RESUMEN
Lipotoxicity has been considered the main cause of pancreatic beta-cell failure during type 2 diabetes development. Lipid droplets (LD) are believed to regulate the beta-cell sensitivity to free fatty acids (FFA), but the underlying molecular mechanisms are largely unclear. Accumulating evidence points, however, to an important role of intracellular sphingosine-1-phosphate (S1P) metabolism in lipotoxicity-mediated disturbances of beta-cell function. In the present study, we compared the effects of an increased irreversible S1P degradation (S1P-lyase, SPL overexpression) with those associated with an enhanced S1P recycling (overexpression of S1P phosphatase 1, SGPP1) on LD formation and lipotoxicity in rat INS1E beta-cells. Interestingly, although both approaches led to a reduced S1P concentration, they had opposite effects on the susceptibility to FFA. Overexpression of SGPP1 prevented FFA-mediated caspase-3 activation by a mechanism involving an enhanced lipid storage capacity and prevention of oxidative stress. In contrast, SPL overexpression limited LD biogenesis, content, and size, while accelerating lipophagy. This was associated with FFA-induced hydrogen peroxide formation, mitochondrial fragmentation, and dysfunction, as well as ER stress. These changes coincided with the upregulation of proapoptotic ceramides but were independent of lipid peroxidation rate. Also in human EndoC-ßH1 beta-cells, suppression of SPL with simultaneous overexpression of SGPP1 led to a similar and even more pronounced LD phenotype as that in INS1E-SGPP1 cells. Thus, intracellular S1P turnover significantly regulates LD content and size and influences beta-cell sensitivity to FFA.
Asunto(s)
Células Secretoras de Insulina , Gotas Lipídicas , Lisofosfolípidos , Esfingosina , Células Secretoras de Insulina/metabolismo , Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Ratas , Animales , Gotas Lipídicas/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Aldehído-Liasas/metabolismo , Aldehído-Liasas/genética , Metabolismo de los Lípidos , Humanos , Línea Celular , Estrés Oxidativo , Espacio Intracelular/metabolismoRESUMEN
Adipose tissue-derived non-esterified saturated long-chain fatty acid palmitate (PA) decisively contributes to ß-cell demise in type 2 diabetes mellitus in part through the excessive generation of hydrogen peroxide (H2O2). The endoplasmic reticulum (ER) as the primary site of oxidative protein folding could represent a significant source of H2O2. Both ER-oxidoreductin-1 (ERO-1) isoenzymes, ERO-1α and ERO-1ß, catalyse oxidative protein folding within the ER, generating equimolar amounts of H2O2 for every disulphide bond formed. However, whether ERO-1-derived H2O2 constitutes a potential source of cytotoxic luminal H2O2 under lipotoxic conditions is still unknown. Here, we demonstrate that both ERO-1 isoforms are expressed in pancreatic ß-cells, but interestingly, PA only significantly induces ERO-1α. Its specific deletion significantly attenuates PA-mediated oxidative ER stress and subsequent ß-cell death by decreasing PA-mediated ER-luminal and mitochondrial H2O2 accumulation, by counteracting the dysregulation of ER Ca2+ homeostasis, and by mitigating the reduction of mitochondrial membrane potential and lowered ATP content. Moreover, ablation of ERO-1α alleviated PA-induced hyperoxidation of the ER redox milieu. Importantly, ablation of ERO-1α did not affect the insulin secretory capacity, the unfolded protein response, or ER redox homeostasis under steady-state conditions. The involvement of ERO-1α-derived H2O2 in PA-mediated ß-cell lipotoxicity was corroborated by the overexpression of a redox-active ERO-1α underscoring the proapoptotic activity of ERO-1α in pancreatic ß-cells. Overall, our findings highlight the critical role of ERO-1α-derived H2O2 in lipotoxic ER stress and ß-cell failure.
Asunto(s)
Apoptosis , Estrés del Retículo Endoplásmico , Peróxido de Hidrógeno , Células Secretoras de Insulina , Palmitatos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Palmitatos/metabolismo , Palmitatos/farmacología , Peróxido de Hidrógeno/metabolismo , Ratones , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Humanos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacosRESUMEN
The endoplasmic reticulum (ER) lumen is not only the major site for the assembly and folding of newly synthesized proteins but also the main intracellular Ca2+ store. Ca2+ ions are involved in versatile biochemical processes, including posttranslational processing and folding of nascent proteins. Disruption of ER Ca2+ homeostasis is usually accompanied by an ER stress response that can ultimately lead to apoptosis if unresolved. Abnormal ER Ca2+ depletion has been linked to pancreatic ß-cell dysfunction and death under lipotoxic conditions. However, the underlying mechanisms how the ß-cell toxic saturated free fatty acid palmitate perturbs ER Ca2+ homeostasis and its interplay with other organelles are not fully understood. In the present study, we demonstrate that treatment of insulin-secreting INS-1E cells with palmitate diminished ER Ca2+ levels, elevated cytosolic/mitochondrial Ca2+ content, lowered the mitochondrial membrane potential, and ATP content. In addition, palmitate-pretreated ß-cells contained significantly less luminal Ca2+ , revealed a severely impaired ER Ca2+ reuptake rate, and substantially lower insulin content. Importantly, detoxification of luminal H2 O2 by expression of the ER-resident glutathione peroxidase 8 (GPx8) abrogated the lipotoxic effects of palmitate. Moreover, GPx8 supported oxidative protein folding and preserved insulin content under lipotoxic conditions. A direct involvement of luminal H2 O2 in palmitate-mediated ER Ca2+ depletion could be corroborated by the ectopic expression of an ER-luminal active catalase. Our data point to the critical role of luminal H2 O2 in palmitate-mediated depletion of ER Ca2+ through redox-dependent impairment of Ca2+ ATPase pump activity upstream of mitochondrial dysfunction in insulin-secreting INS-1E cells.
Asunto(s)
Células Secretoras de Insulina , Palmitatos , Palmitatos/metabolismo , Células Secretoras de Insulina/metabolismo , Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Insulina/metabolismoRESUMEN
Elevated plasma concentrations of saturated free fatty acids (SFAs) are involved in pancreatic ß-cell dysfunction and apoptosis, referred to as lipotoxicity. However, in contrast to apoptosis, the involvement of ferroptosis, as a distinct type of oxidative regulated cell death in ß-cell lipotoxicity remains elusive. Therefore, the aim of this study was to determine the effects of various free fatty acids on ferroptosis induction in rat insulin-producing ß-cells. Herein, rat insulin-producing ß-cells underwent lipid peroxidation in the presence of long-chain SFAs and ω-6-polyunsaturated fatty acids (PUFAs), but only the latter induced ferroptosis. On the other hand, the ω-3-PUFA α-linolenate did not induce ferroptosis but sensitized insulin-producing ß-cells to SFA-mediated lipid peroxidation. While the monounsaturated fatty acid oleate, overexpression of glutathione peroxidase 4, and the specific ferroptosis inhibitor ferrostatin-1 significantly abrogated lipid peroxidation, neither glutathione peroxidase 4 nor ferrostatin-1 affected palmitate-mediated toxicity. Site-specific expression of catalase in cytosol, mitochondria, and ER attenuated lipid peroxidation, indicating the contribution of metabolically generated H2O2 from all three subcellular compartments. These observations suggest that only ω-6-PUFAs reach the thresholds of lipid peroxidation required for ferroptosis, whereas SFAs favour apoptosis in ß-cells. Hence, avoiding an excessive dietary intake of ω-6-PUFAs might be a crucial prerequisite for prevention of reactive oxygen species-mediated ferroptosis in insulin-producing cells.
Asunto(s)
Ácidos Grasos Omega-3 , Ferroptosis , Insulinas , Animales , Ácidos Grasos/farmacología , Ácidos Grasos no Esterificados/farmacología , Peróxido de Hidrógeno , Insulinas/metabolismo , Peroxidación de Lípido , Fosfolípido Hidroperóxido Glutatión Peroxidasa , RatasRESUMEN
AIMS/HYPOTHESIS: The aim of this study was to examine the effects of proinflammatory cytokines on cells of different developmental stages during the generation of stem cell-derived beta cells (SC-beta cells) from human pluripotent stem cells (hPSCs). We wanted to find out to what extent human SC-beta cells are suitable as an experimental cellular model and, with regard to a possible therapeutic use, whether SC-beta cells have a comparable vulnerability to cytokines as bona fide beta cells. METHODS: hPSCs were differentiated towards pancreatic organoids (SC-organoids) using a 3D production protocol. SC-beta cells and non-insulin-producing cells were separated by FACS and differential gene expression profiles of purified human SC-beta cells, progenitor stages and the human beta cell line EndoC-ßH1, as a reference, were determined after 24 h incubation with the proinflammatory cytokines IL-1ß, TNF-α and IFN-γ via a transcriptome microarray. Furthermore, we investigated apoptosis based on caspase cleavage, the generation of reactive oxygen species and activation of mitogen-activated protein-kinase (MAPK) stress-signalling pathways. RESULTS: A 24 h exposure of SC-beta cells to proinflammatory cytokines resulted in significant activation of caspase 3/7 and apoptosis via the extrinsic and intrinsic apoptosis signalling pathways. At this time point, SC-beta cells showed a markedly higher sensitivity towards proinflammatory cytokines than non-insulin-producing cells and EndoC-ßH1 cells. Furthermore, we were able to demonstrate the generation of reactive oxygen species and rule out the involvement of NO-mediated stress. A transient activation of stress-signalling pathways p38 mitogen-activated protein kinases (p38) and c-Jun N-terminal kinase (JNK) was already observed after 10 min of cytokine exposure. The transcriptome analysis revealed that the cellular response to proinflammatory cytokines increased with the degree of differentiation of the cells. Cytokines induced the expression of multiple inflammatory mediators including IL-32, CXCL9 and CXCL10 in SC-beta cells and in non-insulin-producing cells. CONCLUSIONS/INTERPRETATION: Our results indicate that human SC-beta cells respond to proinflammatory cytokines very similarly to human islets. Due to the fast and fulminant cellular response of SC-beta cells, we conclude that SC-beta cells represent a suitable model for diabetes research. In light of the immaturity of SC-beta cells, they may be an attractive model for developmentally young beta cells as they are, for example, present in patients with early-onset type 1 diabetes. The secretion of chemotactic signals may promote communication between SC-beta cells and immune cells, and non-insulin-producing cells possibly participate in the overall immune response and are thus capable of amplifying the immune response and further stimulating inflammation. We demonstrated that cytokine-treated SC-organoids secrete IL-32, which is considered a promising candidate for type 1 diabetes onset. This underlines the need to ensure the survival of SC-beta cells in an autoimmune environment such as that found in type 1 diabetes.
Asunto(s)
Citocinas , Diabetes Mellitus Tipo 1 , Inflamación , Células Secretoras de Insulina , Células Madre Pluripotentes , Apoptosis , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Humanos , Inflamación/metabolismo , Células Secretoras de Insulina/metabolismo , Interleucinas , Óxido Nítrico/metabolismo , Células Madre Pluripotentes/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Differentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. SOX9 plays a crucial role during development of the pancreas and particularly in the development of insulin-producing cells as SOX9+ cells form the source for NEUROG3+ endocrine progenitor cells. For the purpose of easy monitoring of differentiation efficiencies into pancreatic progenitors and insulin-producing cells, we generated new reporter lines by knocking in a P2A-H-2Kk-F2A-GFP2 reporter gene into the SOX9-locus and a P2A-mCherry reporter gene into the INS-locus mediated by CRISPR/CAS9-technology. The knock-ins enabled co-expression of the endogenous and reporter genes and report on the endogenous gene expression. Furthermore, FACS and MACS enabled the purification of pancreatic progenitors and insulin-producing cells. Using these cell lines, we established a new differentiation protocol geared towards SOX9+ cells to efficiently drive human pluripotent stem cells into glucose-responsive beta cells. Our new protocol offers an alternative route towards stem cell-derived beta cells, pointing out the importance of Wnt/beta-catenin inhibition and the efficacy of EGF for the development of pancreatic progenitors, as well as the significance of 3D culture for the functionality of the generated beta cells.
Asunto(s)
Células Secretoras de Insulina , Células Madre Pluripotentes , Diferenciación Celular/genética , Línea Celular , Humanos , Insulina/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismoRESUMEN
During diabetes development insulin production and glucose-stimulated insulin secretion (GSIS) are defective due to inflammation-related, yet not fully understood mechanisms. MCPIP1 (monocyte chemotactic protein-induced protein-1) is a strong regulator of inflammation, and acts predominantly as a specific RNase. The impact of MCPIP1 on insulin secretory capacity is unknown. We show that the expression of the ZC3H12A gene, which encodes MCPIP1, was induced by T1DM- and by T2DM-simulating conditions, with a stronger effect of cytokines. The number of MCPIP1-positive pancreatic islet-cells, including beta-cells, was significantly higher in diabetic compared to nondiabetic individuals. In the 3'UTR regions of mRNAs coding for Pdx1 (pancreatic and duodenal homeobox 1), FoxO1 (forkhead box protein O1), and of a novel regulator of insulin handling, Grp94 (glucose-regulated protein 94), MCPIP1-target structures were detected. Overexpression of the wild type MCPIP1wt, but not of the mutant MCPIP1D141N (lacking the RNase activity), decreased the expression of genes involved in insulin production and GSIS. Additionally INS1-E-MCPIP1wt cells exhibited a higher Ire1 (inositol-requiring enzyme 1) expression. MCPIP1wt overexpression blunted GSIS and glucose-mediated calcium influx with no deleterious effects on glucose uptake or glucokinase activity. We identify MCPIP1 as a new common link between diabetogenic conditions and beta-cell failure. MCPIP1 may serve as an interesting target for novel beta-cell protective approaches.
Asunto(s)
Diabetes Mellitus/metabolismo , Secreción de Insulina/fisiología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Ribonucleasas/metabolismo , Factores de Transcripción/metabolismo , Regiones no Traducidas 3'/fisiología , Animales , Calcio/metabolismo , Línea Celular , Citocinas/metabolismo , Diabetes Mellitus/patología , Proteína Forkhead Box O1/metabolismo , Glucosa/metabolismo , Humanos , Células Secretoras de Insulina/patología , ARN Mensajero/metabolismo , RatasRESUMEN
Pro-inflammatory cytokines are crucial mediators of beta-cell destruction in type 1 diabetes mellitus (T1DM). The involvement of ferroptosis as a form of oxidative non-apoptotic cell death in T1DM pathogenesis has not been elucidated so far. Moreover, the role of glutathione peroxidase 4 (GPx4) as an antioxidative enzyme and a major regulator of ferroptosis remains elusive. Assessment of GPx4 expression in different pancreatic islet cell types revealed a predominant expression in beta-cells. Silencing of GPx4 by RNA interference and exposure to tert-butyl hydroperoxide (tert-BHP) caused ferroptosis in rat pancreatic beta-cells as evidenced by non-apoptotic cell death in association with increased lipid peroxidation, disturbed ATP synthesis, reduced GSH content, and GPx4 degradation. GPx4 overexpression as well as the ferroptosis inhibitor ferrostatin-1 effectively attenuated beta-cell death induced by tert-BHP. Notably, beta-cell toxic cytokines did not induce ferroptosis although beta-cells underwent cell death. Inhibition of iNOS by Nω-nitro-L-arginine however led to a massive lipid peroxidation upon exposure to pro-inflammatory cytokines. Hence, nitric oxide produced during pro-inflammatory cytokine action prevents the induction of ferroptosis, thereby favouring apoptosis as a primary cell death mechanism. The extraordinarily high abundance of the phospholipid hydroperoxidase GPx4 in beta-cells in contrast to the very low expression in other islet cell types points to a susceptibility of beta-cells to the accumulation of toxic lipid peroxides. Overall, these data strongly suggest that GPx4 is indispensable for beta-cell function under physiological conditions. On the other hand, our results exclude an involvement of ferroptosis as an alternative beta-cell death mode under pro-inflammatory cytokine attack.
Asunto(s)
Apoptosis , Citocinas/metabolismo , Ferroptosis , Mediadores de Inflamación/metabolismo , Células Secretoras de Insulina/patología , Peroxidación de Lípido , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Animales , Células Secretoras de Insulina/metabolismo , Masculino , Oxidación-Reducción , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Ratas , Ratas Endogámicas LewRESUMEN
Increased circulating levels of saturated fatty acids (FFAs) and glucose are considered to be major mediators of ß-cell dysfunction and death in T2DM. Although it has been proposed that endoplasmic reticulum (ER) and oxidative stress play a crucial role in gluco/lipotoxicity, their interplay and relative contribution to ß-cell dysfunction and apoptosis has not been fully elucidated. In addition it is still unclear how palmitate - the physiologically most abundant long-chain saturated FFA - elicits ER stress and which immediate signals commit ß-cells to apoptosis. To study the underlying mechanisms of palmitate-mediated ER stress and ß-cell toxicity, we exploited the observation that the recently described ER-resident GPx7 and GPx8 are not expressed in rat ß-cells. Expression of GPx7 or GPx8 attenuated FFAs-mediated H2O2 generation, ER stress, and apoptosis induction. These results could be confirmed by a H2O2-specific inactivating ER catalase, indicating that accumulation of H2O2 in the ER lumen is critical in FFA-induced ER stress. Furthermore, neither the expression of GPx7 nor of GPx8 increased insulin content or facilitated disulfide bond formation in insulin-secreting INS-1E cells. Hence, reduction of H2O2 by ER-GPx isoforms is not rate-limiting in oxidative protein folding in rat ß-cells. These data suggest that FFA-mediated ER stress is partially dependent on oxidative stress and selective expression of GPx7 or GPx8 improves the ER antioxidative capacity of rat ß-cells without compromising insulin production and the oxidative protein folding machinery.
Asunto(s)
Retículo Endoplásmico/efectos de los fármacos , Glucosa/toxicidad , Ácido Palmítico/toxicidad , Peroxidasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Expresión Génica , Glutatión Peroxidasa , Peróxido de Hidrógeno/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Estrés Oxidativo , Peroxidasas/genética , Plásmidos/química , Plásmidos/metabolismo , Ratas , Transfección , TransgenesRESUMEN
BACKGROUND: The fate of hydrogen peroxide (H2O2) in the endoplasmic reticulum (ER) has been inferred indirectly from the activity of ER-localized thiol oxidases and peroxiredoxins, in vitro, and the consequences of their genetic manipulation, in vivo. Over the years hints have suggested that glutathione, puzzlingly abundant in the ER lumen, might have a role in reducing the heavy burden of H2O2 produced by the luminal enzymatic machinery for disulfide bond formation. However, limitations in existing organelle-targeted H2O2 probes have rendered them inert in the thiol-oxidizing ER, precluding experimental follow-up of glutathione's role in ER H2O2 metabolism. RESULTS: Here we report on the development of TriPer, a vital optical probe sensitive to changes in the concentration of H2O2 in the thiol-oxidizing environment of the ER. Consistent with the hypothesized contribution of oxidative protein folding to H2O2 production, ER-localized TriPer detected an increase in the luminal H2O2 signal upon induction of pro-insulin (a disulfide-bonded protein of pancreatic ß-cells), which was attenuated by the ectopic expression of catalase in the ER lumen. Interfering with glutathione production in the cytosol by buthionine sulfoximine (BSO) or enhancing its localized destruction by expression of the glutathione-degrading enzyme ChaC1 in the lumen of the ER further enhanced the luminal H2O2 signal and eroded ß-cell viability. CONCLUSIONS: A tri-cysteine system with a single peroxidatic thiol enables H2O2 detection in oxidizing milieux such as that of the ER. Tracking ER H2O2 in live pancreatic ß-cells points to a role for glutathione in H2O2 turnover.
Asunto(s)
Retículo Endoplásmico/metabolismo , Peróxido de Hidrógeno/metabolismo , Sondas Moleculares/metabolismo , Fenómenos Ópticos , Animales , Catálisis , Línea Celular , Retículo Endoplásmico/efectos de los fármacos , Fluorescencia , Glutatión/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Cinética , Ratones , Oxidación-Reducción , Compuestos de Sulfhidrilo/metabolismoRESUMEN
AIMS/HYPOTHESIS: The aim of this study was to perform a detailed analysis of cytokine toxicity in the new human EndoC-ßH1 beta cell line. METHODS: The expression profile of the antioxidative enzymes in the new human EndoC-ßH1 beta cells was characterised and compared with that of primary beta cells in the human pancreas. The effects of proinflammatory cytokines on reactive oxygen species formation, insulin secretory responsiveness and apoptosis of EndoC-ßH1 beta cells were determined. RESULTS: EndoC-ßH1 beta cells were sensitive to the toxic action of proinflammatory cytokines. Glucose-dependent stimulation of insulin secretion and an increase in the ATP/ADP ratio was abolished by proinflammatory cytokines without induction of IL-1ß expression. Cytokine-mediated caspase-3 activation was accompanied by reactive oxygen species formation and developed more slowly than in rodent beta cells. Cytokines transiently increased the expression of unfolded protein response genes, without inducing endoplasmic reticulum stress-marker genes. Cytokine-mediated NFκB activation was too weak to induce inducible nitric oxide synthase expression. The resultant lack of nitric oxide generation in EndoC-ßH1 cells, in contrast to rodent beta cells, makes these cells dependent on exogenously generated nitric oxide, which is released from infiltrating immune cells in human type 1 diabetes, for full expression of proinflammatory cytokine toxicity. CONCLUSIONS/INTERPRETATION: EndoC-ßH1 beta cells are characterised by an imbalance between H2O2-generating and -inactivating enzymes, and react to cytokine exposure in a similar manner to primary human beta cells. They are a suitable beta cell surrogate for cytokine-toxicity studies.
Asunto(s)
Citocinas/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Western Blotting , Caspasa 3/metabolismo , Línea Celular , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Glucosa/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Insulina/metabolismo , Estrés Oxidativo/efectos de los fármacos , Pancrelipasa/metabolismo , Especies Reactivas de Oxígeno , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Superóxido Dismutasa-1/metabolismoRESUMEN
Brown adipose tissue (BAT) cells have a very high oxidative capacity. On the other hand, in obesity and obesity-related diabetes, levels of pro-inflammatory cytokines are elevated, which might promote BAT dysfunction and consequently impair carbohydrate metabolism and thereby exacerbate cellular dysfunction and promote diabetes progression. Therefore, the antioxidative enzyme status of a brown adipocyte cell line and its susceptibility towards pro-inflammatory cytokines, which participate in the pathogenesis of diabetes, and reactive oxygen species (ROS) were analysed. Mature brown adipocytes exhibited significantly higher levels of expression of mitochondrially and peroxisomally located antioxidative enzymes compared with non-differentiated brown adipocytes. Pro-inflammatory cytokines induced a significant decrease in the viability of differentiated brown adipocytes, which was accompanied by a massive ROS production and down-regulation of BAT-specific markers, such as uncoupling protein 1 (UCP-1) and ß-Klotho. Taken together, the results strongly indicate that pro-inflammatory cytokines cause brown adipocyte dysfunction and death through suppression of BAT-specific proteins, especially of UCP-1 and ß-Klotho, and consequently increased oxidative stress.
Asunto(s)
Adipocitos Marrones/metabolismo , Citocinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Adipocitos Marrones/patología , Animales , Antioxidantes/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Proteínas Klotho , Proteínas de la Membrana/biosíntesis , Ratones , Mitocondrias/metabolismo , Proteína Desacopladora 1/biosíntesisRESUMEN
The endoplasmic reticulum (ER)-localized peroxiredoxin 4 (PRDX4) supports disulfide bond formation in eukaryotic cells lacking endoplasmic reticulum oxidase 1 (ERO1). The source of peroxide that fuels PRDX4-mediated disulfide bond formation has remained a mystery, because ERO1 is believed to be a major producer of hydrogen peroxide (H2O2) in the ER lumen. We report on a simple kinetic technique to track H2O2 equilibration between cellular compartments, suggesting that the ER is relatively isolated from cytosolic or mitochondrial H2O2 pools. Furthermore, expression of an ER-adapted catalase to degrade lumenal H2O2 attenuated PRDX4-mediated disulfide bond formation in cells lacking ERO1, whereas depletion of H2O2 in the cytosol or mitochondria had no similar effect. ER catalase did not effect the slow residual disulfide bond formation in cells lacking both ERO1 and PRDX4. These observations point to exploitation of a hitherto unrecognized lumenal source of H2O2 by PRDX4 and a parallel slow H2O2-independent pathway for disulfide formation.
Asunto(s)
Retículo Endoplásmico/metabolismo , Glicoproteínas/metabolismo , Peróxido de Hidrógeno/metabolismo , Mitocondrias/metabolismo , Peroxirredoxinas/metabolismo , Animales , Catalasa/metabolismo , Línea Celular , Disulfuros/metabolismo , Retículo Endoplásmico/enzimología , Fibroblastos/metabolismo , Glicoproteínas/genética , Humanos , Ratones , Oxidorreductasas , Peroxirredoxinas/genética , Pliegue de ProteínaRESUMEN
Oxidative folding of (pro)insulin is crucial for its assembly and biological function. This process takes place in the endoplasmic reticulum (ER) and is accomplished by protein disulfide isomerase and ER oxidoreductin 1ß, generating stoichiometric amounts of hydrogen peroxide (H2O2) as byproduct. During insulin resistance in the prediabetic state, increased insulin biosynthesis can overwhelm the ER antioxidative and folding capacity, causing an imbalance in the ER redox homeostasis and oxidative stress. Peroxiredoxin 4 (Prdx4), an ER-specific antioxidative peroxidase can utilize luminal H2O2 as driving force for reoxidizing protein disulfide isomerase family members, thus efficiently contributing to disulfide bond formation. Here, we examined the functional significance of Prdx4 on ß-cell function with emphasis on insulin content and secretion during stimulation with nutrient secretagogues. Overexpression of Prdx4 in glucose-responsive insulin-secreting INS-1E cells significantly metabolized luminal H2O2 and improved the glucose-induced insulin secretion, which was accompanied by the enhanced proinsulin mRNA transcription and insulin content. This ß-cell beneficial effect was also observed upon stimulation with the nutrient insulin secretagogue combination of leucine plus glutamine, indicating that the effect is not restricted to glucose. However, knockdown of Prdx4 had no impact on H2O2 metabolism or ß-cell function due to the fact that Prdx4 expression is negligibly low in pancreatic ß-cells. Moreover, we provide evidence that the constitutively low expression of Prdx4 is highly susceptible to hyperoxidation in the presence of high glucose. Overall, these data suggest an important role of Prdx4 in maintaining insulin levels and improving the ER folding capacity also under conditions of a high insulin requirement.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Peroxirredoxinas/biosíntesis , Edulcorantes/farmacología , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Células Hep G2 , Humanos , Peróxido de Hidrógeno/metabolismo , Insulina/genética , Secreción de Insulina , Células Secretoras de Insulina/citología , Oxidación-Reducción/efectos de los fármacos , Peroxirredoxinas/genética , Pliegue de Proteína/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Edulcorantes/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiologíaRESUMEN
Oxidative protein folding in the endoplasmic reticulum (ER) is associated with the formation of native disulfide bonds, which inevitably results in the formation of hydrogen peroxide (H(2)O(2)). Particularly in pancreatic ß-cells with their high secretory activity and extremely low antioxidant capacity, the H(2)O(2) molecules generated during oxidative protein folding could represent a significant oxidative burden. Therefore this study was conducted to elucidate the H(2)O(2) generation during disulfide bond formation in insulin-producing RINm5F cells by targeting and specifically expressing the H(2)O(2)-sensitive biosensor HyPer in the ER (ER-HyPer). In addition the influence of overexpression of the H(2)O(2)-metabolizing ER-resident peroxiredoxin IV (PRDXIV) on H(2)O(2) levels was examined. The ER-HyPer fluorescent protein was completely oxidized, whereas HyPer expressed in cytosol, peroxisomes, and mitochondria was prevalently in the reduced state, indicating a high basal H(2)O(2) concentration in the ER lumen. These results could also be confirmed in non-insulin-producing COS-7 cells. Overexpression of PRDXIV in RINm5F cells effectively protected against H(2)O(2)-mediated toxicity; however, it did not affect the fluorescence signal of ER-HyPer. Moreover, the inhibition of de novo protein synthesis and the associated H(2)O(2) generation by cycloheximide had no influence on the ER-HyPer redox state. Taken together, these findings strongly suggest that the H(2)O(2)-sensitive biosensor reflects exclusively the oxidative milieu in the ER and not the H(2)O(2) concentration in the ER lumen.
Asunto(s)
Disulfuros/metabolismo , Retículo Endoplásmico/metabolismo , Peróxido de Hidrógeno/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Luminiscentes/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Animales , Técnicas Biosensibles , Línea Celular , Cicloheximida/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Fluorescencia , Expresión Génica , Genes Reporteros , Peróxido de Hidrógeno/farmacología , Células Secretoras de Insulina/citología , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Sondas Moleculares , Oxidación-Reducción , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Peroxisomas/efectos de los fármacos , Peroxisomas/metabolismo , Pliegue de Proteína , RatasRESUMEN
Pro-inflammatory cytokine-mediated beta cell apoptosis is activated through multiple signaling pathways involving mitochondria and endoplasmic reticulum. Activation of organelle-specific caspases has been implicated in the progression and execution of cell death. This study was therefore performed to elucidate the effects of pro-inflammatory cytokines on a possible cross-talk between the compartment-specific caspases 9 and 12 and their differential contribution to beta cell apoptosis. Moreover, the occurrence of ROS-mediated mitochondrial damage in response to beta cell toxic cytokines has been quantified. ER-specific caspase-12 was strongly activated in response to pro-inflammatory cytokines; however, its inhibition did not abolish cytokine-induced mitochondrial caspase-9 activation and loss of cell viability. In addition, there was a significant induction of oxidative mitochondrial DNA damage and elevated cardiolipin peroxidation in insulin-producing RINm5F cells and rat islet cells. Overexpression of the H(2)O(2) detoxifying enzyme catalase effectively reduced the observed cytokine-induced oxidative damage of mitochondrial structures. Taken together, the results strongly indicate that mitochondrial caspase-9 is not a downstream substrate of ER-specific caspase-12 and that pro-inflammatory cytokines cause apoptotic beta cell death through activation of caspase-9 primarily by hydroxyl radical-mediated mitochondrial damage.
Asunto(s)
Apoptosis/fisiología , Caspasa 12/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiología , Mitocondrias/metabolismo , Estrés Oxidativo/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Caspasa 12/genética , Caspasa 9/metabolismo , Caspasa 9/fisiología , Catalasa/metabolismo , Células Cultivadas , Citocinas/metabolismo , Citocinas/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Activación Enzimática/fisiología , Mediadores de Inflamación/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Ratas , Ratas Endogámicas LewRESUMEN
Although nitric oxide (NO) and oxidative stress both contribute to proinflammatory cytokine toxicity in pancreatic ß-cells during type 1 diabetes mellitus (T1DM) development, the interactions between NO and reactive oxygen species (ROS) in cytokine-mediated ß-cell death have not been clarified. Exposure of insulin-producing RINm5F cells to IL-1ß generated NO, while exposure to a combination of IL-1ß, TNF-α, and IFN-γ, which simulates T1DM conditions, generated both NO and ROS. In theory, two reactions between NO and ROS are possible, one with the superoxide radical yielding peroxynitrite, and the other with hydrogen peroxide (H(2)O(2)) yielding hydroxyl radicals. Results of the present work exclude peroxynitrite involvement in cytokine toxicity to ß-cells because its generation did not correlate with the toxic action of cytokines. On the other hand, we show that H(2)O(2), produced upon exposure of insulin-producing cell clones and primary rat islet cells to cytokines almost exclusively in the mitochondria, reacted in the presence of trace metal (Fe(++)) with NO forming highly toxic hydroxyl radicals, thus explaining the severe toxicity that causes apoptotic ß-cell death. Expression of the H(2)O(2)-inactivating enzyme catalase in mitochondria protected against cytokine toxicity by preventing hydroxyl radical formation. We therefore conclude that proinflammatory cytokine-mediated ß-cell death is due to nitro-oxidative stress-mediated hydroxyl radical formation in the mitochondria.
Asunto(s)
Citocinas/toxicidad , Radical Hidroxilo/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Mitocondrias/metabolismo , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/metabolismo , Caspasa 3/metabolismo , Línea Celular , Células Clonales , Inhibidores Enzimáticos/farmacología , Fluoresceínas/metabolismo , Peróxido de Hidrógeno/metabolismo , Insulina/biosíntesis , Células Secretoras de Insulina/enzimología , Células Secretoras de Insulina/patología , Mitocondrias/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Nitritos/metabolismo , Oxidación-Reducción/efectos de los fármacos , Ácido Peroxinitroso/biosíntesis , Ratas , Ratas Endogámicas Lew , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Pro-inflammatory cytokines are key mediators in the selective and progressive destruction of insulin-producing beta cells during type 1 diabetes development. However, the mechanisms of cytokine-induced beta cell apoptosis are not fully understood. This study demonstrates that pro-inflammatory cytokines strongly modified the expression of the anti-apoptotic protein Bcl-2 and the pro-apoptotic BH3-only proteins Bad, Bim, and Bid in primary rat islets and insulin-producing RINm5F cells. Overexpression of mitochondrially located catalase (MitoCatalase) specifically increased basal Bcl-2 and decreased basal Bax expression, suppressed cytokine-mediated reduction of Bcl-2, and thereby prevented the release of cytochrome c, Smac/DIABLO and the activation of caspase-9 and -3. Thus, cytokine-mediated decrease of Bcl-2 expression and the sequentially changed Bax/Bcl-2 ratio are responsible for the release of pro-apoptotic mitochondrial factors, activation of caspase-9, and ultimately caspase-3. These results indicate that activation of the intrinsic/mitochondrial apoptosis pathway is essential for cytokine-induced beta cell death and the mitochondrial generation of reactive oxygen species, in particular mitochondrial hydrogen peroxide, differentially regulates the Bax/Bcl-2 ratio.
Asunto(s)
Antioxidantes/metabolismo , Citocinas/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Proteína 11 Similar a Bcl2 , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Catalasa/genética , Catalasa/metabolismo , Línea Celular , Células Secretoras de Insulina/citología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Ratas , Ratas Endogámicas Lew , Especies Reactivas de Oxígeno/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The transcription factor nuclear factor (NF)-κB is known to modulate rates of apoptosis and may therefore play a role in the increased ß-cell death that occurs in type 1 and type 2 diabetes. The aim of the present investigation was to study the expression of NF-κB subunits in human islet cells and whether overexpression of the NF-κB subunit c-Rel affects islet cell survival. We detected expression of p65, Rel-B, p50, p105, p52, and the ribosomal protein S3 (rpS3) in human islet cells. Among these, only p65 and rpS3 were translocated from the cytosolic to the nuclear fraction in response to cytokines. Interestingly, rpS3 participated in p65 binding to the κB-element in gel shift analysis experiments. We observed cytoplasmic c-Rel expression in vivo in 6J mice, and signs of nuclear translocation in ß-cells of infiltrated nonobese diabetic islets. Human islet cells were also dispersed by trypsin treatment and transduced with a c-Rel adenoviral vector. This resulted in increased expression of c-Rel and inhibitory factor κB, increased κB-binding activity, and augmented protein levels of Bcl-X(L,) c-IAP2, and heat shock protein 72. c-Rel expression in human islet cells protected against cytokine-induced caspase 3 activation and cell death. c-Rel protected also against streptozotocin- and H(2)O(2)-induced cell death, in both intact rat islets and human islet cells. We conclude that rpS3 participates in NF-κB signaling and that a genetic increase in the activity of the NF-κB subunit c-Rel results in protection against cell death in human islets.
Asunto(s)
Islotes Pancreáticos/efectos de los fármacos , FN-kappa B/biosíntesis , FN-kappa B/fisiología , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/fisiología , Adenoviridae/genética , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Western Blotting , Muerte Celular/fisiología , Supervivencia Celular/fisiología , Colorantes , Ensayo de Cambio de Movilidad Electroforética , Femenino , Expresión Génica/fisiología , Vectores Genéticos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Microscopía Confocal , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/aislamiento & purificación , Perfusión , Ratas , Ratas Sprague-Dawley , Sales de Tetrazolio , Tiazoles , Transducción GenéticaRESUMEN
Type 1 diabetes may depend on cytokine-induced beta-cell death and therefore the current investigation was performed in order to elucidate this response in Shb-deficient islets. A combination of interleukin-1beta and interferon-gamma caused a diminished beta-cell death response in Shb null islets. Furthermore, the induction of an unfolded protein response (UPR) by adding cyclopiazonic acid did not increase cell death in Shb-deficient islets, despite simultaneous expression of UPR markers. The heat-shock protein Hsp70 was more efficiently induced in Shb knockout islets, providing an explanation for the decreased susceptibility of Shb-deficient islets to cytokines. It is concluded that islets deficient in the Shb protein are less susceptible to cytotoxic conditions, and that this partly depends on their increased ability to induce Hsp70 under such circumstances. Interference with Shb signaling may provide means to improve beta-cell viability under conditions of beta-cell stress.