RESUMEN

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/genética

RESUMEN

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/genética

RESUMEN

Type 1 diabetes (T1D) is an autoimmune disease characterized by islet inflammation and progressive pancreatic ß cell destruction. The disease is triggered by a combination of genetic and environmental factors, but the mechanisms leading to the triggering of early innate and late adaptive immunity and consequent progressive pancreatic ß cell death remain unclear. The insulin-producing ß cells are active secretory cells and are thus particularly sensitive to endoplasmic reticulum (ER) stress. ER stress plays an important role in the pathologic pathway leading to autoimmunity, islet inflammation, and ß cell death. We show here that group B coxsackievirus (CVB) infection, a putative causative factor for T1D, induces a partial ER stress in rat and human ß cells. The activation of the PERK/ATF4/CHOP branch is blunted while the IRE1α branch leads to increased spliced XBP1 expression and c-Jun N-terminal kinase (JNK) activation. Interestingly, JNK1 activation is essential for CVB amplification in both human and rat ß cells. Furthermore, a chemically induced ER stress preceding viral infection increases viral replication, in a process dependent on IRE1α activation. Our findings show that CVB tailors the unfolded protein response in ß cells to support their replication, preferentially triggering the pro-viral IRE1α/XBP1s/JNK1 pathway while blocking the pro-apoptotic PERK/ATF4/CHOP pathway.

Asunto(s)

Infecciones por Coxsackievirus/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Enterovirus Humano B/fisiología , Células Secretoras de Insulina/inmunología , Respuesta de Proteína Desplegada/inmunología , Animales , Línea Celular , Infecciones por Coxsackievirus/inmunología , Diabetes Mellitus Tipo 1/inmunología , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Humanos , Evasión Inmune , Células Secretoras de Insulina/virología , MAP Quinasa Quinasa 4/metabolismo , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Transducción de Señal , Replicación Viral , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismo

RESUMEN

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 Joven

RESUMEN

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 Edad

RESUMEN

In the present study, we investigated the relationship between early life protein malnutrition-induced redox imbalance, and reduced glucose-stimulated insulin secretion. After weaning, male Wistar rats were submitted to a normal-protein-diet (17%-protein, NP) or to a low-protein-diet (6%-protein, LP) for 60 days. Pancreatic islets were isolated and hydrogen peroxide (H2 O2 ), oxidized (GSSG) and reduced (GSH) glutathione content, CuZn-superoxide dismutase (SOD1), glutathione peroxidase (GPx1) and catalase (CAT) gene expression, as well as enzymatic antioxidant activities were quantified. Islets that were pre-incubated with H2 O2 and/or N-acetylcysteine, were subsequently incubated with glucose for insulin secretion measurement. Protein malnutrition increased CAT mRNA content by 100%. LP group SOD1 and CAT activities were 50% increased and reduced, respectively. H2 O2 production was more than 50% increased whereas GSH/GSSG ratio was near 60% lower in LP group. Insulin secretion was, in most conditions, approximately 50% lower in LP rat islets. When islets were pre-incubated with H2 O2 (100 µM), and incubated with glucose (33 mM), LP rats showed significant decrease of insulin secretion. This effect was attenuated when LP islets were exposed to N-acetylcysteine.

Asunto(s)

Glucemia/metabolismo , Dieta con Restricción de Proteínas , Insulina/sangre , Islotes Pancreáticos/metabolismo , Estrés Oxidativo , Desnutrición Proteico-Calórica/metabolismo , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Antioxidantes/farmacología , Catalasa/genética , Catalasa/metabolismo , Modelos Animales de Enfermedad , Regulación Enzimológica de la Expresión Génica , Glutatión/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Masculino , Estado Nutricional , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Desnutrición Proteico-Calórica/sangre , Desnutrición Proteico-Calórica/genética , Desnutrición Proteico-Calórica/fisiopatología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Factores de Tiempo

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 Wistar

RESUMEN

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(•) 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 Señal

RESUMEN

Endurance exercise training as well as leucine supplementation modulates glucose homeostasis and protein turnover in mammals. Here, we analyze whether leucine supplementation alters the effects of endurance exercise on these parameters in healthy mice. Mice were distributed into sedentary (C) and exercise (T) groups. The exercise group performed a 12-week swimming protocol. Half of the C and T mice, designated as the CL and TL groups, were supplemented with leucine (1.5 % dissolved in the drinking water) throughout the experiment. As well known, endurance exercise training reduced body weight and the retroperitoneal fat pad, increased soleus mass, increased VO2max, decreased muscle proteolysis, and ameliorated peripheral insulin sensitivity. Leucine supplementation had no effect on any of these parameters and worsened glucose tolerance in both CL and TL mice. In the soleus muscle of the T group, AS-160(Thr-642) (AKT substrate of 160 kDa) and AMPK(Thr-172) (AMP-Activated Protein Kinase) phosphorylation was increased by exercise in both basal and insulin-stimulated conditions, but it was reduced in TL mice with insulin stimulation compared with the T group. Akt phosphorylation was not affected by exercise but was lower in the CL group compared with the other groups. Leucine supplementation increased mTOR phosphorylation at basal conditions, whereas exercise reduced it in the presence of insulin, despite no alterations in protein synthesis. In trained groups, the total FoxO3a protein content and the mRNA for the specific isoforms E2 and E3 ligases were reduced. In conclusion, leucine supplementation did not potentiate the effects of endurance training on protein turnover, and it also reduced its positive effects on glucose homeostasis.

Asunto(s)

Suplementos Dietéticos/análisis , Glucosa/metabolismo , Leucina/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Femenino , Homeostasis , Humanos , Insulina/metabolismo , Ratones , Músculo Esquelético/metabolismo , Resistencia Física , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Natación , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo

RESUMEN

Mutations in human Gli-similar (GLIS) 3 protein cause neonatal diabetes. The GLIS3 gene region has also been identified as a susceptibility risk locus for both type 1 and type 2 diabetes. GLIS3 plays a role in the generation of pancreatic beta cells and in insulin gene expression, but there is no information on the role of this gene on beta cell viability and/or susceptibility to immune- and metabolic-induced stress. GLIS3 knockdown (KD) in INS-1E cells, primary FACS-purified rat beta cells, and human islet cells decreased expression of MafA, Ins2, and Glut2 and inhibited glucose oxidation and insulin secretion, confirming the role of this transcription factor for the beta cell differentiated phenotype. GLIS3 KD increased beta cell apoptosis basally and sensitized the cells to death induced by pro-inflammatory cytokines (interleukin 1ß + interferon-γ) or palmitate, agents that may contribute to beta cell loss in respectively type 1 and 2 diabetes. The increased cell death was due to activation of the intrinsic (mitochondrial) pathway of apoptosis, as indicated by cytochrome c release to the cytosol, Bax translocation to the mitochondria and activation of caspases 9 and 3. Analysis of the pathways implicated in beta cell apoptosis following GLIS3 KD indicated modulation of alternative splicing of the pro-apoptotic BH3-only protein Bim, favouring expression of the pro-death variant BimS via inhibition of the splicing factor SRp55. KD of Bim abrogated the pro-apoptotic effect of GLIS3 loss of function alone or in combination with cytokines or palmitate. The present data suggest that altered expression of the candidate gene GLIS3 may contribute to both type 1 and 2 type diabetes by favouring beta cell apoptosis. This is mediated by alternative splicing of the pro-apoptotic protein Bim and exacerbated formation of the most pro-apoptotic variant BimS.

Asunto(s)

Proteínas Reguladoras de la Apoptosis/genética , Apoptosis/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genética , Anciano , Empalme Alternativo/genética , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Proteínas de Unión al ADN , Diabetes Mellitus Tipo 1/etiología , Diabetes Mellitus Tipo 2/etiología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Insulina/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Persona de Mediana Edad , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Proteínas Represoras , Transactivadores

RESUMEN

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 Señal

RESUMEN

Endurance training has been shown to increase pancreatic ß-cell function and mass. However, whether exercise modulates ß-cell growth and survival pathways signaling is not completely understood. This study investigated the effects of exercise on growth and apoptotic markers levels in rat pancreatic islets. Male Wistar rats were randomly assigned to 8-wk endurance training or to a sedentary control group. After that, pancreatic islets were isolated; gene expression and the total content and phosphorylation of several proteins related to growth and apoptotic pathways as well as the main antioxidant enzymes were determined by real-time polymerase chain reaction and Western blot analysis, respectively. Reactive oxygen species (ROS) production was measured by fluorescence. Endurance training increased the time to reach fatigue by 50%. Endurance training resulted in increased protein phosphorylation content of AKT (75%), AKT substrate (AS160; 100%), mTOR (60%), p70s6k (90%), and ERK1/2 (50%), compared with islets from control group. Catalase protein content was 50% higher, whereas ROS production was 49 and 77% lower in islets from trained rats under basal and stimulating glucose conditions, respectively. Bcl-2 mRNA and protein levels increased by 46 and 100%, respectively. Bax and cleaved caspase-3 protein contents were reduced by 25 and 50% in islets from trained rats, respectively. In conclusion, these results demonstrate that endurance training favors the ß-cell growth and survival by activating AKT and ERK1/2 pathways, enhancing antioxidant capacity, and reducing ROS production and apoptotic proteins content.

Asunto(s)

Células Secretoras de Insulina/fisiología , Islotes Pancreáticos/fisiología , Resistencia Física/fisiología , Transducción de Señal/fisiología , Animales , Antioxidantes/metabolismo , Apoptosis/fisiología , Peso Corporal , Fatiga/genética , Fatiga/metabolismo , Fatiga/fisiopatología , Expresión Génica , Glucosa/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Masculino , Oxidación-Reducción , Fosforilación , Condicionamiento Físico Animal , Resistencia Física/genética , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

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 Señal

RESUMEN

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.

Asunto(s)

Acetilcolina/metabolismo , Citocinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células Secretoras de Insulina/citología , Fragmentos de Péptidos/farmacología , Receptor Muscarínico M3/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carbacol/farmacología , Dominio Catalítico , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Citocinas/química , Técnicas de Silenciamiento del Gen , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Asociadas a Pancreatitis , Fragmentos de Péptidos/química , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Interferente Pequeño/genética , Ratas , Reproducibilidad de los Resultados , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Elemento de Respuesta al Suero/genética , Factor de Transcripción ReIA/deficiencia , Factor de Transcripción ReIA/genética , Regulación hacia Arriba/efectos de los fármacos