RESUMO
Cell-to-cell interactions mediated by intercellular junctions (IJs) are crucial for beta-cell functioning and proper insulin secretion, however, their role in type-2 diabetes is still unclear. This work aimed to evaluate the cellular distribution and expression of proteins associated with adherens (AJs) and gap junctions (GJs) in pancreatic islets of C57BL6 mice fed a high-fat (HF) diet. The administration of HF diet for 30 days induced an increase in body weight, post-prandial glycemia, insulinemia, glucose intolerance, and moderate insulin resistance associated with mild perturbations in insulin secretion. The intercellular content of the AJ-associated proteins (namely, E-, N-cadherins, and α-, ß-catenins) was significantly higher in islet cells of HF-fed mice. Inversely, the gap junctional content of Cx36 was significantly decreased, as revealed by immunofluorescence, which was paralleled by a reduction in the frequency of calcium oscillations in islets of prediabetic mice. In conclusion, the endocrine pancreas displays significant changes in the content of several junctional proteins at the cell-cell contact region following short-term HF diet administration, indicating that IJs may be involved in the adaptive response of beta cells seen during this state.
Assuntos
Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Moléculas de Adesão Celular/metabolismo , Dieta Hiperlipídica/efeitos adversos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Mitocôndrias/metabolismo , Condicionamento Físico Animal/fisiologia , Esforço Físico/fisiologia , Animais , Metilação de DNA , Epigênese Genética/genética , Feminino , Masculino , Camundongos , Mitocôndrias/fisiologia , Músculo Esquelético/fisiologia , Correpressor 1 de Receptor Nuclear/metabolismo , Consumo de Oxigênio/fisiologia , Herança Paterna/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/fisiologia , Condicionamento Físico Animal/métodos , RNA Mensageiro/genéticaRESUMO
BACKGROUND/AIMS: Epigenetic regulation is considered the main molecular mechanism underlying the developmental origin of health and disease's (DOHAD) hypothesis. Previous studies that have investigated the role of paternal exercise on the metabolic health of the offspring did not control for the amount and intensity of the training or possible effects of adaptation to exercise and produced conflicting results regarding the benefits of parental exercise to the next generation. We employed a precisely regulated exercise regimen to study the transgenerational inheritance of improved metabolic health. METHODS: We subjected male mice to a well-controlled exercise -training program to investigate the effects of paternal exercise on glucose tolerance and insulin sensitivity in their adult progeny. To investigate the molecular mechanisms of epigenetic inheritance, we determined chromatin markers in the skeletal muscle of the offspring and the paternal sperm. RESULTS: Offspring of trained male mice exhibited improved glucose homeostasis and insulin sensitivity. Paternal exercise modulated the DNA methylation profile of PI3Kca and the imprinted H19/Igf2 locus at specific differentially methylated regions (DMRs) in the skeletal muscle of the offspring, which affected their gene expression. Remarkably, a similar DNA methylation profile at the PI3Kca, H19, and Igf2 genes was present in the progenitor sperm indicating that exercise-induced epigenetic changes that occurred during germ cell development contributed to transgenerational transmission. CONCLUSION: Paternal exercise might be considered as a strategy that could promote metabolic health in the offspring as the benefits can be inherited transgenerationally.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Metilação de DNA , Resistência à Insulina/genética , Fator de Crescimento Insulin-Like II/genética , Condicionamento Físico Animal/métodos , RNA Longo não Codificante/genética , Espermatozoides/química , Animais , Epigênese Genética , Feminino , Teste de Tolerância a Glucose , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Modelos Animais , Consumo de Oxigênio , Herança Paterna , Análise de Sequência de DNA , Espermatozoides/metabolismoRESUMO
Abstract Spontaneous physical activity (SPA) consists of all daily living activities other than volitional exercise (e.g. sports and fitness-related activities). SPA is an important component of energy expenditure and may protect from overweight and obesity. Little is known about the biological regulation of SPA, but animal researchhas contributedsignificantly to expand our knowledge in this field. Studies in rodents have shown that SPA is influenced by nutrients and volitional exercise. High-fat diet seems to decrease SPA, which contributes to weigh gain. Volitional exercisemayalso reduce SPA, helping to explain the commonly reported low efficiency of exercise to cause weight loss, and highlighting the need to finda volume/intensity of exercise to maximize total daily energy expenditure. Animal studieshave also allowed for the identification of some brain areas and chemical mediatorsinvolved in SPA regulation. These discoveries could enable the development of new therapeutics aiming to enhance SPA.(AU)
Assuntos
Humanos , Animais , Atividades Cotidianas , Metabolismo Energético/fisiologia , Comportamento SedentárioRESUMO
INTRODUCTION: Endurance training improves peripheral insulin sensitivity in the liver and the skeletal muscle, but the mechanism for this effect is poorly understood. Recently, it was proposed that insulin clearance plays a major role in both glucose homeostasis and insulin sensitivity. Therefore, our goal was to determine the mechanism by which endurance training improves insulin sensitivity and how it regulates insulin clearance in mice. METHODS: Mice were treadmill-trained for 4 weeks at 70-80% of maximal oxygen consumption (VO2 max) for 60 min, 5 days a week. The glucose tolerance and the insulin resistance were determined using an IPGTT and an IPITT, respectively, and the insulin decay rate was calculated from the insulin clearance. Protein expression and phosphorylation in the liver and the skeletal muscle were ascertained by Western blot. RESULTS: Trained mice exhibited an increased VO2 max, time to exhaustion, glucose tolerance and insulin sensitivity. They had smaller fat pads and lower plasma concentrations of insulin and glucose. Endurance training inhibited insulin clearance and reduced expression of IDE in the liver, while also inhibiting insulin secretion by pancreatic islets. There was increased phosphorylation of both the canonical (IR-AKT) and the non-canonical (CaMKII-AMPK-ACC) insulin pathways in the liver of trained mice, whereas only the CaMKII-AMPK pathway was increased in the skeletal muscle. CONCLUSION: Endurance training improved glucose homeostasis not only by increasing peripheral insulin sensitivity but also by decreasing insulin clearance and reducing IDE expression in the liver.
Assuntos
Resistência à Insulina , Insulina/sangue , Insulisina/metabolismo , Esforço Físico , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Glicemia/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Insulina/metabolismo , Insulisina/genética , Ilhotas Pancreáticas/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Músculo Esquelético/metabolismo , Consumo de Oxigênio , Receptor de Insulina/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Suplementos Nutricionais/análise , Glucose/metabolismo , Leucina/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Feminino , Homeostase , Humanos , Insulina/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Resistência Física , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Natação , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Isolated islets from low-protein (LP) diet rats showed decreased insulin secretion in response to glucose and carbachol (Cch). Taurine (TAU) increases insulin secretion in rodent islets with a positive effect upon the cholinergic pathway. Here, we investigated the effect of TAU administration upon glucose tolerance and insulin release in rats fed on a normal protein diet (17%) without (NP) or with 2.5% of TAU in their drinking water (NPT), and LP diet fed rats (6%) without (LP) or with TAU (LPT). Glucose tolerance was found to be higher in LP, compared to NP rats. However, plasma glucose levels, during ipGTT, in LPT rats were similar to those of controls. Isolated islets from LP rats secreted less insulin in response to increasing glucose concentrations (2.8-22.2 mmol/L) and to 100 µmol/L Cch. This lower secretion was accompanied by a reduction in Cch-induced internal Ca(2+) mobilization. TAU supplementation prevents these alterations, as judged by the higher secretion induced by glucose or Cch in LPT islets. In addition, Ach-M3R, syntaxin 1 and synaptosomal associated protein of 25 kDa protein expressions in LP were lower than in NP islets. The expressions of these proteins in LPT were normalized. Finally, the sarcoendoplasmatic reticulum Ca(2+)-ATPase 3 protein expression was higher in LPT and NPT, compared with controls. In conclusion, TAU supplementation to LP rats prevented alterations in glucose tolerance as well as in insulin secretion from isolated islets. The latter effect involves the normalization of the cholinergic pathway, associated with the preservation of exocytotic proteins.
Assuntos
Dieta com Restrição de Proteínas , Suplementos Nutricionais , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Taurina/farmacologia , Animais , Western Blotting , Carbacol/administração & dosagem , Regulação da Expressão Gênica , Glucose/administração & dosagem , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/genética , Sintaxina 1/metabolismo , Taurina/sangueRESUMO
OBJECTIVES: Reactive oxygen species (ROS) are involved in many physiological and pathological processes. In the present study, we analysed whether the synthetic glucocorticoid dexamethasone induces oxidative stress in cultured pancreatic islets and whether the effects of dexamethasone on insulin secretion, gene expression, and viability can be counteracted by concomitant incubation with N-acetylcysteine (NAC). METHODS: ROS production was measured by dichlorofluorescein (DCFH-DA) assay, insulin secretion by radioimmunoassay, intracellular calcium dynamics by fura-2-based fluorescence, gene expression by real-time polymerase chain reaction analyses and cell viability by the MTS assay. RESULTS: Dexamethasone (Dexa) increased ROS production and decreased glucose-stimulated insulin secretion after 72 hours incubation. Intracellular ROS levels were decreased and the insulin secretion capacity was recovered by concomitant treatment with Dexa+NAC. The total insulin content and intracellular Ca2+ levels were not modulated in either Dexa or Dexa+NAC groups. There was a decrease in the NAD(P)H production, used as an indicator of viability, after dexamethasone treatment. Concomitant incubation with NAC returned viability to control levels. Dexa also decreased synaptotagmin VII (SYT VII) gene expression. In contrast, the Dexa+NAC group demonstrated an increased expression of SYT VII compared to controls. Surprisingly, treatment with NAC decreased the gene expression of the antioxidant enzyme copper zinc superoxide dismutase soluble. DISCUSSION: Our results indicate that dexamethasone increases ROS production, decreases viability, and impairs insulin secretion in pancreatic rat islets. These effects can be counteracted by NAC, which not only decreases ROS levels but also modulates the expression of genes involved in the secretory pathway and those coding for antioxidant enzymes.
Assuntos
Acetilcisteína/farmacologia , Dexametasona/antagonistas & inibidores , Glucocorticoides/antagonistas & inibidores , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dexametasona/toxicidade , Sequestradores de Radicais Livres/farmacologia , Radicais Livres/metabolismo , Glucocorticoides/toxicidade , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Sinaptotagminas/efeitos dos fármacos , Sinaptotagminas/metabolismoRESUMO
Our group previously demonstrated that CNTF protects pancreatic islets against apoptosis induced by IL1ß. In addition, it is known that AMPK knockout protects beta cells from IL1ß-mediated apoptosis, however how AMPK activation leads to apoptosis remains unknown. The present study was designed to investigate the possible role of AMPK pathway modulation in CNTF protective effects against apoptosis induced by IL1ß or Alloxan and how AMPK activation leads to beta cells apoptosis. First, we observed that apoptosis of MIN6 cells, induced by Alloxan as well as IL-1ß, requires activation of the AMPK pathway, and also that CNTF protective effects are dependent on downregulation of AMPK. In addition, we found that Alloxan induces AMPK differently from IL1ß, as Alloxan acts mainly through CaMKII while IL1ß acts through LKB1 phosphorylation. Meanwhile, CNTF by itself inhibited the AMPK pathway and protected against AMPK activation induced by Alloxan or IL1ß via downregulation of CaMKII. Finally, AMPK-dependent MIN6 cell apoptosis, induced by IL1ß or Alloxan, required increased iNOS expression, an effect that was reversed by CNTF downregulation of AMPK pathway and iNOS expression. In conclusion, IL1ß upregulates the LKB1-AMPK-INOS pathway, while Alloxan acts through CaMKII-AMPK-INOS, both ultimately leading to beta cell death. In this context, CNTF protects beta cells against apoptosis, induced by either IL1ß or Alloxan, through downregulation of the CaMKII-AMPK-INOS pathway.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aloxano/farmacologia , Apoptose , Fator Neurotrófico Ciliar/farmacologia , Interleucina-1beta/farmacologia , Acetil-CoA Carboxilase/metabolismo , Animais , Western Blotting , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Fragmentação do DNA , Regulação para Baixo , Técnicas de Silenciamento de Genes , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , TransfecçãoRESUMO
Alterations in food intake such as caloric restriction modulate the expression of SIRT1 and SIRT4 proteins that are involved in pancreatic ß-cell function. Here, we search for a possible relationship between insulin secretion and the expression of SIRT1, SIRT4, PKC and PKA in islets from adult rats submitted to CR for 21 days. Rats were fed with an isocaloric diet (CTL) or received 60% (CR) of the food ingested by CTL. The dose-response curve of insulin secretion to glucose was shifted to the right in the CR compared with CTL islets (EC(50) of 15.1±0.17 and 10.5±0.11 mmol/L glucose). Insulin release by the depolarizing agents arginine and KCl was reduced in CR compared with CTL islets. Total islet insulin content and glucose oxidation were also reduced in CR islets. Leucine-stimulated secretion was similar in both groups, slightly reduced in CR islets stimulated by leucine plus glutamine but higher in CR islets stimulated by ketoisocaproate (KIC). Insulin secretion was also higher in CR islets stimulated by carbachol, compared with CTL islets. No differences in the rise of cytosolic Ca(2+) concentrations stimulated by either glucose or KCl were observed between groups of islets. Finally, SIRT1, but not SIRT4, protein expression was lower in CR compared with CTL islets, whereas no differences in the expression of PKC and PKA proteins were observed. In conclusion, the lower insulin secretion in islets from CR rats was, at least in part, due to an imbalance between the expression of SIRT1 and SIRT4.
Assuntos
Restrição Calórica , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Sirtuína 1/genética , Animais , Glicemia/metabolismo , Expressão Gênica , Secreção de Insulina , Masculino , Oxirredução , Ratos , Ratos Wistar , Sirtuína 1/metabolismoRESUMO
Taurine (TAU) supplementation increases insulin secretion in response to high glucose concentrations in rodent islets. This effect is probably due to an increase in Ca2+ handling by the islet cells. Here, we investigated the possible involvement of the cholinergic/phospholipase C (PLC) and protein kinase (PK) A pathways in this process. Adult mice were fed with 2% TAU in drinking water for 30 d. The mice were killed and pancreatic islets isolated by the collagenase method. Islets from TAU-supplemented mice showed higher insulin secretion in the presence of 8.3 mm-glucose, 100 µm-carbachol (Cch) and 1 mm-3-isobutyl-1-methyl-xanthine (IBMX), respectively. The increase in insulin secretion in response to Cch in TAU islets was accompanied by a higher intracellular Ca2+ mobilisation and PLCß2 protein expression. The Ca2+ uptake was higher in TAU islets in the presence of 8.3 mm-glucose, but similar when the islets were challenged by glucose plus IBMX. TAU islets also showed an increase in the expression of PKAα protein. This protein may play a role in cation accumulation, since the amount of Ca2+ in these islets was significantly reduced by the PKA inhibitors: N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89) and PK inhibitor-(6-22)-amide (PKI). In conclusion, TAU supplementation increases insulin secretion in response to glucose, favouring both influx and internal mobilisation of Ca2+, and these effects seem to involve the activation of both PLC-inositol-1,4,5-trisphosphate and cAMP-PKA pathways.
Assuntos
Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Fosfolipase C beta/metabolismo , Taurina/administração & dosagem , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Carbacol/farmacologia , Células Cultivadas , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Citoplasma , Suplementos Nutricionais , Secreção de Insulina , Camundongos , Ésteres de Forbol/farmacologia , Fosfolipase C beta/genética , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Taurina/farmacologiaRESUMO
Pancreatic beta-cells and skeletal muscle act in a synergic way in the control of systemic glucose homeostasis. Several pyruvate-dependent and -independent shuttles enhance tricarboxylic acid cycle intermediate (TACI) anaplerosis and increase beta-cell ATP:ADP ratio, triggering insulin exocytotic mechanisms. In addition, mitochondrial TACI cataplerosis gives rise to the so-called metabolic coupling factors, which are also related to insulin release. Peripheral insulin resistance seems to be related to skeletal muscle fatty acid (FA) accumulation and oxidation imbalance. In this sense, exercise has been shown to enhance skeletal muscle TACI anaplerosis, increasing FA oxidation and by this manner restores insulin sensitivity. Protein malnutrition reduces beta-cell insulin synthesis, release and peripheral sensitivity. Despite little available data concerning mitochondrial metabolism under protein malnutrition, evidence points towards reduced beta-cell and skeletal muscle mitochondrial capacity. The observed decrease in insulin synthesis and release may reflect reduced anaplerotic and cataplerotic capacity. Furthermore, insulin release is tightly coupled to ATP:ADP rise which in turn is related to TACI anaplerosis. The effect of protein malnutrition upon peripheral insulin resistance is time-dependent and directly related to FA oxidation capacity. In contrast to beta-cells, TACI anaplerosis and cataplerosis pathways in skeletal muscle seem to control FA oxidation and regulate insulin resistance.
Assuntos
Proteínas Alimentares , Resistência à Insulina , Insulina/metabolismo , Desnutrição/fisiopatologia , Músculo Esquelético/metabolismo , HumanosRESUMO
The present study was designed to determine the exercise intensity equivalent to the metabolic aerobic/anaerobic transition of alloxan diabetic rats, through lactate minimum test (LMT), and to evaluate the effects of swimming exercise at this intensity (LM) on the glucose and protein metabolism of these animals. Adult male Wistar rats received alloxan (SD, alloxan-injected rats that remained sedentary) intravenously (30 mg kg(-1) body weight) for diabetes induction. As controls (SC, vehicle-injected rats that remained sedentary), vehicle-injected rats were utilized. Two weeks later, the animals were submitted to oral glucose tolerance test (oGTT) and LMT. After the tests, some of the animals were submitted to swimming exercise training [TC (vehicle-injected rats that performed a 6-week exercise program) and TD (alloxan-injected rats that performed a 6-week exercise program)] for 1 h day(-1), 5 days week(-1), with an overload equivalent to LM determined by LMT, for 6 weeks. At the end of the experiment, the animals were submitted to a second LMT and oGTT, and blood and skeletal muscle assessments (protein synthesis and degradation in the isolated soleus muscle) were made. The overload equivalent to LM at the beginning of the experiment was lower in the SD group than in the SC group. After training, the overload equivalent to LM was higher in the TC and TD groups than in the SC and SD groups. The blood glucose of TD rats during oGTT was lower than that of SD rats. Protein degradation was higher in the SD group than in other groups. We conclude that LMT was sensitive to metabolic and physiologic alterations caused by uncontrolled diabetes. Training at LM intensity improved aerobic condition and the glucose and protein metabolism of alloxan diabetic rats.