RESUMEN
Insulin secretion from pancreatic ß-cells is impaired in all forms of diabetes. The resultant hyperglycaemia has deleterious effects on many tissues, including ß-cells. Here we show that chronic hyperglycaemia impairs glucose metabolism and alters expression of metabolic genes in pancreatic islets. In a mouse model of human neonatal diabetes, hyperglycaemia results in marked glycogen accumulation, and increased apoptosis in ß-cells. Sulphonylurea therapy rapidly normalizes blood glucose levels, dissipates glycogen stores, increases autophagy and restores ß-cell metabolism. Insulin therapy has the same effect but with slower kinetics. Similar changes are observed in mice expressing an activating glucokinase mutation, in in vitro models of hyperglycaemia, and in islets from type-2 diabetic patients. Altered ß-cell metabolism may underlie both the progressive impairment of insulin secretion and reduced ß-cell mass in diabetes.
Asunto(s)
Apoptosis/fisiología , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucógeno/metabolismo , Hiperglucemia/metabolismo , Enfermedades del Recién Nacido/metabolismo , Células Secretoras de Insulina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Autofagia/fisiología , Glucemia/efectos de los fármacos , Línea Celular , Modelos Animales de Enfermedad , Glucoquinasa/genética , Humanos , Hipoglucemiantes/farmacología , Técnicas In Vitro , Recién Nacido , Insulina/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Ratones , Mutación , Ratas , Compuestos de Sulfonilurea/farmacologíaRESUMEN
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.
Asunto(s)
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
Fly photoreceptors are polarized cells, each of which has an extended interface between its cell body and the light-signaling compartment, the rhabdomere. Upon intense illumination, rhabdomeric calcium concentration reaches millimolar levels that would be toxic if Ca(2+) diffusion between the rhabdomere and cell body was not robustly attenuated. Yet, it is not clear how such effective attenuation is obtained. Here we show that Ca(2+) homeostasis in the photoreceptor cell relies on the protein calphotin. This unique protein functions as an immobile Ca(2+) buffer localized along the base of the rhabdomere, separating the signaling compartment from the cell body. Generation and analyses of transgenic Drosophila strains, in which calphotin-expression levels were reduced in a graded manner, showed that moderately reduced calphotin expression impaired Ca(2+) homeostasis while calphotin elimination resulted in severe light-dependent photoreceptor degeneration. Electron microscopy, electrophysiology, and optical methods revealed that the degeneration was rescued by prevention of Ca(2+) overload via overexpression of CalX, the Na(+)-Ca(2+) exchanger. In addition, Ca(2+)-imaging experiments showed that reduced calphotin levels resulted in abnormally fast kinetics of Ca(2+) elevation in photoreceptor cells. Together, the data suggest that calphotin functions as a Ca(2+) buffer; a possibility that we directly demonstrate by expressing calphotin in a heterologous expression system. We propose that calphotin-mediated compartmentalization and Ca(2+) buffering constitute an effective strategy to protect cells from Ca(2+) overload and light-induced degeneration.
Asunto(s)
Calcio/metabolismo , Compartimento Celular/fisiología , Adaptación a la Oscuridad/fisiología , Luz/efectos adversos , Degeneración Retiniana/etiología , Degeneración Retiniana/prevención & control , Animales , Animales Modificados Genéticamente , Tampones (Química) , Proteínas de Unión al Calcio/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/patología , Degeneración Retiniana/patologíaRESUMEN
Recent studies revealed a surprising regenerative capacity of insulin-producing ß cells in mice, suggesting that regenerative therapy for human diabetes could in principle be achieved. Physiologic ß cell regeneration under stressed conditions relies on accelerated proliferation of surviving ß cells, but the factors that trigger and control this response remain unclear. Using islet transplantation experiments, we show that ß cell mass is controlled systemically rather than by local factors such as tissue damage. Chronic changes in ß cell glucose metabolism, rather than blood glucose levels per se, are the main positive regulator of basal and compensatory ß cell proliferation in vivo. Intracellularly, genetic and pharmacologic manipulations reveal that glucose induces ß cell replication via metabolism by glucokinase, the first step of glycolysis, followed by closure of K(ATP) channels and membrane depolarization. Our data provide a molecular mechanism for homeostatic control of ß cell mass by metabolic demand.
Asunto(s)
Glucemia/metabolismo , Células Secretoras de Insulina/fisiología , Regeneración , Animales , Membrana Celular/fisiología , Proliferación Celular , Glucoquinasa/antagonistas & inhibidores , Glucoquinasa/metabolismo , Glucólisis , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/trasplante , Canales KATP/metabolismo , RatonesRESUMEN
Transient Receptor Potential channels are polymodal cellular sensors involved in a wide variety of cellular processes, mainly by increasing cellular Ca(2+). In this review we focus on the roles of these channels in: (i) cell death (ii) proliferation and differentiation and (iii) transmitter release. Cell death: Ca(2+) influx participates in apoptotic and necrotic cell death. The Ca(2+) permeability and high sensitivity of part of these channels to oxidative/metabolic stress make them important participants in cell death. Several examples are given. Transient Receptor Potential Melastatin 2 is activated by H(2)O(2), inducing cell death through an increase in cellular Ca(2+) and activation of Poly ADP-Ribose Polymerase. Exposure of cultured cortical neurons to oxygen-glucose deprivation, in vitro, causes cell death via cation influx, mediated by Transient Receptor Potential Melastatin 7. Metabolic stress constitutively activates the Ca(2+) permeable Transient Receptor Potential channels of Drosophila photoreceptor in the dark, potentially leading to retinal degeneration. Similar sensitivity to metabolic stress characterizes several mammalian Transient Receptor Potential Canonical channels. Proliferation and differentiation: The rise in cytosolic Ca(2+) induces cell growth, differentiation and proliferation via activation of several transcription factors. Activating a variety of store operated and Transient Receptor Potential channels cause a rise in cytosolic Ca(2+), making these channels components involved in proliferation and differentiation. Transmitter release: Transient Receptor Potential Melastatin 7 channels reside in synaptic vesicles and regulate neurotransmitter release by a mechanism that is not entirely clear. All the above features of Transient Receptor Potential channels make them crucial components in important, sometimes conflicting, cellular processes that still need to be explored.
Asunto(s)
Muerte Celular/fisiología , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Calcio/metabolismo , Muerte Celular/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proliferación Celular , Humanos , Modelos Biológicos , Vesículas Sinápticas/metabolismo , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
Open channel block is a process in which ions bound to the inside of a channel pore block the flow of ions through that channel. Repulsion of the blocking ions by depolarization is a known mechanism of open channel block removal. For the NMDA channel, this mechanism is necessary for channel activation and is involved in neuronal plasticity. Several types of transient receptor potential (TRP) channels, including the Drosophila TRP and TRP-like (TRPL) channels, also exhibit open channel block. Therefore, removal of open channel block is necessary for the production of the physiological response to light. Because there is no membrane depolarization before the light response develops, it is not clear how the open channel block is removed, an essential step for the production of a robust light response under physiological conditions. Here we present a novel mechanism to alleviate open channel block in the absence of depolarization by membrane lipid modulations. The results of this study show open channel block removal by membrane lipid modulations in both TRPL and NMDA channels of the photoreceptor cells and CA1 hippocampal neurons, respectively. Removal of open channel block is characterized by an increase in the passage-rate of the blocking cations through the channel pore. We propose that the profound effect of membrane lipid modulations on open channel block alleviation, allows the productions of a robust current in response to light in the absence of depolarization.
Asunto(s)
Activación del Canal Iónico/efectos de los fármacos , Lípidos de la Membrana/farmacología , Receptores de N-Metil-D-Aspartato/fisiología , Canales de Potencial de Receptor Transitorio/fisiología , Animales , Animales Modificados Genéticamente , Biofisica , Calcio/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Estimulación Eléctrica , Proteínas Fluorescentes Verdes/genética , Hipocampo/citología , Técnicas In Vitro , Activación del Canal Iónico/genética , Activación del Canal Iónico/fisiología , Luz , Ácido Linoleico/farmacología , Magnesio/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Mutación/genética , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Placa-Clamp/métodos , Células Fotorreceptoras de Invertebrados/metabolismo , Ratas , Receptores de N-Metil-D-Aspartato/genética , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
Transient receptor potential (TRP) channels are essential components of biological sensors that detect changes in the environment in response to a myriad of stimuli. A major difficulty in the study of TRP channels is the lack of pharmacological agents that modulate most members of the TRP superfamily. Notable exceptions are the thermoTRPs, which respond to either cold or hot temperatures and are modulated by a relatively large number of chemical agents. In the present study we demonstrate by patch clamp whole cell recordings from Schneider 2 and Drosophila photoreceptor cells that carvacrol, a known activator of the thermoTRPs, TRPV3 and TRPA1 is an inhibitor of the Drosophila TRPL channels, which belongs to the TRPC subfamily. We also show that additional activators of TRPV3, thymol, eugenol, cinnamaldehyde and menthol are all inhibitors of the TRPL channel. Furthermore, carvacrol also inhibits the mammalian TRPM7 heterologously expressed in HEK cells and ectopically expressed in a primary culture of CA3-CA1 hippocampal brain neurons. This study, thus, identifies a novel inhibitor of TRPC and TRPM channels. Our finding that the activity of the non-thermoTRPs, TRPL and TRPM7 channels is modulated by the same compound as thermoTRPs, suggests that common mechanisms of channel modulation characterize TRP channels.