracelulares/metabolismo , Islotes Pancreáticos/enzimología , Mibefradil/farmacología , Ratones , FN-kappa B/metabolismo , Nimodipina/farmacología , Ratas , Ratas Endogámicas WF , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
We here show that GLP-1 and the long-acting GLP-1 analogue, liraglutide, interfere with diabetes-associated apoptotic processes in the beta-cell. Studies using primary neonatal rat islets showed that native GLP-1 and liraglutide inhibited both cytokine- and free fatty acid-induced apoptosis in a dose-dependent manner. The anti-apoptotic effect of liraglutide was mediated by the GLP-1 receptor as the specific GLP-1 receptor antagonist, exendin(9-39), blocked the effects. The adenylate cyclase activator, forskolin, had an anti-apoptotic effect similar to those of GLP-1 and liraglutide indicating that the effect was cAMP-mediated. Blocking the PI3 kinase pathway using wortmannin but not the MAP kinase pathways by PD98059 inhibited the effects of liraglutide. In conclusion, GLP-1 receptor activation has anti-apoptotic effect on both cytokine, and free fatty acid-induced apoptosis in primary islet-cells, thus suggesting that the long-acting GLP-1 analogue, liraglutide, may be useful for retaining beta-cell mass in both type 1 and type 2 diabetic patients.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácidos Grasos no Esterificados/farmacología , Glucagón/análogos & derivados , Glucagón/química , Glucagón/farmacología , Islotes Pancreáticos/efectos de los fármacos , Fragmentos de Péptidos/química , Precursores de Proteínas/química , Animales , AMP Cíclico/metabolismo , Citocinas/antagonistas & inhibidores , Péptido 1 Similar al Glucagón , Islotes Pancreáticos/citología , Liraglutida , Óxido Nítrico/antagonistas & inhibidores , Oxidorreductasas/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Precursores de Proteínas/farmacología , Ratas , Ratas Wistar , Transducción de SeñalRESUMEN
Type 1 diabetes (T1D) is the result of selective destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. T1D is due to a complex interplay between the beta-cell, the immune system, and the environment in genetically susceptible individuals. The initiating mechanism(s) behind the development of T1D are largely unknown, and no genes or proteins are specific for most T1D cases. Different pro-apoptotic cytokines, IL-1 beta in particular, are present in the islets during beta-cell destruction and are able to modulate beta-cell function and induce beta-cell death. In beta-cells exposed to IL-1 beta, a race between destructive and protective events are initiated and in susceptible individuals the deleterious events prevail. Proteins are involved in most cellular processes, and it is thus expected that their cumulative expression profile reflects the specific activity of cells. Proteomics may be useful in describing the protein expression profile and thus the diabetic phenotype. Relatively few studies using proteomics technologies to investigate the T1D pathogenesis have been published to date despite the defined target organ, the beta-cell. Proteomics has been applied in studies of differentiating beta-cells, cytokine exposed islets, dietary manipulated islets, and in transplanted islets. Although that the studies have revealed a complex and detailed picture of the protein expression profiles many functional implications remain to be answered. In conclusion, a rather detailed picture of protein expression in beta-cell lines, islets, and transplanted islets both in vitro and in vivo have been described. The data indicate that the beta-cell is an active participant in its own destruction during diabetes development. No single protein alone seems to be responsible for the development of diabetes. Rather the cumulative pattern of changes seems to be what favors a transition from dynamic stability in the unperturbed beta-cell to dynamic instability and eventually to beta-cell destruction.
Asunto(s)
Diabetes Mellitus Tipo 1/etiología , Proteómica/métodos , Proteómica/tendencias , Animales , Línea Celular , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Predicción , HumanosRESUMEN
Malignant insulinoma is a critical cancer form with a poor prognosis. Because cure by surgery is infrequent, effective chemotherapy is in demand. Induction of cell death in tumor cells by proteasome inhibitors is emerging as a potential strategy in cancer therapy. Here we investigated whether inhibition of the proteasome has an antitumorigenic potential in insulinoma cells. Exposure of mouse betaTC3 insulinoma cells to the proteasome inhibitor N-Acetyl-Leu-Leu-Nle-CHO (ALLN) reduced cell viability, activated caspase-3, induced apoptosis, and suppressed insulin release. Treatment with ALLN also resulted in phosphorylation of c-jun N-terminal kinase (JNK) and an increase in in vitro phosphorylation of c-jun. In insulinoma cells with impaired JNK signaling, ALLN-induced apoptosis was significantly suppressed. Another proteasome inhibitor, lactacystin, also stimulated JNK activation, caused activation of caspase-3, suppressed cell viability, and induced apoptosis in betaTC3 and rat INS-1E cells. Both ALLN and lactacystin caused a marked decrease in the cellular amount of the JNK scaffold protein JNK-interacting protein 1/islet-brain-1. In primary pancreatic rat islet cells, proteasome inhibition reduced insulin secretion but had no impact on cell viability and even partially protected against the toxic effect of proinflammatory cytokines. Our findings demonstrate that proteasome inhibitors possess antitumorigenic and antiinsulinogenic effects on insulinoma cells.
Asunto(s)
Acetilcisteína/análogos & derivados , Antineoplásicos/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Insulinoma/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de Proteasoma , Acetilcisteína/farmacología , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Línea Celular Tumoral , Insulinoma/patología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Leupeptinas/farmacología , Ratones , Neoplasias Pancreáticas/patología , Ratas , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Complex traits like type 1 diabetes mellitus (T1DM) are generally taken to be under the influence of multiple genes interacting with each other to confer disease susceptibility and/or protection. Although novel methods are being developed, analyses of whole-genome scans are most often performed with multipoint methods that work under the assumption that multiple trait loci are unrelated to each other; that is, most models specify the effect of only one locus at a time. We have applied a novel approach, which includes decision-tree construction and artificial neural networks, to the analysis of T1DM genome-scan data. We demonstrate that this approach (1) allows identification of all major susceptibility loci identified by nonparametric linkage analysis, (2) identifies a number of novel regions as well as combinations of markers with predictive value for T1DM, and (3) may be useful in characterizing markers in linkage disequilibrium with protective-gene variants. Furthermore, the approach outlined here permits combined analyses of genetic-marker data and information on environmental and clinical covariates.
Asunto(s)
Árboles de Decisión , Diabetes Mellitus Tipo 1/genética , Predisposición Genética a la Enfermedad/genética , Genoma Humano , Redes Neurales de la Computación , Marcadores Genéticos/genética , Humanos , Desequilibrio de Ligamiento , Modelos GenéticosRESUMEN
The cytokine IL-1beta suppresses rodent islets of Langerhans in vitro. Presently we used inhibitors of the proteasome to investigate if these compounds could counteract the suppressive effects of the cytokine. Thus, isolated rat islets were cultured and pre-treated with proteasome inhibitors and subsequently exposed for 48 h to 25 U/ml human IL-1beta. After this period functional tests were carried out. The rate of glucose oxidation (pmol/10 islets x 90 min) was suppressed by IL-1beta (115 +/- 17 vs. control 380 +/- 57). Pre-treatment with 10 microM of the proteasome inhibitor MG115 (N-carbobenzoxyl-leu-leu-norvalinal) and 100 microM of the calpain inhibitor norLEU (N-acetyl-leu-leu-norleucinal; known to affect proteasome activity) counteracted the suppressive effects (253 +/- 17 and 262 +/- 10 respectively). The calpain inhibitor alIMET (N-acetyl-leu-leu-methional) had no effect. MG115 (10 microM) and norLEU (100 microM) blocked nitric oxide formation induced by IL-1beta, while alIMET was without effect. We also investigated if IL-1beta could influence the expression of two inducible proteasome subunits, namely LMP2 and LMP7, and found that the cytokine increased the mRNA expression of the proteasome subunit LMP2 in islets, and that the proteasome inhibitor MG115 prevented this increase. In conclusion our study shows that IL-1beta increases the transcription of the proteasome subunit LMP2, and that the proteasome is involved in IL-1beta induced suppression of islet function. Moreover, the observation that inhibitors of the proteasome protect islets against IL-1beta induced inhibition of glucose metabolism, suggests that these compounds might be worthwile to explore in future therapies against the development of type 1 diabetes.
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Cisteína Endopeptidasas/metabolismo , Interleucina-1/farmacología , Islotes Pancreáticos/efectos de los fármacos , Complejos Multienzimáticos/metabolismo , Animales , Secuencia de Bases , Inhibidores de Cisteína Proteinasa/farmacología , ADN/metabolismo , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Glucosa/metabolismo , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Leupeptinas/farmacología , Masculino , Complejos Multienzimáticos/antagonistas & inhibidores , Nitritos/metabolismo , Complejo de la Endopetidasa Proteasomal , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The free radical nitric oxide (NO) has been implicated in cytokine mediated destruction of rat beta-cells in islets of Langerhans. Cytokine mediated NO production is associated with increased expression of the inducible nitric oxide synthase (iNOS). We have previously shown a strain dependent difference between Wistar Kyoto (WKY) and Brown Norway (BN) rats of IL-1beta mediated destruction of islets of Langerhans to be related to expression levels of iNOS and NO production. The aim of the present study was to clone and screen the iNOS gene promoter region from WKY and BN rats for polymorphisms and to functionally test such nucleotide differences. Within the total 2077 bp sequenced from both rat strains we identified three polymorphisms in two separate areas: (i) a GT-repeat polymorphism linked to (ii) a C/T polymorphisms, leading to a WT1 binding site approximately 1650bp upstream the BN iNOS promoter and (iii) a G/A SNP in exon 1. Apart from these polymorphisms the homology between all published rat iNOS sequences including the presently described are about 96%. Promoter activity was detected for both genes in a luciferase assay followed cloning of 2012 bp fragments and transient transfection into RIN cells. For both strains IL-1beta induced dose-dependent activity and strain dependent iNOS promoter activity was demonstrated when WT1 was co-expressed. To our knowledge, this is the first demonstration of functional WT1/iNOS promoter interaction. We conclude that the iNOS promoter is strain-dependently regulated which may relate to quantitatively as well as qualitatively strain dependent differences in transcription factor expression, in this study exemplified by WT1.
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Óxido Nítrico Sintasa/genética , Regiones Promotoras Genéticas , Proteínas WT1/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/fisiología , Genes Reporteros , Datos de Secuencia Molecular , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , RatasRESUMEN
UNLABELLED: Type 1 Diabetes Mellitus is characterized by selective destruction of the pancreatic beta-cells in the islets of Langerhans and insulitis. Subcutaneous insulin injections given to diabetes prone BioBreeding (BB-DP) rats reduce diabetes incidence. The underlying mechanism(s) are not known in detail. Previously, we showed that transplantation of 200 syngeneic neonatal islets under the kidney capsule is useful for studying molecular events during diabetes development in BB-DP rats. In the present study we tested if prophylactic insulin treatment of syngeneically transplanted BB-DP rats would protect both islets in situ and transplanted islets from destruction. METHODS: BB-DP rats received transplants of 200 syngeneic neonatal islets under the kidney capsule at 30 days of age. They were given a subcutaneous insulin or placebo implant and were compared to control rats. Blood glucose was measured three times weekly. In total, 193 rats were transplanted and rats were sacrificed 7, 23, 50, 90 days post-transplantation or at onset of diabetes. Pancreatic and transplant sections were stained for insulin and mononuclear cell infiltration and insulitis was graded. RESULTS: Eight (19%) rats developed diabetes in the insulin-treated group and 19 (63%) and 19 (65%) rats in the control and placebo, respectively (p = 0.0002 and p = 0.0001). Onset of diabetes in the insulin treated group was delayed compared to control and placebo, (102, 77 and 81 days of age, respectively (p = 0.0001 and p = 0.0001)). Insulin treatment diminished mononuclear cell infiltration in the islets at day 50 after transplantation compared to placebo. Infiltration pattern in islets in situ correlates with infiltration in transplants (r is 0.9076 and p < 0.001). CONCLUSION/INTERPRETATION: These results suggest that insulin-treatment of syngeneically transplanted BB-DP rats considerably decreases the incidence of diabetes and that this model is well suited for studying molecular changes in the islet transplants.
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Diabetes Mellitus Tipo 1/prevención & control , Insulina/farmacología , Islotes Pancreáticos/inmunología , Estado Prediabético/prevención & control , Animales , Diabetes Mellitus Tipo 1/etiología , Diabetes Mellitus Tipo 1/patología , Femenino , Islotes Pancreáticos/patología , Trasplante de Islotes Pancreáticos/inmunología , Trasplante de Islotes Pancreáticos/patología , Masculino , Estado Prediabético/etiología , Estado Prediabético/patología , Ratas , Ratas Endogámicas BB , Trasplante IsogénicoRESUMEN
The type 1 diabetes mellitus (T1DM) candidate gene SNP IL6-174G/C was genotyped in 253 Danish T1DM families (1129 individuals). TDT analysis demonstrated linkage in the presence of association between the IL6-174C allele and T1DM in the 416 T1DM offspring, P(tdt)=0.04. Gender conditioned TDT analyses revealed that linkage and association with T1DM were present in females exclusively; P(tdt)=6.5 x 10(-4) and P(tdt)=2.4 x 10(-4), respectively. Random transmission of the IL6-174C/G alleles was found in T1DM males, non-T1DM males and non-T1DM females; all P(tdt)>/=0.37. Heterogeneity analyses (T1DM versus non-T1DM females) excluded preferential meiotic segregation in females, P=4.6 x 10(-3), and demonstrated differences in the transmission patterns between female and male T1DM offspring, P=5.1 x 10(-3). The IL6-174 CC genotype was associated with younger age at onset of T1DM in females (P=0.002). The impact of 17beta-estradiol (E(2)) on the IL6-174G/C variants was investigated by reporter studies. The PMA stimulated activity of the T1DM risk IL6-174C variant exceeded that of the T1DM protective IL6-174G variant by approximately 70% in the absence of E(2) (P(c)=0.004), but not with E(2) present (P(c)=0.12). The PMA stimulated activity of the IL6-174G variant was repressed without E(2) present, but was derepressed by addition of E(2), P(c)=0.024. In contrast, the PMA stimulated IL6-174C activity was unaffected by E(2) as were the constitutive activities of the IL6-174G/C variants. In conclusion, higher IL6 promoter activity may confer risk to T1DM in very young females. This excess risk is negated with increasing age, possibly by the increasing E(2) levels in puberty.
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Diabetes Mellitus Tipo 1/genética , Estradiol/metabolismo , Interleucina-6/genética , Polimorfismo Genético , Regiones Promotoras Genéticas , Edad de Inicio , Animales , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Humanos , Interleucina-6/metabolismo , Masculino , Ratones , Factores SexualesRESUMEN
GH is an important regulator of cell growth and metabolism. In the pancreas, GH stimulates mitogenesis as well as insulin production in beta-cells. The cellular effects of GH are exerted mainly through activation of the Janus kinase-signal transducer and activator of transcription (STAT) pathway. Recently it has been found that suppressors of cytokine signaling (SOCS) proteins are able to inhibit GH-induced signal transduction. In the present study, the role of SOCS-3 in GH signaling was investigated in the pancreatic beta-cell lines RIN-5AH and INS-1 by means of inducible expression systems. Via stable transfection of the beta-cell lines with plasmids expressing SOCS-3 under the control of an inducible promoter, a time- and dose-dependent expression of SOCS-3 in the cells was obtained. EMSA showed that SOCS-3 is able to inhibit GH-induced DNA binding of both STAT3 and STAT5 in RIN-5AH cells. Furthermore, using Northern blot analysis it was shown that SOCS-3 can completely inhibit GH-induced insulin production in these cells. Finally, 5-bromodeoxyuridine incorporation followed by fluorescence-activated cell sorting analysis showed that SOCS-3 inhibits GH-induced proliferation of INS-1 cells. These findings support the hypothesis that SOCS-3 is a major regulator of GH signaling in insulin-producing cells.