RESUMEN
OBJECTIVE: Type 2 diabetes (T2D) is characterised by the loss of first-phase insulin secretion. We studied mice with ß-cell selective loss of the glucagon receptor (Gcgrfl/fl X Ins-1Cre), to investigate the role of intra-islet glucagon receptor (GCGR) signalling on pan-islet [Ca2+]I activity and insulin secretion. METHODS: Metabolic profiling was conducted on Gcgrß-cell-/- and littermate controls. Crossing with GCaMP6f (STOP flox) animals further allowed for ß-cell specific expression of a fluorescent calcium indicator. These islets were functionally imaged in vitro and in vivo. Wild-type mice were transplanted with islets expressing GCaMP6f in ß-cells into the anterior eye chamber and placed on a high fat diet. Part of the cohort received a glucagon analogue (GCG-analogue) for 40 days and the control group were fed to achieve weight matching. Calcium imaging was performed regularly during the development of hyperglycaemia and in response to GCG-analogue treatment. RESULTS: Gcgrß-cell-/- mice exhibited higher glucose levels following intraperitoneal glucose challenge (control 12.7 mmol/L ± 0.6 vs. Gcgrß-cell-/- 15.4 mmol/L ± 0.0 at 15 min, p = 0.002); fasting glycaemia was not different to controls. In vitro, Gcgrß-cell-/- islets showed profound loss of pan-islet [Ca2+]I waves in response to glucose which was only partially rescued in vivo. Diet induced obesity and hyperglycaemia also resulted in a loss of co-ordinated [Ca2+]I waves in transplanted islets. This was reversed with GCG-analogue treatment, independently of weight-loss (n = 8). CONCLUSION: These data provide novel evidence for the role of intra-islet GCGR signalling in sustaining synchronised [Ca2+]I waves and support a possible therapeutic role for glucagonergic agents to restore the insulin secretory capacity lost in T2D.
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Diabetes Mellitus Tipo 2 , Glucagón , Glucosa , Homeostasis , Secreción de Insulina , Células Secretoras de Insulina , Receptores de Glucagón , Transducción de Señal , Animales , Glucagón/metabolismo , Ratones , Células Secretoras de Insulina/metabolismo , Glucosa/metabolismo , Receptores de Glucagón/metabolismo , Receptores de Glucagón/genética , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Masculino , Islotes Pancreáticos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Dieta Alta en Grasa , Glucemia/metabolismo , FemeninoRESUMEN
Diabetes mellitus is a growing worldwide epidemic disease, currently affecting 1 in 12 adults. Treatment of disease complications typically consumes â¼10% of healthcare budgets in developed societies. Whilst immune-mediated destruction of insulin-secreting pancreatic ß cells is responsible for Type 1 diabetes, both the loss and dysfunction of these cells underly the more prevalent Type 2 diabetes. The establishment of robust drug development programmes aimed at ß-cell restoration is still hampered by the absence of means to measure ß-cell mass prospectively in vivo, an approach which would provide new opportunities for understanding disease mechanisms and ultimately assigning personalized treatments. In the present review, we describe the progress towards this goal achieved by the Innovative Medicines Initiative in Diabetes, a collaborative public-private consortium supported by the European Commission and by dedicated resources of pharmaceutical companies. We compare several of the available imaging methods and molecular targets and provide suggestions as to the likeliest to lead to tractable approaches. Furthermore, we discuss the simultaneous development of animal models that can be used to measure subtle changes in ß-cell mass, a prerequisite for validating the clinical potential of the different imaging tracers.
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Diabetes Mellitus/patología , Células Secretoras de Insulina/patología , Imagen Molecular/métodos , Adulto , Animales , Adhesión Celular , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Mediciones Luminiscentes , Manganeso , Glicoproteínas de Membrana/metabolismo , Ratones , Ratas , Receptores de Sulfonilureas/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , ZincRESUMEN
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
Whilst the heritable nature of Type 2 diabetes has been recognized for many years, only in the past two decades have linkage analyses in families and genome-wide association studies in large populations begun to reveal the genetic landscape of the disease in detail. Whilst the former have provided a powerful means of identifying the genes responsible for monogenic forms of the disease, the latter highlight relatively large genomic regions. These often harbour multiple genes, whose relative contribution to exaggerated disease risk is uncertain. In the present study, the approaches that have been used to dissect the role of just a few (TCF7L2, SLC30A8, ADCY5, MTNR1B and CDKAL1) of the ~ 500 genes identified at dozens of implicated loci are described. These are usually selected based on the strength of their effect on disease risk, and predictions as to their likely biological role. Direct determination of the effects of identified polymorphisms on gene expression in disease-relevant tissues, notably the pancreatic islet, are then performed to identify genes whose expression is affected by a particular polymorphism. Subsequent functional analyses then involve perturbing gene expression in vitro in ß-cell lines or isolated islets and in vivo in animal models. Although the majority of polymorphisms affect insulin production rather than action, and mainly affect the ß cell, effects via other tissues may also contribute, requiring careful consideration in the design and interpretation of experiments in model systems. These considerations illustrate the scale of the task needed to exploit genome-wide association study data for the development of new therapeutic strategies.
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Diabetes Mellitus Tipo 2/genética , Estudio de Asociación del Genoma Completo , Adenilil Ciclasas/genética , Proteínas de Transporte de Catión/genética , Quinasa 5 Dependiente de la Ciclina/genética , Humanos , Receptor de Melatonina MT2/genética , Proteína 2 Similar al Factor de Transcripción 7/genética , Transportador 8 de Zinc , ARNt MetiltransferasasRESUMEN
AIMS/HYPOTHESIS: Type 2 diabetes is characterised by progressive beta cell dysfunction, with changes in gene expression playing a crucial role in its development. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and therefore alterations in miRNA levels may be involved in the deterioration of beta cell function. METHODS: Global TaqMan arrays and individual TaqMan assays were used to measure islet miRNA expression in discovery (n = 20) and replication (n = 20) cohorts from individuals with and without type 2 diabetes. The role of specific dysregulated miRNAs in regulating insulin secretion, content and apoptosis was subsequently investigated in primary rat islets and INS-1 cells. Identification of miRNA targets was assessed using luciferase assays and by measuring mRNA levels. RESULTS: In the discovery and replication cohorts miR-187 expression was found to be significantly increased in islets from individuals with type 2 diabetes compared with matched controls. An inverse correlation between miR-187 levels and glucose-stimulated insulin secretion (GSIS) was observed in islets from normoglycaemic donors. This correlation paralleled findings in primary rat islets and INS-1 cells where overexpression of miR-187 markedly decreased GSIS without affecting insulin content or apoptotic index. Finally, the gene encoding homeodomain-interacting protein kinase-3 (HIPK3), a known regulator of insulin secretion, was identified as a direct target of miR-187 and displayed reduced expression in islets from individuals with type 2 diabetes. CONCLUSIONS/INTERPRETATION: Our findings suggest a role for miR-187 in the blunting of insulin secretion, potentially involving regulation of HIPK3, which occurs during the pathogenesis of type 2 diabetes.
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Diabetes Mellitus Tipo 2/metabolismo , Glucosa/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , MicroARNs/metabolismo , Adulto , Anciano , Animales , Línea Celular , Células Cultivadas , Humanos , Secreción de Insulina , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , MicroARNs/genética , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
We describe a one-pot strategy for the high yielding, operationally simple synthesis of fluorescent probes for Zn2+ that bear biological targeting groups and exemplify the utility of our method through the preparation of a small library of sensors. Investigation of the fluorescence behaviour of our library revealed that although all behaved as expected in MeCN, under biologically relevant conditions in HEPES buffer, a plasma membrane targeting sensor displayed a dramatic switch on response to excess Zn2+ as a result of aggregation phenomena. Excitingly, in cellulo studies in mouse pancreatic islets demonstrated that this readily available sensor was indeed localised to the exterior of the plasma membrane and clearly responded to the Zn2+ co-released when the pancreatic beta cells were stimulated to release insulin. Conversely, sensors that target intracellular compartments were unaffected. These results demonstrate that this sensor has the potential to allow the real time study of insulin release from living cells and exemplifies the utility of our simple synthetic approach.
RESUMEN
Outside of the biological arena the term 'repression' often has a negative connotation. However, in the pancreatic ß-cell a small group of genes, which are abundantly expressed in most if not all other mammalian tissues, are highly selectively repressed, with likely functional consequences. The two 'founder' members of this group, lactate dehydrogenase A (Ldha) and monocarboxylate transporter-1 (MCT-1/Slc16a1), are inactivated by multiple mechanisms including histone modifications and microRNA-mediated silencing. Their inactivation ensures that pyruvate and lactate, derived from muscle during exercise, do not stimulate insulin release inappropriately. Correspondingly, activating mutations in the MCT-1 promoter underlie 'exercise-induced hyperinsulinism' (EIHI) in man, a condition mimicked by forced over-expression of MCT-1 in the ß-cell in mice. Furthermore, LDHA expression in the ß-cell is upregulated in both human type 2 diabetes and in rodent models of the disease. Recent work by us and by others has identified a further â¼60 genes which are selectively inactivated in the ß-cell, a list which we refine here up to seven by detailed comparison of the two studies. These genes include key regulators of cell proliferation and stimulus-secretion coupling. The present, and our earlier results, thus highlight the probable importance of shutting down a subset of 'disallowed' genes for the differentiated function of ß-cells, and implicate previously unsuspected signalling pathways in the control of ß-cell expansion and insulin secretion. Targeting of deregulated 'disallowed' genes in these cells may thus, in the future, provide new therapeutic avenues for type 2 diabetes.
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Diabetes Mellitus Tipo 2/genética , Marcación de Gen/métodos , Hiperinsulinismo/genética , Células Secretoras de Insulina/metabolismo , L-Lactato Deshidrogenasa/genética , Transportadores de Ácidos Monocarboxílicos/genética , Simportadores/genética , Animales , Proliferación Celular , Diabetes Mellitus Tipo 2/fisiopatología , Epigenómica , Ejercicio Físico , Femenino , Silenciador del Gen , Humanos , Hiperinsulinismo/etiología , Hiperinsulinismo/fisiopatología , Isoenzimas/genética , Lactato Deshidrogenasa 5 , Masculino , Ratones , Mutación , Estrés OxidativoRESUMEN
AIMS/HYPOTHESIS: Individuals carrying type 2 diabetes risk alleles in TCF7L2 display decreased beta cell levels of T cell factor 7 like-2 (TCF7L2) immunoreactivity, and impaired insulin secretion and beta cell sensitivity to glucagon-like peptide 1 (GLP-1). Here, we sought to determine whether selective deletion of Tcf7l2 in mouse pancreas impairs insulin release and glucose homeostasis. METHODS: Pancreas-specific Tcf7l2-null (pTcf7l2) mice were generated by crossing mice carrying conditional knockout alleles of Tcf7l2 (Tcf7l2-flox) with mice expressing Cre recombinase under the control of the Pdx1 promoter (Pdx1.Cre). Gene expression was assessed by real-time quantitative PCR and beta cell mass by optical projection tomography. Glucose tolerance, insulin secretion from isolated islets, and plasma insulin, glucagon and GLP-1 content were assessed by standard protocols. RESULTS: From 12 weeks of age, pTcf7l2 mice displayed decreased oral glucose tolerance vs control littermates; from 20 weeks they had glucose intolerance upon administration of glucose by the intraperitoneal route. pTcf7l2 islets displayed impaired insulin secretion in response to 17 (vs 3.0) mmol/l glucose (54.6 ± 4.6%, p < 0.01) or to 17 mmol/l glucose plus 100 nmol/l GLP-1 (44.3 ± 4.9%, p < 0.01) compared with control islets. Glp1r (42 ± 0.08%, p < 0.01) and Ins2 (15.4 ± 4.6%, p < 0.01) expression was significantly lower in pTcf7l2 islets than in controls. Maintained on a high-fat (but not on a normal) diet, pTcf7l2 mice displayed decreased expansion of pancreatic beta cell volume vs control littermates. No differences were observed in plasma insulin, proinsulin, glucagon or GLP-1 concentrations. CONCLUSIONS/INTERPRETATION: Selective deletion of Tcf7l2 in the pancreas replicates key aspects of the altered glucose homeostasis in human carriers of TCF7L2 risk alleles, indicating the direct role of this factor in controlling beta cell function.
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Glucosa/metabolismo , Homeostasis/fisiología , Insulina/metabolismo , Páncreas/metabolismo , Proteína 2 Similar al Factor de Transcripción 7/deficiencia , Animales , Células Cultivadas , Glucagón/sangre , Péptido 1 Similar al Glucagón/sangre , Péptido 1 Similar al Glucagón/farmacología , Prueba de Tolerancia a la Glucosa , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/fisiología , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ratones , Ratones Noqueados , Páncreas/citología , Páncreas/efectos de los fármacos , Proinsulina/sangre , Proteína 2 Similar al Factor de Transcripción 7/genética , Proteína 2 Similar al Factor de Transcripción 7/metabolismoRESUMEN
AIMS/HYPOTHESIS: Intronic single nucleotide polymorphisms within the transcription factor 7-like 2 (TCF7L2) gene are associated with risk of type 2 diabetes. It is widely hypothesised that the predisposing variation is involved in cis-regulation of TCF7L2 activity. The aim of this study was to seek evidence for the existence of novel TCF7L2 isoforms encoded within the type 2 diabetes-associated genomic region. METHODS: We searched expressed sequence tag (EST) databases for novel TCF7L2 transcripts and sought to validate the function and integrity of any isoforms found using a combination of RT-PCR, western blotting and reporter gene techniques. RESULTS: Analysis of EST databases suggested the presence of an alternative polyadenylation site located in intron 4 of TCF7L2. We used 3' rapid amplification of cDNA ends and real-time PCR to validate the integrity of this polyadenylation signal and show its wide use across human tissues. Western blotting results are consistent with the use of this polyadenylation signal to generate novel protein isoforms. The alternative polyadenylation signal results in the production of isoforms that retain the ß-catenin binding domain but do not possess the high-mobility group box DNA-binding domain. Promoter-reporter gene assays suggest that these isoforms inhibit TCF7L2-dependent target genes by sequestering ß-catenin. CONCLUSIONS/INTERPRETATION: We have identified a novel polyadenylation signal within TCF7L2 that can result in the production of isoforms that act to repress TCF/LEF-dependent target genes. These findings may provide new insights into the association of TCF7L2 with susceptibility to type 2 diabetes.
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Regulación de la Expresión Génica , Poliadenilación , Isoformas de Proteínas/metabolismo , Transducción de Señal , Factores de Transcripción TCF/metabolismo , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Empalme Alternativo , Secuencia de Bases , Línea Celular , Bases de Datos Genéticas , Diabetes Mellitus Tipo 2/genética , Exones , Etiquetas de Secuencia Expresada , Humanos , Intestino Delgado/metabolismo , Intrones , Datos de Secuencia Molecular , Páncreas/metabolismo , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Factores de Transcripción TCF/genética , beta Catenina/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
AIM/HYPOTHESIS: AMP-activated protein kinase (AMPK), encoded by Prkaa genes, is emerging as a key regulator of overall energy homeostasis and the control of insulin secretion and action. We sought here to investigate the role of AMPK in controlling glucagon secretion from pancreatic islet alpha cells. METHODS: AMPK activity was modulated in vitro in clonal alphaTC1-9 cells and isolated mouse pancreatic islets using pharmacological agents and adenoviruses encoding constitutively active or dominant negative forms of AMPK. Glucagon secretion was measured during static incubation by radioimmunoassay. AMPK activity was assessed by both direct phosphotransfer assay and by western (immuno-)blotting of the phosphorylated AMPK α subunits and the downstream target acetyl-CoA carboxylase 1. Intracellular free [Ca²(+)] was measured using Fura-Red. RESULTS: Increasing glucose concentrations strongly inhibited AMPK activity in clonal pancreatic alpha cells. Forced increases in AMPK activity in alphaTC1-9 cells, achieved through the use of pharmacological agents including metformin, phenformin and A-769662, or via adenoviral transduction, resulted in stimulation of glucagon secretion at both low and high glucose concentrations, whereas AMPK inactivation inhibited both [Ca²(+)](i) increases and glucagon secretion at low glucose. Transduction of isolated mouse islets with an adenovirus encoding AMPK-CA under the control of the preproglucagon promoter increased glucagon secretion selectively at elevated glucose concentrations. CONCLUSIONS/INTERPRETATION: AMPK is strongly regulated by glucose in pancreatic alpha cells, and increases in AMPK activity are sufficient and necessary for the stimulation of glucagon release in vitro. Modulation of AMPK activity in alpha cells may therefore provide a novel approach to controlling blood glucose concentrations.
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Proteínas Quinasas Activadas por AMP/metabolismo , Células Secretoras de Glucagón/enzimología , Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Acetiltransferasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Compuestos de Bifenilo , Western Blotting , Calcio/metabolismo , Línea Celular , Activación Enzimática/efectos de los fármacos , Femenino , Células Secretoras de Glucagón/efectos de los fármacos , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Inmunohistoquímica , Metformina/farmacología , Ratones , Fenformina/farmacología , Fosforilación/efectos de los fármacos , Pironas/farmacología , Tiofenos/farmacologíaRESUMEN
Despite advanced knowledge on the genetic basis of oxidative phosphorylation-related diseases, the molecular and/or cellular determinants for tissue-specific dysfunction are not completely understood. Here, we report the cellular events associated with mitochondrial respiratory Complex II deficiency occurring before cell death. Mutation or chronic inhibition of Complex II determined a large increase of basal and agonist-evoked Ca(2+) signals in the cytosol and the mitochondria, in parallel with mitochondrial dysfunction characterized by membrane potential (Δψ(mit)) loss, [ATP] reduction and increased reactive oxygen species production. Cytosolic and mitochondrial Ca(2+) overload are linked to increased endoplasmic reticulum (ER) Ca(2+) leakage, and to SERCA2b and PMCA proteasome-dependent degradation. Increased [Ca(2+)](mit) is also contributed by decreased mitochondrial motility and increased ER-mitochondria contact sites. Interestingly, increased intracellular [Ca(2+)] activated on the one hand a compensatory Ca(2+)-dependent glycolytic ATP production and determined on the second hand mitochondrial pathology. These results revealed the primary function for Ca(2+) signalling in the control of mitochondrial dysfunction and cellular bioenergetics outcomes linked to respiratory chain Complex II deficiency.
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Señalización del Calcio , Calcio/metabolismo , Complejo II de Transporte de Electrones/metabolismo , Metabolismo Energético , Mitocondrias/metabolismo , Adenosina Trifosfato/metabolismo , Apoptosis , Células Cultivadas , Regulación hacia Abajo , Complejo II de Transporte de Electrones/deficiencia , Complejo II de Transporte de Electrones/genética , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Nitrocompuestos/farmacología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Propionatos/farmacología , Piridonas/farmacología , Especies Reactivas de Oxígeno/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
AIMS/HYPOTHESIS: Zinc is highly concentrated in pancreatic beta cells, is critical for normal insulin storage and may regulate glucagon secretion from alpha cells. Zinc transport family member 8 (ZnT8) is a zinc efflux transporter that is highly abundant in beta cells. Polymorphisms of ZnT8 (also known as SLC30A8) gene in man are associated with increased risk of type 2 diabetes. While global Znt8 knockout (Znt8KO) mice have been characterised, ZnT8 is also present in other islet cell types and extra-pancreatic tissues. Therefore, it is important to find ways of understanding the role of ZnT8 in beta and alpha cells without the difficulties caused by the confounding effects of ZnT8 in these other tissues. METHODS: We generated mice with beta cell-specific (Znt8BKO) and alpha cell-specific (Znt8AKO) knockout of Znt8, and performed in vivo and in vitro characterisation of the phenotypes to determine the functional and anatomical impact of ZnT8 in these cells. Thus we assessed zinc accumulation, insulin granule morphology, insulin biosynthesis and secretion, and glucose homeostasis. RESULTS: Znt8BKO mice are glucose-intolerant, have reduced beta cell zinc accumulation and atypical insulin granules. They also display reduced first-phase glucose-stimulated insulin secretion, reduced insulin processing enzyme transcripts and increased proinsulin levels. In contrast, Znt8AKO mice show no evident abnormalities in plasma glucagon and glucose homeostasis. CONCLUSIONS/INTERPRETATION: This is the first report of specific beta and alpha cell deletion of Znt8. Our data indicate that while, under the conditions studied, ZnT8 is absolutely essential for proper beta cell function, it is largely dispensable for alpha cell function.
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Proteínas de Transporte de Catión/genética , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Zinc/metabolismo , Análisis de Varianza , Animales , Western Blotting , Proteínas de Transporte de Catión/metabolismo , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/metabolismo , Células Secretoras de Glucagón/metabolismo , Inmunohistoquímica , Insulina/genética , Secreción de Insulina , Ratones , Ratones Noqueados , Microscopía Confocal , Microscopía Inmunoelectrónica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transportador 8 de ZincRESUMEN
AIMS/HYPOTHESIS: AMP-activated protein kinase (AMPK) is an evolutionarily conserved enzyme and a target of glucose-lowering agents, including metformin. However, the precise role or roles of the enzyme in controlling insulin secretion remain uncertain. METHODS: The catalytic alpha1 and alpha2 subunits of AMPK were ablated selectively in mouse pancreatic beta cells and hypothalamic neurons by breeding Ampkalpha1 [also known as Prkaa1]-knockout mice, bearing floxed Ampkalpha2 [also known as Prkaa2] alleles (Ampkalpha1 ( -/- ),alpha2( fl/fl ),), with mice expressing Cre recombinase under the rat insulin promoter (RIP2). RIP2 was used to express constitutively activated AMPK selectively in beta cells in transgenic mice. Food intake, body weight and urinary catecholamines were measured using metabolic cages. Glucose and insulin tolerance were determined after intraperitoneal injection. Beta cell mass and morphology were analysed by optical projection tomography and confocal immunofluorescence microscopy, respectively. Granule docking, insulin secretion, membrane potential and intracellular free Ca(2+) were measured with standard techniques. RESULTS: Trigenic Ampkalpha1 ( -/- ),alpha2( fl/fl ) expressing Cre recombinase and lacking both AMPKalpha subunits in the beta cell, displayed normal body weight and increased insulin sensitivity, but were profoundly insulin-deficient. Secreted catecholamine levels were unchanged. Total beta cell mass was unaltered, while mean islet and beta cell volume were reduced. AMPK-deficient beta cells displayed normal glucose-induced changes in membrane potential and intracellular free Ca(2+), while granule docking and insulin secretion were enhanced. Conversely, betaAMPK transgenic mice were glucose-intolerant and displayed defective insulin secretion. CONCLUSIONS/INTERPRETATION: Inhibition of AMPK activity within the beta cell is necessary, but not sufficient for stimulation of insulin secretion by glucose to occur. AMPK activation in extrapancreatic RIP2.Cre-expressing cells might also influence insulin secretion in vivo.
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Proteínas Quinasas Activadas por AMP/metabolismo , Hipotálamo/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Neuronas/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Análisis de Varianza , Animales , Glucemia/metabolismo , Peso Corporal/genética , Grasas de la Dieta , Ingestión de Alimentos/genética , Electrofisiología , Técnica del Anticuerpo Fluorescente , Prueba de Tolerancia a la Glucosa , Hiperglucemia/genética , Hiperglucemia/metabolismo , Insulina/genética , Secreción de Insulina , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas/genética , RatasRESUMEN
Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout (ZnT8(-/-)) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8(-/-) beta cells and were replaced by immature, pale insulin "progranules," which were larger than in ZnT8(+/+) islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8(-/-) mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8(-/-) genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.
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Proteínas de Transporte de Catión/metabolismo , Insulina/química , Insulina/metabolismo , Animales , Calcio/metabolismo , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Cristalización , Glucosa/administración & dosificación , Glucosa/metabolismo , Intolerancia a la Glucosa/inducido químicamente , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/metabolismo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Zinc/metabolismo , Transportador 8 de ZincRESUMEN
AIMS/HYPOTHESIS: Changes in the activity of glucose-excited and glucose-inhibited neurons within the basomedial hypothalamus are key to the central regulation of satiety. However, the molecular mechanisms through which these cells respond to extracellular stimuli remain poorly understood. Here, we investigate the role of 5'-AMP-activated protein kinase (AMPK), a trimeric complex encoded by seven distinct genes of the PRKA family, in the responses to glucose and leptin of each cell type. METHODS: The activity of isolated rat basomedial hypothalamic neurons was assessed by: (1) recording cellular voltage responses under current clamp; (2) measuring intracellular free Ca(2+) with fluo-3 or fura-2; and (3) developing a neuropeptide Y (NPY) promoter-driven adenovirally produced ratiometric 'pericam' (a green fluorescent protein-based Ca(2+) sensor) to monitor [Ca(2+)] changes selectively in NPY-positive neurons. RESULTS: The stimulatory effects of decreased (0 or 1.0 vs 15 mmol/l) glucose on glucose-inhibited neurons were mimicked by the AMPK activator, 5-amino-imidazole-4-carboxamide riboside (AICAR) and blocked by the inhibitor Compound C. Similarly, AICAR reversed the inhibitory effects of leptin in the majority of glucose-inhibited neurons. The responses to glucose of Npy-expressing cells, which represented approximately 40 % of all glucose-inhibited neurons, were also sensitive to Compound C or AICAR. Forced changes in AMPK activity had no effect on glucose-excited and non-glucose-responsive neurons. CONCLUSIONS/INTERPRETATION: Changes in AMPK activity are involved in the responses of glucose-inhibited neurons to large fluctuations in glucose concentration, and possibly also to leptin. This mechanism may contribute to the acute reduction of electrical activity and Ca(2+) oscillation frequency in these, but not other neurons, in the basomedial hypothalamus.
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Glucosa/farmacología , Hipotálamo/citología , Leptina/farmacología , Complejos Multienzimáticos/metabolismo , Neuronas/metabolismo , Neuropéptido Y/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Calcio/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Hipoglucemiantes/farmacología , Hipotálamo/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Complejos Multienzimáticos/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Ratas , Ratas Wistar , Ribonucleótidos/farmacologíaRESUMEN
Minute-to-minute control of the release of insulin by pancreatic beta-cells in response to glucose or other stimuli requires the precise delivery of large dense-core vesicles to the plasma membrane and regulated exocytosis. At present, the precise spatial organization at the cell surface and the nature of these events ('transient' versus 'full fusion') are debated. In order to monitor secretory events simultaneously over most of the surface of clusters of single MIN6 beta-cells, we have expressed recombinant neuropeptide Y-Venus (an enhanced and vesicle-targeted form of yellow fluorescent protein) as an insulin surrogate. Individual exocytotic events were monitored using Nipkow spinning disc confocal microscopy, with acquisition of a three-dimensional complete image (eight to twelve confocal slices) in <1 s, in response to cell depolarization. Corroborating earlier studies using TIRF (total internal reflection fluorescence) microscopy, this approach indicates that events occur with roughly equal probability over the entire cell surface, with only minimal clustering in individual areas, and provides no evidence for multiple events at the same site. Nipkow disc confocal imaging may thus provide a useful tool to determine whether event types occur at different sites at the cell surface and to explore the role of endocytic proteins including dynamin-1 and -2 in terminating individual exocytotic events.
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Exocitosis/fisiología , Insulina/metabolismo , Microscopía Confocal/métodos , Animales , Línea Celular , Membrana Celular/metabolismo , Procesamiento de Imagen Asistido por Computador , Secreción de Insulina , Células Secretoras de Insulina/metabolismoRESUMEN
The transcription factor PDX-1 (pancreatic duodenal homeobox-1) is required for normal pancreatic development and for the function of insulin-producing islet beta-cells in mammals. We have shown previously that glucose regulates insulin gene expression in part through the activation and translocation of PDX-1 from the nuclear periphery to the nucleoplasm. We have also found that PASK [PAS (Per-Arnt-Sim) kinase], a member of the nutrient-regulated family of protein kinases, is activated in response to glucose challenge in beta-cells and is involved in the regulation of expression of PDX-1. Purified PASK efficiently phosphorylated recombinant PDX-1 in vitro on a single site (Thr-152). To determine the impact of phosphorylation at this site, we generated wild-type and mutant (T152A, T152D and T152E) forms of PDX-1 and examined the distribution of each of these in clonal MIN6 beta-cells by immunocytochemical analysis. Unexpectedly, only the T152D mutation significantly affected subcellular distribution, increasing the ratio of nuclear/cytosolic labelling at low and high glucose concentrations, suggesting that phosphorylation at Thr-152 inhibits nuclear uptake in response to glucose. Based on these results, experiments to examine the contribution of Thr-152 to the overall phosphorylation of PDX-1 in intact cells will be undertaken.
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Núcleo Celular/metabolismo , Duodeno/fisiología , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/fisiología , Células Secretoras de Insulina/fisiología , Páncreas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Transactivadores/metabolismo , Proteínas de Homeodominio/genética , Homeostasis , Humanos , Mutación , Transporte de Proteínas , Transactivadores/genéticaRESUMEN
MOTIVATION: Many standard statistical techniques are effective on data that are normally distributed with constant variance. Microarray data typically violate these assumptions since they come from non-Gaussian distributions with a non-trivial mean-variance relationship. Several methods have been proposed that transform microarray data to stabilize variance and draw its distribution towards the Gaussian. Some methods, such as log or generalized log, rely on an underlying model for the data. Others, such as the spread-versus-level plot, do not. We propose an alternative data-driven multiscale approach, called the Data-Driven Haar-Fisz for microarrays (DDHFm) with replicates. DDHFm has the advantage of being 'distribution-free' in the sense that no parametric model for the underlying microarray data is required to be specified or estimated; hence, DDHFm can be applied very generally, not just to microarray data. RESULTS: DDHFm achieves very good variance stabilization of microarray data with replicates and produces transformed intensities that are approximately normally distributed. Simulation studies show that it performs better than other existing methods. Application of DDHFm to real one-color cDNA data validates these results. AVAILABILITY: The R package of the Data-Driven Haar-Fisz transform (DDHFm) for microarrays is available in Bioconductor and CRAN.
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Algoritmos , Interpretación Estadística de Datos , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Colorimetría/métodos , Colorimetría/normas , Simulación por Computador , Bases de Datos Genéticas , Almacenamiento y Recuperación de la Información/métodos , Modelos Genéticos , Modelos Estadísticos , Valores de Referencia , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
AIMS/HYPOTHESIS: ATP-sensitive K(+) (K(ATP)) channels located on the beta cell plasma membrane play a critical role in regulating insulin secretion and are targets for the sulfonylurea class of antihyperglycaemic drugs. Recent reports suggest that these channels may also reside on insulin-containing dense-core vesicles and mitochondria. The aim of this study was to explore these possibilities and to test the hypothesis that vesicle-resident channels play a role in the control of organellar Ca(2+) concentration or pH. METHODS: To quantify the subcellular distribution of the pore-forming subunit Kir6.2 and the sulfonylurea binding subunit SUR1 in isolated mouse islets and clonal pancreatic MIN6 beta cells, we used four complementary techniques: immunoelectron microscopy, density gradient fractionation, vesicle immunopurification and fluorescence-activated vesicle isolation. Intravesicular and mitochondrial concentrations of free Ca(2+) were measured in intact or digitonin-permeabilised MIN6 cells using recombinant, targeted aequorins, and intravesicular pH was measured with the recombinant fluorescent probe pHluorin. RESULTS: SUR1 and Kir6.2 immunoreactivity were concentrated on dense-core vesicles and on vesicles plus the endoplasmic reticulum/Golgi network, respectively, in both islets and MIN6 cells. Reactivity to neither subunit was detected on mitochondria. Glibenclamide, tolbutamide and diazoxide all failed to affect Ca(2+) uptake into mitochondria, and K(ATP) channel regulators had no significant effect on intravesicular free Ca(2+) concentrations or vesicular pH. CONCLUSIONS/INTERPRETATION: A significant proportion of Kir6.2 and SUR1 subunits reside on insulin-secretory vesicles and the distal secretory pathway in mouse beta cells but do not influence intravesicular ion homeostasis. We propose that dense-core vesicles may serve instead as sorting stations for the delivery of channels to the plasma membrane.