RESUMEN
The presence of sugar in the gut causes induction of SGLT1, the sodium/glucose cotransporter in intestinal epithelial cells (enterocytes), and this is accompanied by stimulation of sugar absorption. Sugar sensing was suggested to involve a G-protein coupled receptor and cAMP - protein kinase A signalling, but the sugar receptor has remained unknown. We show strong expression and co-localization with SGLT1 of the ß2-adrenergic receptor (ß 2-AR) at the enterocyte apical membrane and reveal its role in stimulating glucose uptake from the gut by the sodium/glucose-linked transporter, SGLT1. Upon heterologous expression in different reporter systems, the ß 2-AR responds to multiple sugars in the mM range, consistent with estimated gut sugar levels after a meal. Most adrenergic receptor antagonists inhibit sugar signaling, while some differentially inhibit epinephrine and sugar responses. However, sugars did not inhibit binding of I125-cyanopindolol, a ß 2-AR antagonist, to the ligand-binding site in cell-free membrane preparations. This suggests different but interdependent binding sites. Glucose uptake into everted sacs from rat intestine was stimulated by epinephrine and sugars in a ß 2-AR-dependent manner. STD-NMR confirmed direct physical binding of glucose to the ß 2-AR. Oral administration of glucose with a non-bioavailable ß 2-AR antagonist lowered the subsequent increase in blood glucose levels, confirming a role for enterocyte apical ß 2-ARs in stimulating gut glucose uptake, and suggesting enterocyte ß 2-AR as novel drug target in diabetic and obese patients. Future work will have to reveal how glucose sensing by enterocytes and neuroendocrine cells is connected, and whether ß 2-ARs mediate glucose sensing also in other tissues.
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OBJECTIVE: The aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63+ basal cells reportedly do not exist in the normal pancreas. DESIGN: We evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed. RESULTS: In normal human pancreas, rare ΔNp63+ cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63+ cells are atypical KRT19+ duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme. CONCLUSION: In larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models.
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Autoantibodies against islet cell antigens are routinely used to identify subjects at increased risk of symptomatic type 1 diabetes, but their relation to the intra-islet pathogenetic process that leads to positivity for these markers is poorly understood. We screened 556 non-diabetic organ donors (3 months to 24 years) for five different autoantibodies and found positivity in 27 subjects, 25 single- and two double autoantibody-positive donors. Histopathological screening of pancreatic tissue samples showed lesion characteristic for recent-onset type 1 diabetes in the two organ donors with a high-risk profile, due to their positivity for multiple autoantibodies and HLA-inferred risk. Inflammatory infiltrates (insulitis) were found in a small fraction of islets (<5%) and consisted predominantly of CD3+CD8+ T-cells. Islets with insulitis were found in close proximity to islets devoid of insulin-positivity; such pseudo-atrophic islets were present in multiple small foci scattered throughout the pancreatic tissue or were found to be distributed with a lobular pattern. Relative beta cell area in both single and multiple autoantibody-positive donors was comparable to that in autoantibody-negative controls. In conclusion, in organ donors under age 25 years, insulitis and pseudo-atrophic islets were restricted to multiple autoantibody-positive individuals allegedly at high risk of developing symptomatic type 1 diabetes, in line with reports in older age groups. These observations may give further insight into the early pathogenetic events that may culminate in clinically overt disease.
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Antígenos/inmunología , Autoanticuerpos/sangre , Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Células Secretoras de Insulina/inmunología , Trasplante de Islotes Pancreáticos , Donantes de Tejidos , Adolescente , Factores de Edad , Biomarcadores/sangre , Estudios de Casos y Controles , Proliferación Celular , Niño , Preescolar , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/patología , Selección de Donante , Femenino , Humanos , Lactante , Células Secretoras de Insulina/patología , Masculino , Adulto JovenRESUMEN
Insulitis is a characteristic inflammatory lesion consisting of immune cell infiltrates around and within the pancreatic islets of patients with recent-onset type 1 diabetes (T1D). The infiltration is typically mild, both in terms of the number of infiltrating cells and the number of islets affected. Here, we present an unusual histopathological case study of a 66-year-old female patient with long-standing T1D, insulitis, and islet-associated lymphoid tissue. Most islets in the head of the pancreas of this patient were insulin-deficient, whereas the islets in the tail appeared normal. Insulitis was present in 0.84% of the insulin-containing islets and three islets had large lymphocytic infiltrates resembling tertiary lymphoid structures (TLS). Of note, this is the first description of potential TLS in the endocrine pancreas of a patient with T1D. Their association with a marked residual beta cell mass is of interest and may hint at new insights into disease progression and regulation of autoimmunity.
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Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Islotes Pancreáticos/patología , Ganglios Linfáticos/patología , Anciano , Autoinmunidad/inmunología , Diabetes Mellitus Tipo 1/complicaciones , Progresión de la Enfermedad , Femenino , Humanos , Insulina/metabolismo , Islotes Pancreáticos/inmunología , Ganglios Linfáticos/inmunologíaRESUMEN
M2 macrophages play an important role in tissue repair and regeneration. They have also been found to modulate ß-cell replication in mouse models of pancreatic injury and disease. We previously reported that ß-cell replication is strongly increased in a subgroup of human organ donors characterized by prolonged duration of stay in an intensive care unit (ICU) and increased number of leukocytes in the pancreatic tissue. In the present study we investigated the relationship between duration of stay in the ICU, M2 macrophages, vascularization, and pancreatic cell replication. Pancreatic organs from 50 donors without diabetes with different durations of stay in the ICU were analyzed by immunostaining and digital image analysis. The number of CD68+CD206+ M2 macrophages increased three- to sixfold from ≥6 days' duration of stay in the ICU onwards. This was accompanied by a threefold increased vascular density and a four- to ninefold increase in pancreatic cells positive for the replication marker Ki67. A strong correlation was observed between the number of M2 macrophages and ß-cell replication. These results show that a prolonged duration of stay in the ICU is associated with an increased M2 macrophage number, increased vascular density, and an overall increase in replication of all pancreatic cell types. Our data show evidence of marked levels of tissue repair in the human donor pancreas.
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Proliferación Celular/fisiología , Unidades de Cuidados Intensivos , Tiempo de Internación , Macrófagos/patología , Páncreas/fisiología , Regeneración/fisiología , Donantes de Tejidos , Adolescente , Adulto , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Femenino , Humanos , Antígeno Ki-67/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/metabolismo , Persona de Mediana Edad , Neovascularización Fisiológica/fisiología , Páncreas/metabolismo , Páncreas/patología , Receptores de Superficie Celular/metabolismo , Adulto JovenRESUMEN
CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3'-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.
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Sistemas CRISPR-Cas , Edición Génica/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Mioblastos/metabolismo , Distrofia Miotónica/genética , Expansión de Repetición de Trinucleótido/genética , Células Cultivadas , Niño , Femenino , Humanos , Persona de Mediana Edad , Desarrollo de Músculos/genética , Distrofia Miotónica/metabolismo , Distrofia Miotónica/patologíaRESUMEN
The specific phenotype of mature differentiated beta cells not only depends on the specific presence of genes that allow beta cell function but also on the selective absence of housekeeping genes ("disallowed genes") that would interfere with this function. Recent studies have shown that both histone modifications and DNA methylation via the de novo methyltransferase DNMT3A are involved in repression of disallowed genes in neonatal beta cells when these cells acquire their mature phenotype. It is unknown, however, if the environmental influence of advanced age, pregnancy and the metabolic stress of high fat diet or diabetes could alter the repression of disallowed genes in beta cells. In the present study, we show that islet disallowed genes-which are also deeply repressed in FACS-purified beta cells-remain deeply repressed in animals of advanced age and in pregnant females. Moreover, the stability of this repression was correlated with strong and stable histone repression marks that persisted in islets isolated from 2 year old mice and with overall high expression of Dnmt3a in islets. Furthermore, repression of disallowed genes was unaffected by the metabolic stress of high fat diet. However, repression of about half of the disallowed genes was weakened in 16 week-old diabetic db/db mice. In conclusion, we show that the disallowed status of islet genes is stable under physiological challenging conditions (advanced age, pregnancy, high fat diet) but partially lost in islets from diabetic animals.
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Envejecimiento/fisiología , Metilación de ADN/genética , Diabetes Mellitus/metabolismo , Dieta Alta en Grasa , Código de Histonas/genética , Células Secretoras de Insulina/metabolismo , Estrés Fisiológico/fisiología , Animales , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Femenino , Prueba de Tolerancia a la Glucosa , Insulina/metabolismo , Células Secretoras de Insulina/citología , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , EmbarazoRESUMEN
Transplantation of islet allografts into type 1 diabetic recipients usually requires multiple pancreas donors to achieve insulin independence. This adds to the challenges of immunological monitoring of islet transplantation currently relying on surrogate immune markers in peripheral blood. We investigated donor origin and infiltration of islets transplanted in the liver of a T1D patient who died of hemorrhagic stroke 4 months after successful transplantation with two intraportal islet grafts combining six donors. Immunohistological staining for donor HLA using a unique panel of human monoclonal HLA-specific alloantibodies was performed on liver cryosections after validation on cryopreserved kidney, liver, and pancreas and compared with auto- and alloreactive T-cell immunity in peripheral blood. HLA-specific staining intensity and signal-to-noise ratio varied between tissues from very strong on kidney glomeruli, less in liver, kidney tubuli, and endocrine pancreas to least in exocrine pancreas, complicating the staining of inflamed islets in an HLA-disparate liver. Nonetheless, five islets from different liver lobes could be attributed to donors 1, 2, and 5 by staining patterns with multiple HLA types. All islets showed infiltration with CD8+ cytotoxic T cells that was mirrored by progressive alloreactive responses in peripheral blood mononuclear cells (PBMCs) to donors 1, 2, and 5 after transplantation. Stably low rates of peripheral islet autoreactive T-cell responses after islet infusion fit with a complete HLA mismatch between grafts and recipient and exclude the possibility that the islet-infiltrating CD8 T cells were autoreactive. HLA-specific immunohistochemistry can identify donor origin in situ and differentiate graft dysfunction and immunological destruction.
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Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/cirugía , Trasplante de Islotes Pancreáticos/inmunología , Donantes de Tejidos , Autoinmunidad/inmunología , Linfocitos T CD8-positivos/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Femenino , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Hígado/metabolismo , Persona de Mediana Edad , Páncreas/inmunología , Páncreas/metabolismo , Trasplante HomólogoRESUMEN
ß-Cell failure in type 2 diabetes (T2D) was recently proposed to involve dedifferentiation of ß-cells and ectopic expression of other islet hormones, including somatostatin and glucagon. Here we show that gastrin, a stomach hormone typically expressed in the pancreas only during embryogenesis, is expressed in islets of diabetic rodents and humans with T2D. Although gastrin in mice is expressed in insulin+ cells, gastrin expression in humans with T2D occurs in both insulin+ and somatostatin+ cells. Genetic lineage tracing in mice indicates that gastrin expression is turned on in a subset of differentiated ß-cells after exposure to severe hyperglycemia. Gastrin expression in adult ß-cells does not involve the endocrine progenitor cell regulator neurogenin3 but requires membrane depolarization, calcium influx, and calcineurin signaling. In vivo and in vitro experiments show that gastrin expression is rapidly eliminated upon exposure of ß-cells to normal glucose levels. These results reveal the fetal hormone gastrin as a novel marker for reversible human ß-cell reprogramming in diabetes.
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Diabetes Mellitus Tipo 2/metabolismo , Gastrinas/metabolismo , Células Secretoras de Insulina/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Estudios de Casos y Controles , Diabetes Mellitus/metabolismo , Regulación del Desarrollo de la Expresión Génica , Gerbillinae , Humanos , Inmunohistoquímica , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Proteínas del Tejido Nervioso/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Secretoras de Somatostatina/metabolismo , Células Madre/metabolismoRESUMEN
Somatic mutations in the epidermal growth factor receptor-tyrosine kinase (EGFR-TK) domain of non-small cell lung cancer (NSCLC) influence the responsiveness of these tumors to EGFR-TK inhibitors, indicating their usefulness as a predictive molecular marker. However, for mutation analysis, the amount of clinical material available from NSCLC patients is often very limited, suboptimally preserved, and composed of both normal and tumor cells. As a consequence, the total amount of recovered DNA is frequently very limited, with mutant alleles being often strongly underrepresented, and thus requiring highly sensitive methods for the detection of mutations. In the present study, EGFR mutation screening was performed on 210 NSCLC clinical samples by heminested polymerase chain reaction (PCR), followed by denaturing gradient gel electrophoresis (DGGE). Candidate mutations were further characterized by sequencing. Seventeen different types of pathogenic EGFR-TK domain mutations were detected in 55 of the 210 samples (26%). We reanalyzed 149 of the 155 samples in which no mutation was found by real-time PCR for the presence of recurrent exon 21 and exon 19 mutations using peptide nucleic acid probes in the PCR mix to increase sensitivity by mutant allele enrichment. Four additional samples with exon 19 mutations were detected. Thus, it is found that the relatively simple and inexpensive PCR-DGGE assay is already very sensitive for the detection of mutations in clinical samples, including samples with low tumor cellularity (10% or higher tumor cell content), although the sensitivity and speed of the assay can be further increased for a restricted panel of mutations by introducing peptide nucleic acid probes in the DGGE or real-time PCR-based assay.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutación , Reacción en Cadena de la Polimerasa/métodos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Activación Enzimática/genética , Receptores ErbB/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Estructura Terciaria de ProteínaRESUMEN
CONTEXT AND OBJECTIVE: Intraportal islet transplantation can restore insulin production in type 1 diabetes patients, but its effect is subject to several interfering processes. To assess the influence of ß-cell loss before and during engraftment, we searched for a real-time marker of ß-cell destruction. Previous studies showed that 65-kDa isoform of glutamate decarboxylase (GAD65) is discharged by chemically damaged rat ß-cells. We therefore examined the utility of the GAD65 assay to detect and quantify destruction of human ß-cells in vitro and in vivo. DESIGN AND PARTICIPANTS: A time-resolved fluorescence immunoassay was used to measure GAD65 discharge from ß-cells after administration of toxins or after intraportal transplantation. The study in patients involved type 1 diabetes recipients of 56 implants. RESULTS: GAD65 was discharged from cultured human ß-cells between 4 and 24 hours after acute insult and proportional to the number of dying cells. It was also detected in plasma during the first 24 hours after intraportal transplantation of human islet cell grafts. Diabetic nude rat recipients without hyperglycemic correction exhibited higher plasma GAD65 levels than those with normalization. In type 1 diabetes recipients of grafts with 2-5 × 10(6) ß-cells per kilogram of body weight, five of six with plasma GAD65 greater than 1 ng/mL failed to increase plasma C-peptide by greater than 0.5 ng/mL at posttransplant month 2, whereas five of six with undetectable plasma GAD 65 and 15 of 19 with intermediate levels did result in such increase. CONCLUSION: Plasma GAD65 qualifies as a marker for early ß-cell loss after intraportal transplantation. Further studies are needed to extend its clinical utility.
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Biomarcadores/sangre , Diabetes Mellitus Tipo 1/terapia , Glutamato Descarboxilasa/sangre , Células Secretoras de Insulina/fisiología , Trasplante de Islotes Pancreáticos/métodos , Animales , Muerte Celular , Células Cultivadas , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/diagnóstico , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/diagnóstico , Supervivencia de Injerto , Humanos , Masculino , Vena Porta , Pronóstico , Ratas , Ratas Desnudas , Ratas Wistar , EstreptozocinaRESUMEN
Pregnancy requires a higher functional beta cell mass and this is associated with profound changes in the gene expression profile of pancreatic islets. Taking Tph1 as a sensitive marker for pregnancy-related islet mRNA expression in female mice, we previously identified prolactin receptors and placental lactogen as key signalling molecules. Since beta cells from male mice also express prolactin receptors, the question arose whether male and female islets have the same phenotypic resilience at the mRNA level during pregnancy. We addressed this question in vitro, by stimulating cultured islets with placental lactogen and in vivo, by transplanting male or female islets into female acceptor mice. Additionally, the islet mRNA expression pattern of pregnant prolactin receptor deficient mice was compared with that of their pregnant wild-type littermates. When cultured with placental lactogen, or when transplanted in female recipients that became pregnant (day 12.5), male islets induced the 'islet pregnancy gene signature', which we defined as the 12 highest induced genes in non-transplanted female islets at day 12.5 of pregnancy. In addition, serotonin immunoreactivity and beta cell proliferation was also induced in these male transplanted islets at day 12.5 of pregnancy. In order to further investigate the importance of prolactin receptors in these mRNA changes we used a prolactin receptor deficient mouse model. For the 12 genes of the signature, which are highly induced in control pregnant mice, no significant induction of mRNA transcripts was found at day 9.5 of pregnancy. Together, our results support the key role of placental lactogen as a circulating factor that can trigger the pregnancy mRNA profile in both male and female beta cells.
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Células Secretoras de Insulina/trasplante , Lactógeno Placentario/sangre , Embarazo/genética , ARN Mensajero/genética , Receptores de Prolactina/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Ratones , Lactógeno Placentario/farmacología , Embarazo/sangre , Receptores de Prolactina/genéticaRESUMEN
PURPOSE OF REVIEW: The purpose of this article is to summarize recent developments in the histopathology of recent-onset type 1 diabetes. RECENT FINDINGS: Insulitis is considered to be the defining lesion in young type 1 diabetic patients, and insight into its pathogenic mechanism is crucial for the development of effective new therapies. The present overview highlights some recent developments including an international consensus guideline for the diagnosis of insulitis in type 1 diabetic patients, immunophenotyping of the insulitic lesions, evidence for a heterogeneity of the disease process and a discussion on the differences and similarities between insulitis in patients and in rodent models of the disease. SUMMARY: We have reviewed recent data, published over the last year, on the nature and mechanism of insulitis in both patients and the nonobese diabetic mouse model.
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Autoinmunidad , Diabetes Mellitus Tipo 1/inmunología , Páncreas/inmunología , Linfocitos T/inmunología , Animales , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/fisiopatología , Modelos Animales de Enfermedad , Humanos , Ratones Endogámicos NOD , Páncreas/metabolismo , Páncreas/fisiopatología , Pronóstico , Especificidad de la Especie , Linfocitos T/metabolismo , Investigación Biomédica TraslacionalRESUMEN
The human growth hormone (hGH) minigene is frequently used in the derivation of transgenic mouse lines to enhance transgene expression. Although this minigene is present in the transgenes as a secondcistron, and thus not thought to be expressed, we found that three commonly used lines, Pdx1-Cre(Late), RIP-Cre, and MIP-GFP, each expressed significant amounts of hGH in pancreatic islets. Locally secreted hGH binds to prolactin receptors on ß cells, activates STAT5 signaling, and induces pregnancy-like changes in gene expression, thereby augmenting pancreatic ß cell mass and insulin content. In addition, islets of Pdx1-Cre(Late) mice have lower GLUT2 expression and reduced glucose-induced insulin release and are protected against the ß cell toxin streptozotocin. These findings may be important when interpreting results obtained when these and other hGH minigene-containing transgenic mice are used.
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Hormona de Crecimiento Humana/metabolismo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Animales , Femenino , Hormona de Crecimiento Humana/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Receptores de Prolactina/genética , Receptores de Prolactina/metabolismo , Triptófano Hidroxilasa/genética , Triptófano Hidroxilasa/metabolismoRESUMEN
Human type 1 diabetes (T1D) is considered to be an autoimmune disease, with CD8+ T-cell-mediated cytotoxicity being directed against the insulin-producing beta cells, leading to a gradual decrease in beta cell mass and the development of chronic hyperglycemia. The histopathologically defining lesion in recent-onset T1D patients is insulitis, a relatively subtle leucocytic infiltration present in approximately 10% of the islets of Langerhans from children with recent-onset (<1 year) disease. Due to the transient nature of the infiltrate, its heterogeneous distribution in the pancreas and the nature of the patient population, material for research is extremely rare and limited to a cumulative total of approximately 150 cases collected over the past century. Most studies on the etiopathogenesis of T1D have therefore focused on the non-obese diabetic (NOD) mouse model, which shares many genetic and immunological disease characteristics with human T1D, although its islet histopathology is remarkably different. In view of these differences and in view of the limited success of clinical immune interventions based on observations in the NOD mouse, there is a renewed focus on studying the pathogenetic process in patient material.
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Diabetes Mellitus Tipo 1/etiología , Inflamación/inmunología , Islotes Pancreáticos/inmunología , Animales , Autoinmunidad , Diabetes Mellitus Tipo 1/patología , Modelos Animales de Enfermedad , Humanos , Inflamación/patología , Islotes Pancreáticos/patología , Ratones , Ratones Endogámicos NODRESUMEN
ß cell failure in type 2 diabetes (T2D) is associated with hyperglycemia, but the mechanisms are not fully understood. Congenital hyperinsulinism caused by glucokinase mutations (GCK-CHI) is associated with ß cell replication and apoptosis. Here, we show that genetic activation of ß cell glucokinase, initially triggering replication, causes apoptosis associated with DNA double-strand breaks and activation of the tumor suppressor p53. ATP-sensitive potassium channels (KATP channels) and calcineurin mediate this toxic effect. Toxicity of long-term glucokinase overactivity was confirmed by finding late-onset diabetes in older members of a GCK-CHI family. Glucagon-like peptide-1 (GLP-1) mimetic treatment or p53 deletion rescues ß cells from glucokinase-induced death, but only GLP-1 analog rescues ß cell function. DNA damage and p53 activity in T2D suggest shared mechanisms of ß cell failure in hyperglycemia and CHI. Our results reveal membrane depolarization via KATP channels, calcineurin signaling, DNA breaks, and p53 as determinants of ß cell glucotoxicity and suggest pharmacological approaches to enhance ß cell survival in diabetes.
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Hiperinsulinismo Congénito/complicaciones , Roturas del ADN de Doble Cadena , Diabetes Mellitus Tipo 2/complicaciones , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Biomarcadores/metabolismo , Calcineurina/metabolismo , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Hiperinsulinismo Congénito/enzimología , Hiperinsulinismo Congénito/patología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Ayuno/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Glucoquinasa/biosíntesis , Glucosa/toxicidad , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/enzimología , Potenciales de la Membrana/efectos de los fármacos , Ratones , TransgenesRESUMEN
BACKGROUND: Rapid revascularization of islet cell implants is important for engraftment and subsequent survival and function. Development of an adequate vascular network is expected to allow adaptive growth of the ß-cell mass. The present study compares omentum and kidney capsule as sites for growth and differentiation of immature ß-cell grafts. METHODS: Perinatal porcine islet cell grafts were implanted in omentum or under kidney capsule of nondiabetic nude rats. Implants were compared over 10 weeks for their respective growth, cellular composition, number and size of ß cells, their proliferative activity, and implant blood vessel density. RESULTS: In both sites, the ß-cell volume increased fourfold between weeks 1 and 10 reflecting a rise in ß-cell number. In the omental implants, however, the cellular insulin reserves and the percent of proliferating cells were twofold higher than in kidney implants. In parallel, the blood vessel density in omental implants increased twofold, reaching a density comparable with islets in adult pig pancreas. A positive correlation was found between the percent bromodeoxyuridine-positive ß cells and the vessel density. CONCLUSIONS: Growth of the ß-cell volume proceeds similarly in the omentum and under the kidney capsule. However, the omentum leads to higher insulin reserves and an increased pool of proliferating cells, which might be related to a more extended vascular network. Our observations support the omentum as an alternative site for immature porcine islet cells, with beneficial effects on proliferation and implant revascularization.
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Células Secretoras de Insulina/citología , Insulina/metabolismo , Trasplante de Islotes Pancreáticos/métodos , Riñón/patología , Epiplón/patología , Animales , Vasos Sanguíneos/patología , Bromodesoxiuridina , Proliferación Celular , Supervivencia Celular , Células Secretoras de Glucagón/citología , Inmunohistoquímica , Masculino , Ratas , Ratas Desnudas , Ratas Wistar , PorcinosRESUMEN
BACKGROUND: Pancreatic-tail duct ligation (PDL) in adult rodents has been reported to induce beta cell generation and increase beta cell mass but increases in beta cell number have not been demonstrated. This study examines whether PDL increases beta cell number and whether this is caused by neogenesis of small clusters and/or their growth to larger aggregates. METHODOLOGY: Total beta cell number and its distribution over small (<50 µm), medium, large (>100 µm) clusters was determined in pancreatic tails of 10-week-old mice, 2 weeks after PDL or sham. PRINCIPAL FINDINGS: PDL increased total beta cell mass but not total beta cell number. It induced neogenesis of small beta cell clusters (2.2-fold higher number) which contained a higher percent proliferating beta cells (1.9% Ki67+cells) than sham tails (<0.2%); their higher beta cell number represented <5% of total beta cell number and was associated with a similar increase in alpha cell number. It is unknown whether the regenerative process is causally related to the inflammatory infiltration in PDL-tails. Human pancreases with inflammatory infiltration also exhibited activation of proliferation in small beta cell clusters. CONCLUSIONS/SIGNIFICANCE: The PDL model illustrates the advantage of direct beta cell counts over beta cell mass measurements when assessing and localizing beta cell regeneration in the pancreas. It demonstrates the ability of the adult mouse pancreas for neogenesis of small beta cell clusters with activated beta cell proliferation. Further studies should investigate conditions under which neoformed small beta cell clusters grow to larger aggregates and hence to higher total beta cell numbers.