RESUMEN
Nuclear lamins form the lamina on the interior of the nuclear envelope, and are involved in the regulation of various cellular processes, including DNA replication and chromatin organization. Despite this evidence, little is known about potential alterations in nuclear metabolism, specifically lamin structure and integrity in isolated ß-cells subjected to stress conditions, including chronic exposure to hyperglycemia (i.e., glucotoxicity). Herein, we investigated effects of glucotoxic conditions on the catalytic activation of caspase 3 and the associated degradation of one of its substrate proteins, namely lamin-B. We report that incubation of insulin-secreting INS-1 832/13 cells, normal rat islets or human islets under glucotoxic conditions (20 mM; 12-48 h) results in the degradation of native lamin B leading to accumulation of the degraded products in non-relevant cellular compartments, including cytosol. Moreover, the effects of high glucose on caspase 3 activation and lamin B degradation were mimicked by thapsigargin, a known inducer of endoplasmic reticulum stress (ER stress). Nifedipine, a known blocker of calcium channel activation, inhibited high glucose-induced caspase 3 activation and lamin B degradation in these cells. 4-Phenyl butyric acid, a known inhibitor of ER stress, markedly attenuated glucose-induced CHOP expression (ER stress marker), caspase 3 activation and lamin B degradation. We conclude that glucotoxic conditions promote caspase 3 activation and lamin B degradation, which may, in part, be due to increased ER stress under these conditions. We also provide further evidence to support beneficial effects of calcium channel blockers against metabolic dysfunction of the islet ß-cell induced by hyperglycemic conditions.
Asunto(s)
Caspasa 3/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Lamina Tipo B/metabolismo , Nifedipino/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Citosol/efectos de los fármacos , Citosol/metabolismo , Glucosa/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Masculino , Fenilbutiratos/farmacología , Ratas , Ratas Sprague-Dawley , Tapsigargina/farmacología , Factor de Transcripción CHOP/metabolismoRESUMEN
Several cellular proteins undergo post-translational lipidation, including prenylation, palmitoylation and myristoylation, which are felt to promote intracellular targeting, membrane association and interaction with effector partner proteins. Recent findings implicate definitive roles of isoprenylation in islet ß-cell function including glucose-stimulated insulin secretion [GSIS]. Published evidence also suggests novel regulatory roles for protein palmitoylation not only in GSIS but also in the metabolic dysfunction induced by proinflammatory cytokines and lipotoxic conditions. Herein, we overviewed the existing evidence on the regulatory roles of protein palmitoylation in the metabolic [dys]regulation of the islet ß-cell and highlighted the developments in this area, specifically on potential identity of palmitoylated proteins, and on the utility of two structurally distinct inhibitors of palmitoylation [e.g., cerulenin and 2-bromopalmitate] in halting the metabolic dysfunction of the islet ß-cell known to occur following exposure to proinflammatory cytokines and lipotoxic conditions. Potential avenues for future research, including the immediate need for discovery of novel small molecule compounds as inhibitors of palmitoyl transferases to attenuate deleterious consequences of proinflammatory cytokines and glucolipotoxicity are discussed. Furthermore, some relevant patents are also highlighted in this review.
Asunto(s)
Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiología , Lipoilación/fisiología , Páncreas/metabolismo , Páncreas/fisiopatología , Proteínas/fisiología , Animales , Proteínas de Unión al GTP/metabolismo , Glucosa/farmacología , Humanos , Insulina/metabolismo , Lipoilación/efectos de los fármacos , Patentes como Asunto , Proteínas/metabolismoRESUMEN
Several lines of recent evidence provided compelling evidence to suggest increased generation of reactive oxygen species (ROS) as causal for mitochondrial dysregulation and apoptosis in islet ß-cells exposed to noxious stimuli including high glucose, lipids and proinflammatory cytokines. Studies along these lines are also suggestive of a significant contributory role for NADPH oxidase in the generation of ROS under the above conditions. We have recently reported a marked increase in the expression and activation of cytosolic components of NADPH oxidase (p47phox, Rac1) in cell culture models of glucotoxicity and in islets from T2DM animals (Zucker Diabetic Fatty rat) and humans. In this communication, we provide further evidence indicating significant activation of NADPH activity (~2-fold) in INS-1 832/13 cells exposed to chronic hyperglycemic conditions (20 mM; 48 h). We also report marked attenuation of this activity, by apocynin, a selective inhibitor of phagocyte-like NADPH oxidase (Nox2) activity. Together, our findings implicate Nox2 as a source for ROS generation in ß-cells exposed to glucotoxic conditions.
Asunto(s)
Diabetes Mellitus Tipo 2/enzimología , Activación Enzimática , Hiperglucemia/metabolismo , Células Secretoras de Insulina/enzimología , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Acetofenonas/farmacología , Animales , Línea Celular Tumoral , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glucosa/metabolismo , Hiperglucemia/etiología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/metabolismo , Glicoproteínas de Membrana/antagonistas & inhibidores , NADPH Oxidasa 2 , NADPH Oxidasas/antagonistas & inhibidores , Concentración Osmolar , Ratas , Ratas Zucker , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Phagocyte-like NADPH oxidase (Nox2) has been shown to play regulatory roles in the metabolic dysfunction of the islet ß-cell under the duress of glucolipotoxic conditions and exposure to proinflammatory cytokines. However, the precise mechanisms underlying Nox2 activation by these stimuli remain less understood. To this end, we report a time-dependent phosphorylation of p47phox, a cytosolic subunit of Nox2, by cytomix (IL-1ß+TNFα+IFNγ) in insulin-secreting INS-1 832/13 cells. Furthermore, cytomix induced the expression of gp91phox, a membrane component of Nox2. 2-Bromopalmitate (2-BP), a known inhibitor of protein palmitoylation, markedly attenuated cytokine-induced, Nox2-mediated reactive oxygen species (ROS) generation and inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) generation. However, 2-BP failed to exert any significant effects on cytomix-induced CHOP expression, a marker for endoplasmic reticulum stress. Together, our findings identify palmitoyltransferase as a target for inhibition of cytomix-induced oxidative (ROS generation) and nitrosative (NO generation) stress in the pancreatic ß-cell.
Asunto(s)
Citocinas/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasas/metabolismo , Óxido Nítrico/biosíntesis , Palmitatos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Línea Celular , Estrés del Retículo Endoplásmico , Células Secretoras de Insulina/metabolismo , Interferón gamma/farmacología , Interleucina-1beta/farmacología , Lipoilación , NADPH Oxidasa 2 , Fosforilación , Ratas , Factor de Transcripción CHOP/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia ArribaRESUMEN
Emerging evidence implicates novel roles for post-translational prenylation (i.e., farnesylation and geranylgeranylation) of various signaling proteins in a variety of cellular functions including hormone secretion, survival and apoptosis. In the context of cellular apoptosis, it has been shown previously that caspase-3 activation, a hallmark of mitochondrial dysregulation, promotes hydrolysis of several key cellular proteins. We report herein that exposure of insulin-secreting INS 832/13 cells or normal rat islets to etoposide leads to significant activation of caspase-3 and subsequent degradation of the common α-subunit of farnesyl/geranylgeranyl transferases (FTase/GGTase). Furthermore, the above stated signaling steps were prevented by Z-DEVD-FMK, a known inhibitor of caspase-3. In addition, treatment of cell lysates with recombinant caspase-3 also caused FTase/GGTase α-subunit degradation. Moreover, nifedipine, a calcium channel blocker, markedly attenuated etoposide-induced caspase-3 activation, FTase/GGTase α-subunit degradation in INS 832/13 cells and normal rat islets. Further, nifedipine significantly restored etoposide-induced loss in metabolic cell viability in INS 832/13 cells. Based on these findings, we conclude that etoposide induces loss in cell viability by inducing mitochondrial dysfunction, caspase-3 activation and degradation of FTase/GGTase α-subunit. Potential significance of these findings in the context of protein prenylation and ß-cell survival are discussed.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Caspasa 3/metabolismo , Etopósido/antagonistas & inhibidores , Farnesiltransferasa/metabolismo , Células Secretoras de Insulina/metabolismo , Nifedipino/farmacología , Animales , Inhibidores de Caspasas/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Etopósido/farmacología , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Oligopéptidos/farmacología , Prenilación de Proteína/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Mephedrone (4-methylmethcathinone) is a ß-ketoamphetamine with close structural analogy to substituted amphetamines and cathinone derivatives. Abuse of mephedrone has increased dramatically in recent years and has become a significant public health problem in the United States and Europe. Unfortunately, very little information is available on the pharmacological and neurochemical actions of mephedrone. In light of the proven abuse potential of mephedrone and considering its similarity to methamphetamine and methcathinone, it is particularly important to know if mephedrone shares with these agents an ability to cause damage to dopamine nerve endings of the striatum. Accordingly, we treated mice with a binge-like regimen of mephedrone (4 × 20 or 40 mg/kg) and examined the striatum for evidence of neurotoxicity 2 or 7 days after treatment. While mephedrone caused hyperthermia and locomotor stimulation, it did not lower striatal levels of dopamine, tyrosine hydroxylase or the dopamine transporter under any of the treatment conditions used presently. Furthermore, mephedrone did not cause microglial activation in striatum nor did it increase glial fibrillary acidic protein levels. Taken together, these surprising results suggest that mephedrone, despite its numerous mechanistic overlaps with methamphetamine and the cathinone derivatives, does not cause neurotoxicity to dopamine nerve endings of the striatum.