RESUMO
Pancreatic beta cell failure is the central event leading to diabetes. Beta cells share many phenotypic traits with neurons, and proper beta cell function relies on the activation of several neuron-like transcription programs. Regulation of gene expression by alternative splicing plays a pivotal role in brain, where it affects neuronal development, function, and disease. The role of alternative splicing in beta cells remains unclear, but recent data indicate that splicing alterations modulated by both inflammation and susceptibility genes for diabetes contribute to beta cell dysfunction and death. Here we used RNA sequencing to compare the expression of splicing-regulatory RNA-binding proteins in human islets, brain, and other human tissues, and we identified a cluster of splicing regulators that are expressed in both beta cells and brain. Four of them, namely Elavl4, Nova2, Rbox1, and Rbfox2, were selected for subsequent functional studies in insulin-producing rat INS-1E, human EndoC-ßH1 cells, and in primary rat beta cells. Silencing of Elavl4 and Nova2 increased beta cell apoptosis, whereas silencing of Rbfox1 and Rbfox2 increased insulin content and secretion. Interestingly, Rbfox1 silencing modulates the splicing of the actin-remodeling protein gelsolin, increasing gelsolin expression and leading to faster glucose-induced actin depolymerization and increased insulin release. Taken together, these findings indicate that beta cells share common splicing regulators and programs with neurons. These splicing regulators play key roles in insulin release and beta cell survival, and their dysfunction may contribute to the loss of functional beta cell mass in diabetes.
Assuntos
Células Secretoras de Insulina/citologia , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Animais , Apoptose , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Proteína Semelhante a ELAV 4/genética , Proteína Semelhante a ELAV 4/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/genética , RatosRESUMO
Alternative splicing (AS) is a fundamental mechanism for the regulation of gene expression. It affects more than 90% of human genes but its role in the regulation of pancreatic beta cells, the producers of insulin, remains unknown. Our recently published data indicated that the 'neuron-specific' Nova1 splicing factor is expressed in pancreatic beta cells. We have presently coupled specific knockdown (KD) of Nova1 with RNA-sequencing to determine all splice variants and downstream pathways regulated by this protein in beta cells. Nova1 KD altered the splicing of nearly 5000 transcripts. Pathway analysis indicated that these genes are involved in exocytosis, apoptosis, insulin receptor signaling, splicing and transcription. In line with these findings, Nova1 silencing inhibited insulin secretion and induced apoptosis basally and after cytokine treatment in rodent and human beta cells. These observations identify a novel layer of regulation of beta cell function, namely AS controlled by key splicing regulators such as Nova1.
Assuntos
Processamento Alternativo , Células Secretoras de Insulina/metabolismo , Proteínas de Ligação a RNA/fisiologia , Animais , Apoptose , Cálcio/metabolismo , Citocinas/farmacologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Antígeno Neuro-Oncológico Ventral , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Ratos Wistar , Receptor de Insulina/genética , Receptor de Insulina/metabolismoRESUMO
AIMS/HYPOTHESIS: Proinflammatory cytokines contribute to beta cell damage in type 1 diabetes in part through activation of endoplasmic reticulum (ER) stress. In rat beta cells, cytokine-induced ER stress involves NO production and consequent inhibition of the ER Ca(2+) transporting ATPase sarco/endoplasmic reticulum Ca(2+) pump 2 (SERCA2B). However, the mechanisms by which cytokines induce ER stress and apoptosis in mouse and human pancreatic beta cells remain unclear. The purpose of this study is to elucidate the role of ER stress on cytokine-induced beta cell apoptosis in these three species and thus solve ongoing controversies in the field. METHODS: Rat and mouse insulin-producing cells, human pancreatic islets and human EndoC-ßH1 cells were exposed to the cytokines IL-1ß, TNF-α and IFN-γ, with or without NO inhibition. A global comparison of cytokine-modulated gene expression in human, mouse and rat beta cells was also performed. The chemical chaperone tauroursodeoxycholic acid (TUDCA) and suppression of C/EBP homologous protein (CHOP) were used to assess the role of ER stress in cytokine-induced apoptosis of human beta cells. RESULTS: NO plays a key role in cytokine-induced ER stress in rat islets, but not in mouse or human islets. Bioinformatics analysis indicated greater similarity between human and mouse than between human and rat global gene expression after cytokine exposure. The chemical chaperone TUDCA and suppression of CHOP or c-Jun N-terminal kinase (JNK) protected human beta cells against cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: These observations clarify previous results that were discrepant owing to the use of islets from different species, and confirm that cytokine-induced ER stress contributes to human beta cell death, at least in part via JNK activation.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Ratos , Ratos Wistar , Ácido Tauroquenodesoxicólico/farmacologia , Fator de Transcrição CHOP/farmacologia , ômega-N-Metilarginina/farmacologiaRESUMO
Pancreatic ß-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and KATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5% Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced KATP inhibition and Cav activity. RH islets secreted less insulin at high K(+) concentration and showed enhanced KATP activity. Tau supplementation normalized K(+)-induced secretion and enhanced glucose-induced Ca(2+) influx in RHT islets. R islets presented lower Ca(2+) influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the KATP. Tau increased the protein content of the α1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the KATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the KATP channels and enhancing Synt-1 islet content.
Assuntos
Cálcio/metabolismo , Gorduras na Dieta/farmacologia , Suplementos Nutricionais , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Desnutrição/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Taurina/farmacologia , Animais , Humanos , Secreção de Insulina , Masculino , Camundongos , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
Type 1 diabetes is caused by an autoimmune assault that induces progressive beta-cell dysfunction and dead. Pro-inflammatory cytokines, such as interleukin 1 beta (IL1B), tumor necrosis factor (TNF) and interferon gamma (IFNG) contribute for beta-cell death, which involves the activation of the nuclear factor kappa B (NFκB) and c- Jun N-terminal kinase (JNK). Prolactin (PRL), a physiological mediator for beta-cell proliferation, was shown to protect beta cells against cytokines pro-apoptotic effects. We presently investigated the mechanisms involved in the protective effects of prolactin against cytokine-induced beta-cell death. The findings obtained indicate that STAT3 activation is involved in the anti-apoptotic role of PRL in rat beta cells. PRL prevents the activation of JNK via AKT and promotes a shift from expression of pro- to anti-apoptotic proteins downstream of the JNK cascade. Furthermore, PRL partially prevents the activation of NFκB and the transcription of its target genes IkBa, Fas, Mcp1, A20 and Cxcl10 and also decreases NO production. On the other hand, the pro-survival effects of PRL do not involve modulation of cytokine-induced endoplasmic reticulum stress. These results suggest that the beneficial effects of PRL in beta cells involve augmentation of anti-apoptotic mechanisms and, at the same time, reduction of pro-apoptotic effectors, rendering beta cells better prepared to deal with inflammatory insults. The better understanding of the pro-survival mechanisms modulated by PRL in beta cells can provide tools to prevent cell demise during an autoimmune attack or following islet transplantation.
Assuntos
Apoptose/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Prolactina/farmacologia , Animais , Western Blotting , Células Cultivadas , Feminino , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
Nonalcoholic fatty liver disease (NAFLD) is the principal manifestation of liver disease in obesity and metabolic syndrome. By comparing hypertriglyceridemic transgenic mice expressing apolipoprotein (apo) CIII with control nontransgenic (NTg) littermates, we demonstrated that overexpression of apoCIII, independent of a high-fat diet (HFD), produces NAFLD-like features, including increased liver lipid content; decreased antioxidant power; increased expression of TNFα, TNFα receptor, cleaved caspase-1, and interleukin-1ß; decreased expression of adiponectin receptor-2; and increased cell death. This phenotype is aggravated and additional NAFLD features are differentially induced in apoCIII mice fed a HFD. HFD induced glucose intolerance together with increased gluconeogenesis, indicating hepatic insulin resistance. Additionally, the HFD led to marked increases in plasma TNFα (8-fold) and IL-6 (60%) in apoCIII mice. Cell death signaling (Bax/Bcl2), effector (caspase-3), and apoptosis were augmented in apoCIII mice regardless of whether a HFD or a low-fat diet was provided. Fenofibrate treatment reversed several of the effects associated with diet and apoCIII expression but did not normalize inflammatory traits even when liver lipid content was fully corrected. These results indicate that apoCIII and/or hypertriglyceridemia plays a major role in liver inflammation and cell death, which in turn increases susceptibility to and the severity of diet-induced NAFLD.
Assuntos
Apolipoproteína C-III/biossíntese , Hipertrigliceridemia/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Apolipoproteína C-III/metabolismo , Morte Celular/fisiologia , Dieta Hiperlipídica , Inflamação/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The aim of this study was to investigate the insulin clearance in diet-induced obese (DIO) mice submitted to acute endurance exercise (3h of treadmill exercise at 60-70% VO2max). Glucose-stimulated insulin secretion in isolated islets; ipGTT; ipITT; ipPTT; in vivo insulin clearance; protein expression in liver, skeletal muscle, and adipose tissue (insulin degrading enzyme (IDE), insulin receptor subunitß(IRß), phospho-Akt (p-Akt) and phospho-AMPK (p-AMPK)), and the activity of IDE in the liver and skeletal muscle were accessed. In DIO mice, acute exercise reduced fasting glycemia and insulinemia, improved glucose and insulin tolerance, reduced hepatic glucose production, and increased p-Akt protein levels in liver and skeletal muscle and p-AMPK protein levels in skeletal muscle. In addition, insulin secretion was reduced, whereas insulin clearance and the expression of IDE and IRß were increased in liver and skeletal muscle. Finally, IDE activity was increased only in skeletal muscle. In conclusion, we propose that the increased insulin clearance and IDE expression and activity, primarily, in skeletal muscle, constitute an additional mechanism, whereby physical exercise reduces insulinemia in DIO mice.
Assuntos
Insulina/metabolismo , Obesidade/metabolismo , Obesidade/terapia , Condicionamento Físico Animal/fisiologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Peptídeo C/sangue , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Insulina/sangue , Insulisina/metabolismo , Fígado/metabolismo , Masculino , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Músculo Esquelético/metabolismo , Obesidade/etiologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/metabolismoRESUMO
The effects of exercise on insulin clearance and IDE expression are not yet fully elucidated. Here, we have explored the effect of acute exercise on insulin clearance and IDE expression in lean mice. Male Swiss mice were subjected to a single bout of exercise on a speed/angle controlled treadmill for 3-h at approximately 60-70% of maximum oxygen consumption. As expected, acute exercise reduced glycemia and insulinemia, and increased insulin tolerance. The activity of AMPK-ACC, but not of IR-Akt, pathway was increased in the liver and skeletal muscle of trained mice. In an apparent contrast to the reduced insulinemia, glucose-stimulated insulin secretion was increased in isolated islets of these mice. However, insulin clearance was increased after acute exercise and was accompanied by increased expression of the insulin-degrading enzyme (IDE), in the liver and skeletal muscle. Finally, C2C12, but not HEPG2 cells, incubated at different concentrations of 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) for 3-h, showed increased expression of IDE. In conclusion, acute exercise increases insulin clearance, probably due to an augmentation of IDE expression in the liver and skeletal muscle. The elevated IDE expression, in the skeletal muscle, seems to be mediated by activation of AMPK-ACC pathway, in response to exercise. We believe that the increase in the IDE expression, comprise a safety measure to maintain glycemia at or close to physiological levels, turning physical exercise more effective and safe.