RESUMEN
The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.
Asunto(s)
Proteínas Portadoras/farmacocinética , Membrana Celular/metabolismo , Células Madre Embrionarias/metabolismo , Proteínas Fluorescentes Verdes/farmacocinética , Proteínas Estructurales Virales/farmacocinética , Animales , Western Blotting , Pulpa Dental/citología , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Estructurales Virales/genéticaRESUMEN
The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.
Asunto(s)
Animales , Humanos , Ratones , Proteínas Portadoras/farmacocinética , Membrana Celular/metabolismo , Células Madre Embrionarias/metabolismo , Proteínas Fluorescentes Verdes/farmacocinética , Proteínas Estructurales Virales/farmacocinética , Western Blotting , Pulpa Dental/citología , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Estructurales Virales/genéticaRESUMEN
Growing evidence indicates that the regulation of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels is essential for maintaining normal ß-cell glucose responsiveness. While long-term exposure to high glucose induces oxidative stress in ß cells, conflicting results have been published regarding the impact of ROS on acute glucose exposure and their role in glucose stimulated insulin secretion (GSIS). Although ß cells are considered to be particularly vulnerable to oxidative damage, as they express relatively low levels of some peroxide-metabolizing enzymes such as catalase and glutathione (GSH) peroxidase, other less known GSH-based antioxidant systems are expressed in ß cells at higher levels. Herein, we discuss the key mechanisms of ROS/RNS production and their physiological function in pancreatic ß cells. We also hypothesize that specific interactions between RNS and ROS may be the cause of the vulnerability of pancreatic ß cells to oxidative damage. In addition, using a hypothetical metabolic model based on the data available in the literature, we emphasize the importance of amino acid availability for GSH synthesis and for the maintenance of ß-cell function and viability during periods of metabolic disturbance before the clinical onset of diabetes.
Asunto(s)
Aminoácidos/metabolismo , Antioxidantes/metabolismo , Células Secretoras de Insulina/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Glutatión/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Modelos BiológicosRESUMEN
It is now widely accepted that reactive oxygen species (ROS) contribute to cell and tissue dysfunction and damage in diabetes. The source of ROS in the insulin secreting pancreatic beta cells has traditionally been considered to be the mitochondrial electron transport chain. While this source is undoubtedly important, we fully describe in this article recent information and evidence of NADPH oxidase-dependent generation of ROS in pancreatic beta cells and identify the various isoforms that contribute to O(2)(*-) and H(2)O(2) production in various conditions. While glucose-stimulated ROS generation may be important for acute regulation of insulin secretion, at higher levels ROS may disrupt mitochondrial energy metabolism. However, ROS may alter other cellular processes such as signal transduction, ion fluxes and/or cell proliferation/death. The various beta cell isoforms of NADPH oxidase (described in this review) may, via differences in the kinetics and species of ROS generated, positively and negatively regulate insulin secretion and cell survival.
Asunto(s)
Células Secretoras de Insulina/enzimología , NADPH Oxidasas/metabolismo , Membrana Celular/enzimología , Diabetes Mellitus/fisiopatología , Glucosa/metabolismo , Homeostasis , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/fisiología , Isoenzimas/metabolismo , Oxidación-Reducción , Fosforilación , Especies Reactivas de Oxígeno/metabolismoRESUMEN
We previously described the presence of nicotinamide adenine dinucleotide phosphate reduced form [NAD(P)H]oxidase components in pancreatic beta-cells and its activation by glucose, palmitic acid, and proinflammatory cytokines. In the present study, the importance of the NAD(P)H oxidase complex for pancreatic beta-cell function was examined. Rat pancreatic islets were incubated in the presence of glucose plus diphenyleneiodonium, a NAD(P)H oxidase inhibitor, for 1 h or with the antisense oligonucleotide for p47(PHOX) during 24 h. Reactive oxygen species (ROS) production was determined by a fluorescence assay using 2,7-dichlorodihydrofluorescein diacetate. Insulin secretion, intracellular calcium responses, [U-(14)C]glucose oxidation, and expression of glucose transporter-2, glucokinase and insulin genes were examined. Antisense oligonucleotide reduced p47(PHOX) expression [an important NAD(P)H oxidase cytosolic subunit] and similarly to diphenyleneiodonium also blunted the enzyme activity as indicated by reduction of ROS production. Suppression of NAD(P)H oxidase activity had an inhibitory effect on intracellular calcium responses to glucose and glucose-stimulated insulin secretion by isolated islets. NAD(P)H oxidase inhibition also reduced glucose oxidation and gene expression of glucose transporter-2 and glucokinase. These findings indicate that NAD(P)H oxidase activation plays an important role for ROS production by pancreatic beta-cells during glucose-stimulated insulin secretion. The importance of this enzyme complex for the beta-cell metabolism and the machinery involved in insulin secretion were also shown.
Asunto(s)
Glucosa/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , NADPH Oxidasas/fisiología , Animales , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Peróxido de Hidrógeno/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , Compuestos Onio/farmacología , Oxidación-Reducción/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
It is now widely accepted, given the current weight of experimental evidence, that reactive oxygen species (ROS) contribute to cell and tissue dysfunction and damage caused by glucolipotoxicity in diabetes. The source of ROS in the insulin secreting pancreatic beta-cells and in the cells which are targets for insulin action has been considered to be the mitochondrial electron transport chain. While this source is undoubtably important, we provide additional information and evidence for NADPH oxidase-dependent generation of ROS both in pancreatic beta-cells and in insulin sensitive cells. While mitochondrial ROS generation may be important for regulation of mitochondrial uncoupling protein (UCP) activity and thus disruption of cellular energy metabolism, the NADPH oxidase associated ROS may alter parameters of signal transduction, insulin secretion, insulin action and cell proliferation or cell death. Thus NADPH oxidase may be a useful target for intervention strategies based on reversing the negative impact of glucolipotoxicity in diabetes.