RESUMEN
Prolonged exercise has positive metabolic effects in obese or diabetic individuals. These effects are usually ascribed to improvements in insulin sensitivity. We evaluated whether exercise also generates circulating signals that protect human and rodent ß cells against endoplasmic reticulum (ER) stress and apoptosis. For this purpose, we obtained serum from humans or mice before and after an 8 wk training period. Exposure of human islets or mouse or rat ß cells to human or rodent sera, respectively, obtained from trained individuals reduced cytokine (IL-1ß+IFN-γ)- or chemical ER stressor-induced ß-cell ER stress and apoptosis, at least in part via activation of the transcription factor STAT3. These findings indicate that exercise training improves human and rodent ß-cell survival under diabetogenic conditions and support lifestyle interventions as a protective approach for both type 1 and 2 diabetes.-Paula, F. M. M., Leite, N. C., Borck, P. C., Freitas-Dias, R., Cnop, M., Chacon-Mikahil, M. P. T., Cavaglieri, C. R., Marchetti, P., Boschero, A. C., Zoppi, C. C., Eizirik, D. L. Exercise training protects human and rodent ß cells against endoplasmic reticulum stress and apoptosis.
Asunto(s)
Apoptosis/fisiología , Estrés del Retículo Endoplásmico/fisiología , Ejercicio Físico/fisiología , Células Secretoras de Insulina/metabolismo , Condicionamiento Físico Animal/fisiología , Animales , Femenino , Humanos , Células Secretoras de Insulina/citología , Masculino , Ratones , Ratas , Ratas WistarRESUMEN
Type 1 diabetes (T1D) is provoked by an autoimmune assault against pancreatic ß cells. Exercise training enhances ß-cell mass in T1D. Here, we investigated how exercise signals ß cells in T1D condition. For this, we used several approaches. Wild-type and IL-6 knockout (KO) C57BL/6 mice were exercised. Afterward, islets from control and trained mice were exposed to inflammatory cytokines (IL-1ß plus IFN-γ). Islets from control mice and ß-cell lines (INS-1E and MIN6) were incubated with serum from control or trained mice or medium obtained from 5-aminoimidazole-4 carboxamide1-ß-d-ribofuranoside (AICAR)-treated C2C12 skeletal muscle cells. Subsequently, islets and ß cells were exposed to IL-1ß plus IFN-γ. Proteins were assessed by immunoblotting, apoptosis was determined by DNA-binding dye propidium iodide fluorescence, and NO(â¢) was estimated by nitrite. Exercise reduced 25, 75, and 50% of the IL-1ß plus IFN-γ-induced iNOS, nitrite, and cleaved caspase-3 content, respectively, in pancreatic islets. Serum from trained mice and medium from AICAR-treated C2C12 cells reduced ß-cell death, induced by IL-1ß plus IFN-γ treatment, in 15 and 38%, respectively. This effect was lost in samples treated with IL-6 inhibitor or with serum from exercised IL-6 KO mice. In conclusion, muscle contraction signals ß-cell survival in T1D through IL-6.
Asunto(s)
Apoptosis , Diabetes Mellitus Tipo 1/patología , Células Secretoras de Insulina/patología , Interleucina-6/fisiología , Islotes Pancreáticos/patología , Músculo Esquelético/patología , Condicionamiento Físico Animal , Animales , Western Blotting , Proliferación Celular , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/terapia , Glucosa/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Interferón gamma/farmacología , Interleucina-1beta/farmacología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Óxido Nítrico/metabolismo , ARN Mensajero/genética , Radioinmunoensayo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Aim Investigate whether inheritance of improved skeletal muscle mitochondrial function and its association with glycemic control are multigenerational benefits of exercise. MAIN METHODS: Male Swiss mice were subjected to 8 weeks of endurance training and mated with untrained females. KEY FINDINGS: Trained fathers displayed typical endurance training-induced adaptations. Remarkably, offspring from trained fathers also exhibited higher endurance performance, mitochondrial oxygen consumption, glucose tolerance and insulin sensitivity. However, PGC-1α expression was not increased in the offspring. In the offspring, the expression of the co-repressor NCoR1 was reduced, increasing activation of PGC-1α target genes. These effects correlated with higher DNA methylation at the NCoR1 promoter in both, the sperm of trained fathers and in the skeletal muscle of their offspring. SIGNIFICANCE: Higher skeletal muscle mitochondrial function is inherited by epigenetic de-activation of a key PGC-1α co-repressor.
Asunto(s)
Mitocondrias/metabolismo , Condicionamiento Físico Animal/fisiología , Esfuerzo Físico/fisiología , Animales , Metilación de ADN , Epigénesis Genética/genética , Femenino , Masculino , Ratones , Mitocondrias/fisiología , Músculo Esquelético/fisiología , Co-Represor 1 de Receptor Nuclear/metabolismo , Consumo de Oxígeno/fisiología , Herencia Paterna/fisiología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Receptores Activados del Proliferador del Peroxisoma/fisiología , Condicionamiento Físico Animal/métodos , ARN Mensajero/genéticaRESUMEN
Chronic administration of glucocorticoids induces insulin resistance that is compensated by an increase in p-cell function and mass. Since insulin signaling is involved in the control of p-cell function and mass, we investigated the content of insulin pathway proteins in pancreatic islets. Rats were made insulin resistant by daily administration of dexamethasone (1mg/kg, b.w., i.p.) for 5 consecutive days (DEX), whilst control rats received saline (CTL). Circulating insulin and insulin released from isolated islets were measured by radioimmunoassay whereas the content of proteins was analyzed by Western blotting. DEX rats were hyperinsulinemic and exhibited augmented insulin secretion in response to glucose (P < 0.01). The IRa-subunit, IRS-1, Shc, AKT, p-p70S6K, ERK1/2, p-ERK1/2, and glucocorticoid receptor protein levels were similar between DEX and CTL islets. However, the IRp-subunit, p-IRp-subunit, IRS-2, PI3-K, p-AKT and p70S6K protein contents were increased in DEX islets (P < 0.05). We conclude that IRS-2 may have a major role, among the immediate substrates of the insulin receptor, to link activated receptors to downstream signaling components related to islet function and growth in this insulin-resistant rat model.
Asunto(s)
Dexametasona/efectos adversos , Glucocorticoides/efectos adversos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Resistencia a la Insulina , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Animales , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Masculino , Ratas , Ratas Wistar , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Transducción de SeñalRESUMEN
Most obese and insulin-resistant individuals do not develop diabetes. This is the result of the capacity of ß-cells to adapt and produce enough insulin to cover the needs of the organism. The underlying mechanism of ß-cell adaptation in obesity, however, remains unclear. Previous studies have suggested a role for STAT3 in mediating ß-cell development and human glucose homeostasis, but little is known about STAT3 in ß-cells in obesity. We observed enhanced cytoplasmic expression of STAT3 in severely obese subjects with diabetes. To address the functional role of STAT3 in adult ß-cells, we generated mice with tamoxifen-inducible partial or full deletion of STAT3 in ß-cells and fed them a high-fat diet before analysis. Interestingly, ß-cell heterozygous and homozygous STAT3-deficient mice showed glucose intolerance when fed a high-fat diet. Gene expression analysis with RNA sequencing showed that reduced expression of mitochondrial genes in STAT3 knocked down human EndoC-ß1H cells, confirmed in FACS-purified ß-cells from obese STAT3-deficient mice. Moreover, silencing of STAT3 impaired mitochondria activity in EndoC-ß1H cells and human islets, suggesting a mechanism for STAT3-modulated ß-cell function. Our study postulates STAT3 as a novel regulator of ß-cell function in obesity.
Asunto(s)
Intolerancia a la Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Mitocondrias/metabolismo , Obesidad/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Glucemia/metabolismo , Dieta Alta en Grasa , Genes Mitocondriales , Intolerancia a la Glucosa/genética , Humanos , Insulina/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/genética , Obesidad/genética , Factor de Transcripción STAT3/genéticaRESUMEN
BACKGROUND: Antibodies targeting PD-1 and its ligand PDL1 are used in cancer immunotherapy but may lead to autoimmune diseases, including type 1 diabetes (T1D). It remains unclear whether PDL1 is expressed in pancreatic islets of people with T1D and how is it regulated. METHODS: The expression of PDL1, IRF1, insulin and glucagon was evaluated in samples of T1D donors by immunofluorescence. Cytokine-induced PDL1 expression in the human beta cell line, EndoC-ßH1, and in primary human pancreatic islets was determined by real-time RT-PCR, flow cytometry and Western blot. Specific and previously validated small interference RNAs were used to inhibit STAT1, STAT2, IRF1 and JAK1 signaling. Key results were validated using the JAK inhibitor Ruxolitinib. FINDINGS: PDL1 was present in insulin-positive cells from twelve T1D individuals (6 living and 6 deceased donors) but absent from insulin-deficient islets or from the islets of six non-diabetic controls. Interferons-α and -γ, but not interleukin-1ß, induced PDL1 expression in vitro in human islet cells and EndoC-ßH1 cells. Silencing of STAT1 or STAT2 individually did not prevent interferon-α-induced PDL1, while blocking of JAKs - a proposed therapeutic strategy for T1D - or IRF1 prevented PDL1 induction. INTERPRETATION: These findings indicate that PDL1 is expressed in beta cells from people with T1D, possibly to attenuate the autoimmune assault, and that it is induced by both type I and II interferons via IRF1.
Asunto(s)
Antígeno B7-H1/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Regulación de la Expresión Génica , Factor 1 Regulador del Interferón/metabolismo , Interferón-alfa/metabolismo , Interferón gamma/metabolismo , Islotes Pancreáticos/metabolismo , Adolescente , Adulto , Biomarcadores , Línea Celular , Niño , Preescolar , Humanos , Células Secretoras de Insulina/metabolismo , Persona de Mediana Edad , Adulto JovenRESUMEN
It has been reported that the incretin system, including regulated GLP-1 secretion and locally expressed DPP-4, is present in pancreatic islets. In this study we comprehensively evaluated the expression and role of DPP-4 in islet alpha and beta cells from non-diabetic (ND) and type 2 diabetic (T2D) individuals, including the effects of its inhibition on beta cell function and survival. Isolated islets were prepared from 25 ND and 18 T2D organ donors; studies were also performed with the human insulin-producing EndoC-ßH1 cells. Morphological (including confocal microscopy), ultrastructural (electron microscopy, EM), functional (glucose-stimulated insulin secretion), survival (EM and nuclear dyes) and molecular (RNAseq, qPCR and western blot) studies were performed under several different experimental conditions. DPP-4 co-localized with glucagon and was also expressed in human islet insulin-containing cells. Furthermore, DPP-4 was expressed in EndoC-ßH1 cells. The proportions of DPP-4 positive alpha and beta cells and DPP-4 gene expression were significantly lower in T2D islets. A DPP-4 inhibitor protected ND human beta cells and EndoC-ßH1 cells against cytokine-induced toxicity, which was at least in part independent from GLP1 and associated with reduced NFKB1 expression. Finally, DPP-4 inhibition augmented glucose-stimulated insulin secretion, reduced apoptosis and improved ultrastructure in T2D beta cells. These results demonstrate the presence of DPP-4 in human islet alpha and beta cells, with reduced expression in T2D islets, and show that DPP-4 inhibition has beneficial effects on human ND and T2D beta cells. This suggests that DPP-4, besides playing a role in incretin effects, directly affects beta cell function and survival.
Asunto(s)
Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/patología , Dipeptidil Peptidasa 4/metabolismo , Células Secretoras de Insulina/enzimología , Anciano , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citocinas/toxicidad , Citoprotección/efectos de los fármacos , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Regulación hacia Abajo/efectos de los fármacos , Femenino , Humanos , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/ultraestructura , Masculino , Persona de Mediana EdadRESUMEN
Peroxiredoxins are a family of six antioxidant enzymes (PRDX1-6), and may be an alternative system for the pancreatic beta cells to cope with oxidative stress. This study investigated whether the main diabetogenic pro-inflammatory cytokines or the anti-inflammatory cytokine IL-4 modulate PRDXs levels and putative intracellular pathways important for this process in the insulin-producing RINm5F cells. RINm5F cells expressed significant amounts of PRDX1, PRDX3 and PRDX6 enzymes. Only PRDX6 was modulated by cytokines, showing both mRNA and protein down-regulation following incubation of RINm5F cells with TNF-alpha and IFN-gamma but not with IL-1beta. Separately IFN-gamma or TNF-alpha decreased PRDX6 protein but not mRNA levels. The blockage of the JNK signalling and of the calpains and proteasome proteolysis systems restored PRDX6 protein levels. IL-4 alone did not modulate PRDXs levels. However, pre/co-incubation with IL-4 substantially prevented the decrease in PRDX6 induced by pro-inflammatory cytokines. Knockdown of PRDX6 increased susceptibility of RINm5F cells to the deleterious effects of pro-inflammatory cytokines and to oxidative stress. These results show that, from the PRDXs significantly expressed in RINm5F cells, only PRDX6 is modulated by the diabetogenic cytokines IFN-gamma and TNF-alpha. This PRDX6 down-regulation depends on the calpain and proteasome systems and JNK signalling. PRDX6 is an important enzyme for protection against oxidative stress and the interaction between pro- and anti-inflammatory cytokines might be important to determine the antioxidant capacity of the cells.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Insulina/biosíntesis , Interferón gamma/farmacología , Peroxiredoxina VI/genética , Factor de Necrosis Tumoral alfa/farmacología , Animales , Calpaína/antagonistas & inhibidores , Calpaína/genética , Calpaína/metabolismo , Línea Celular , Regulación de la Expresión Génica , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Interleucina-1beta/farmacología , Interleucina-4/farmacología , MAP Quinasa Quinasa 4/antagonistas & inhibidores , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Estrés Oxidativo , Peroxiredoxina VI/antagonistas & inhibidores , Peroxiredoxina VI/metabolismo , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Transducción de SeñalRESUMEN
The pentadecapeptide comprising the 104-118 amino acid sequence of the ilotropin-derived Reg3-related islet neogenesis-associated protein (INGAP-PP) has been implicated in beta cell neogenesis and enhancement of insulin secretion in pancreatic islets. The aim of this study was to investigate intracellular pathways by which INGAP-PP signals in insulin-producing cells. Treatment with INGAP-PP increased insulin secretion and intracellular calcium levels in MIN6 cells. INGAP-PP exposure activated c-Myc, serum and particularly nuclear factor-kappaB (NF-kappaB) response elements in insulin-producing cells (1.7+/-0.1, 1.8+/-0.1, 2.4+/-0.3 for RINm5F, and 1.3+/-0.1, 1.3+/-0.1 and 1.6+/-0.1 fold for MIN6 cells compared to controls, respectively). There was an increase in the proliferation rate of viable cells (162+/-17% for RINm5F and 155+/-13% for MIN6) that was accompanied by an increase in proliferating cell nuclear antigen (PCNA) protein expression (187+/-19% and 170+/-8% for RINm5F and MIN6 cells respectively) following INGAP-PP treatment. INGAP-PP increased the expression of the muscarinic M(3) receptor subtype (169+/-4% for RINm5F and 222+/-20% for MIN6 cells). Activation of multiple serum response elements by foetal calf serum also increased muscarinic M(3) receptor expression (173+/-9% for RINm5F and 140+/-7% for MIN6 cells). The blockade of NF-kappaB signalling pathway strongly decreased muscarinic M(3) receptor expression in response to both stimuli. In summary, a network of intracellular signals that includes activation of c-Myc signalling pathway and increased PCNA expression might be related to the increased proliferation rate of insulin-producing cells following incubation with INGAP-PP. NF-kappaB signalling plays an essential role in controlling the expression of the muscarinic M(3) receptor.