Exercício físico melhora a via de sinalização da insulina através da proteína ROCK-2 no músculo esquelético em ratos de meia-idade

O envelhecimento está associado com mudanças na composição corporal e na função metabólica. Dentre as alterações pode-se destacar o aumento da adiposidade corporal e redução da massa muscular esquelética, que é acompanhada pelo declínio na taxa metabólica. Além disso, com o envelhecimento ocorre redução na taxa de secreção de insulina e/ou na sensibilidade periférica a este hormônio, com alterações na glicemia. Tal prejuízo na homeostase glicêmica pode estar relacionado a defeitos na via de sinalização da insulina, induzida por biomoléculas secretadas pelos adipócitos ou atrelados a diminuição no conteúdo de proteínas com papel chave na transmissão intracelular do sinal da insulina em organismos envelhecidos. Por outro lado, o exercício físico aumenta a captação de glicose com efeitos sobre as proteínas envolvidas na via de sinalização da insulina em organismos jovens e idosos. No entanto, os efeitos moleculares do exercício físico na transdução do sinal da insulina ainda não são totalmente compreendidos, especialmente quando se trata de idades mais avançadas. Recentemente, uma nova proteína foi classificada com o papel de mediar a sinalização da insulina, potencializando seus efeitos. Esta proteína foi denominada Rho-quinase (ROCK), de ação tanto em tecidos periféricos (músculo esquelético, fígado e tecido adiposo) como também no hipotálamo. Sabe-se que a ROCK contribui em até 50% na ação da insulina no meio intracelular. Isto foi observado a partir de estudos com animais que tiveram a deleção, ou inibição da proteína ROCK, os quais desenvolveram resistência à insulina. Deste modo, se faz necessário à realização de mais estudos sobre esta nova proteína, visando evidenciar um melhor entendimento da ação dela na sinalização da insulina em organismos no processo de senescência. Sendo assim, o objetivo deste estudo foi investigar o efeito do envelhecimento sobre o conteúdo proteico de ROCK e sobre a via molecular de sinalização da insulina no músculo esquelético de ratos de meia-idade sedentários ou submetidos ao protocolo de exercício agudo de natação. Foram utilizados ratos Wistar distribuídos nos seguintes grupos: Jovem Sedentário (JSed): ratos com 5 meses sedentários (n=7); Meia-Idade Sedentário (MISed): ratos com 17 meses sedentários (n=7) e Meia-Idade Exercitado (MIExe): ratos com 17 meses submetidos ao exercício agudo de natação (n=7). Os animais foram submetidos ao teste de tolerância à insulina e submetidos ao protocolo de exercício de natação para extração do tecido muscular para posterior análise proteica. O protocolo de exercício físico agudo consistiu em duas sessões de 3 horas de nado sem carga separadas por 45 minutos de descanso. Foram mensuradas as proteínas ROCK-1, ROCK-2 e pAKT através da técnica de immunoblot. Os resultados obtidos demonstraram a instalação da resistência à insulina em animais de meia-idade sedentários. Este achado pode ter sido em parte, devido a diminuição do conteúdo proteico de ROCK-2 no músculo esquelético e diminuição da fosforilação da AKT. Em contrapartida, o grupo exercitado demonstrou melhora na sensibilidade à insulina acompanhado ao aumento de ROCK-2 e fosforilação da AKT após o protocolo de exercício agudo. Com isso, o exercício físico contribui para a homeostase glicêmica através do metabolismo da proteína ROCK em animais de meia-idade. Ademais, esses achados revelam um novo mecanismo no qual o exercício físico colabora com a captação de glicose no músculo esquelético de animais na meia-idade, prevenindo o agravamento da resistência à insulina em idososAging is associated with changes in body composition and metabolic function. Among the changes it may be highlighted the increase in body mass and reduction in skeletal muscle mass, which is accompanied by the decline in the metabolic rate. Furthermore, it is considered that with the aging there is a reduction in insulin secretion rate and / or in peripheral sensitivity to this hormone with changes in blood glucose. Such impaired glucose homeostasis may be related to the defects in insulin signaling pathway, induced by bio-molecules secreted by adipocytes or linked to the decrease in protein content with a key role in the transmission of intracellular insulin signal in aged organisms. Moreover, the exercise increases the glucose uptake with effects on the proteins involved in insulin signaling pathway in young and elderly organisms. However, the molecular effects of exercise in insulin signal transduction are not yet fully understood, especially when dealing with older ages. Recently, a new protein has been classified to the role of mediating the insulin signaling, enhancing their effects. This new protein was named as Rho kinase (ROCK), with action on both in peripheral tissues (skeletal muscle, liver and adipose tissue) as well as the hypothalamus. It is known that ROCK contributes up to 50% in insulin action intracellularly. This has been observed from studies with animals that had the deletion or inhibition of ROCK protein, which have developed resistance to insulin. Thus, it is necessary to carry out more studies on this new protein, in order to highlight a better understanding of its action in insulin signaling in aged organisms. Thus being, the aim of this study was to investigate the effect of aging over the ROCK protein content and over the molecular pathway of insulin signaling in skeletal muscle of sedentary middle-aged rats and submitted to the acute swimming exercise protocol. Wistar rats divided into the following groups were used: Young Sedentary (JSed): sedentary rats 5 months old (n = 7); Middle-Aged Sedentary (MISed): sedentary rats 17 months old (n = 7) and Middle-Aged exercised (MIExe): rats 17 months old and submitted to the acute swimming exercise (n = 7). The acute physical exercise protocol consists of two swimming sessions of three hours without charge separated by 45 minutes of rest. The animals were submitted to the insulin tolerance test or submitted to swimming exercise protocol for extraction of muscle tissue for subsequent protein analysis. There were measured the ROCK-1, ROCK-2, and pAKT proteins through the immunoblot technic. The obtained results showed the installation of insulin resistance in sedentary middle-aged animals. This finding was, in part, due to the decreased ROCK-2 protein content in skeletal muscle and the decrease of AKT phosphorylation. On the other hand, the exercised group showed an improvement in insulin sensitivity accompanied with the increase of ROCK-2 and pAKT after the acute exercise protocol. Thereby, the exercise contributes to the glucose homeostasis through the metabolism of ROCK protein in middle-aged animals. In addition, these findings reveal a new mechanism where the exercise collaborates with the glucose uptake in skeletal muscle of middle-aged animals, preventing from worsening the insulin resistance in the elderl

The role of rock protein on glucose uptake in the skeletal muscle of exercised rodents during aging process

Orientadores: José Rodrigo Pauli, Leandro Pereira de MouraDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências AplicadasResumo: A senescência está associada a diversos distúrbios metabólicos, incluindo a resistência à insulina (RI). O exercício físico tem um papel importante como agente não farmacológico na prevenção e tratamento da RI e suas complicações associadas. A Rho-quinase (Rock) tem sido postulada como uma proteína importante que age diretamente na fosforilação do IRS1, contribuindo com até 50% da captação de glicose em músculo esquelético. Portanto, o objetivo deste estudo foi avaliar o papel do exercício físico sobre a atividade da proteína Rock e via de sinalização da insulina em ratos jovens, de meia-idade e idosos. Primeiramente, o papel da proteína Rock foi confirmado em células musculares C2C12 através da sua inibição (Y-27632) e avaliação da via de sinalização da insulina. Em seguida, ratos Fischer 344 e ratos Wistar foram distribuídos em três grupos: Jovem Sedentário (5 meses), Meia Idade (17 meses) e Idosos (26 meses). Os animais de meia-idade foram submetidos ao treinamento físico de natação de curto período (7 dias), no qual cada sessão de exercício foi composta por 4 séries de 30 minutos, com pausa de 10 minutos entre cada série. Sendo que na primeira série os ratos realizaram o exercício de natação sem carga adicional e nas últimas três séries ocorreu a adição de carga acoplada a cauda, que foi equivalente a 1.5% do peso corporal. Já os animais idosos foram submetidos ao protocolo de treinamento físico em esteira por 5 dias, sendo cada dia composto por 2 sessões de 30 minutos (com intervalo de 10 minutos). Os animais foram submetidos ao teste de tolerância à insulina e ao teste de tolerância à glicose. Dezesseis horas após a última sessão de exercício físico os animais foram eutanasiados para avaliar a via de sinalização da insulina e o metabolismo da proteína Rock através das técnicas de imunoprecipitação, immunblot e ensaio de atividade da Rock em tecido muscular e adiposo branco. Nos resultados, ratos Wistar e Fischer de meia-idade demonstraram prejuízos em proteínas relacionadas com o metabolismo da Rock e da via de sinalização da insulina no músculo esquelético. Porém, apenas os ratos Wistar apresentaram um aumento no conteúdo de Rock no tecido adiposo branco (onde essa proteína apresenta um papel negativo), assim como um quadro de resistência à insulina. Em seguida, ao submeter animais jovens ao exercício físico, foi observado um aumento da atividade da Rock, bem como aumento da fosforilação de proteínas envolvidas com a via de sinalização da insulina, e consequentemente o aumento da translocação de GLUT4 para a membrana celular. Por outro lado, a administração do inibidor farmacológico da Rock (Y-27632), suprimiu os efeitos do exercício físico sobre a via de sinalização da insulina e captação de glicose no músculo esquelético. Por fim, em ratos Fischer de meia-idade e idosos não foi observado um grande prejuízo referente ao metabolismo da Rock e via da insulina no músculo sóleo, no entanto, o exercício físico foi capaz de aumentar o conteúdo de Rock2 e a fosforilação de proteínas da via de sinalização da insulina. A luz desses achados, a proteína Rock se mostrou relevante para a sinalização da insulina em roedores. Sendo que o envelhecimento está associado com declínio e o exercício físico com aumento na atividade da Rock no músculo esqeulético e consequentemente na homeostase da glicose do organismo de ratos Wistar e Fischer 344Abstract: The aging process is associated with different metabolic disorders, including the insulin resistance (IR). The physical exercise presents an important non-pharmacological role in the IR treatment and associated health complications. The Rho-kinase (Rock) protein is proposed as an important molecule involved in the IRS1 phosphorylation contributing with ~50% of the skeletal muscle glucose uptake in the skeletal muscle. Thus, the aim of this study was to evaluate the role of physical exercise in the Rock activity and insulin-signaling pathway in young, middle-aged and aged rats. Firstly, the role of Rock protein was confirmed in C2C12 myotube cells through Rock inhibition (Y-27632) and insulin-signaling pathway evaluation. Then, Fischer 344 and Wistar rats were distributed in three groups: Young Sedentary (5 months), Middle-aged (17 months), and Aged (26 months). The middle-aged groups were submitted to swimming physical training protocol during 7 days, composed by 4 sessions of 30 minutes swimming separated by 10 minutes of rest. After the first session, it was utilized the load of 1,5% of the body weight in the last three sessions. The aged animals were submitted to treadmill physical training protocol during 5 days, composed by 2 sessions of 30 minutes separated by 10 minutes. The animals were submitted to insulin tolerance test and glucose tolerance test. Sixteen hours after the physical exercise the animals were euthanized to evaluate the insulin-signaling pathway and proteins involved with Rock metabolism through immunoprecipitation, immunoblot and Rock activity assay in the skeletal muscle and white adipose tissue. In the results, middle-aged Fischer and Wistar demonstrated impairments in the Rock metabolism and insulin-signaling pathway in the skeletal muscle; however, only middle-aged Wistar rats presented increased Rock content in the white adipose tissue (where this protein shows a negative role), and an insulin resistant state. In the next step, we found the role of physical exercise to increase the Rock activity in young rats, and increase the phosphorylation in proteins involved with insulin pathway, also increasing the GLUT4 translocation to plasmatic membrane. Otherwise, the inhibition of Rock activity (Y-27632), suppressed the effect from the physical exercise to improve the insulin sensitivity and the insulin-signaling pathway to increase the glucose uptake in the skeletal muscle. Finally, both in the middle-aged and aged Fischer rats, it was not found great impairments related to Rock metabolism and insulin-signaling pathway in the soleus muscle; however, the physical exercise was able to increase Rock2 content and phosphorylation of insulin-signaling pathway proteins. In summary, the Rock protein seems to show an important participation in the insulin sensitivity improvement after the physical exercise, also, the activity of this protein is impaired in the middle-aged condition, and the physical exercise is one effective intervention to improve its activity in the skeletal muscle, and consequently contributing to the glucose homeostasis in Wistar and Fischer 344 ratsMestradoMetabolismo e Biologia MolecularMestre em Ciências da Nutrição e do Esporte e Metabolismo2015/26000-2FAPES

Regulation of hepatic TRB3/Akt interaction induced by physical exercise and its effect on the hepatic glucose production in an insulin resistance state

To maintain euglycemia in healthy organisms, hepatic glucose production is increased during fasting and decreased during the postprandial period. This whole process is supported by insulin levels. These responses are associated with the insulin signaling pathway and the reduction in the activity of key gluconeogenic enzymes, resulting in a decrease of hepatic glucose production. On the other hand, defects in the liver insulin signaling pathway might promote inadequate suppression of gluconeogenesis, leading to hyperglycemia during fasting and after meals. The hepatocyte nuclear factor 4, the transcription cofactor PGC1-α, and the transcription factor Foxo1 have fundamental roles in regulating gluconeogenesis. The loss of insulin action is associated with the production of pro-inflammatory biomolecules in obesity conditions. Among the molecular mechanisms involved, we emphasize in this review the participation of TRB3 protein (a mammalian homolog of Drosophila tribbles), which is able to inhibit Akt activity and, thereby, maintain Foxo1 activity in the nucleus of hepatocytes, inducing hyperglycemia. In contrast, physical exercise has been shown as an important tool to reduce insulin resistance in the liver by reducing the inflammatory process, including the inhibition of TRB3 and, therefore, suppressing gluconeogenesis. The understanding of these new mechanisms by which physical exercise regulates glucose homeostasis has critical importance for the understanding and prevention of diabetes

Immune‐endocrine responses and physical performance of master athletes during the sports season

The purpose of this study was to investigate the impact of a training season (approximately 7 months) on physiological and salivary immune‐endocrine markers in master athletes. Nine male master athletes were evaluated at the beginning of the season (M1) and a week after the main official competition at the end of the sports season (M2). The controlled variables included Maximal oxygen consumption, anthropometric, physiological, and salivary immune‐endocrine markers. Master athletes presented a reduced percentage of fat mass and increased lean body mass at the end of the season. VO2max values were similar at M1 and M2, while the maximal heart rate and lactate were lower at M2. No differences were observed in Immunoglobulin A and cortisol levels between moments, whereas testosterone levels and the testosterone/cortisol ratio were significantly lower at the end of the season. The results suggest that maintaining regular training throughout life has positive effects on body composition and improves physiological fitness. However, care should be taken to avoid fatigue as indicated by lower testosterone levels at the end of the season120455515557CAPES - Coordenação de Aperfeiçoamento de Pessoal e Nível Superior13642/13‐8; 1417/13‐

Acute physical exercise increases leptin‐induced hypothalamic extracellular signal‐regulated kinase1/2 phosphorylation and thermogenesis of obese mice

The obesity is a result of energy imbalance and the increase in thermogenesis seems an interesting alternative for the treatment of this disease. The mechanism of energy expenditure through thermogenesis is tightly articulated in the hypothalamus by leptin. The hypothalamic extracellular signal‐regulated kinase‐1/2 (ERK1/2) is a key mediator of the thermoregulatory effect of leptin and mediates the sympathetic signal to the brown adipose tissue (BAT). In this context, physical exercise is one of the main interventions for the treatment of obesity. Thus, this study aimed to verify the effects of acute physical exercise on leptin‐induced hypothalamic ERK1/2 phosphorylation and thermogenesis in obese mice. Here we showed that acute physical exercise reduced the fasting glucose of obese mice and increased leptin‐induced hypothalamic p‐ERK1/2 and uncoupling protein 1 (UCP1) content in BAT ( P < 0.05). These molecular changes are accompanied by an increased oxygen uptake (VO 2) and heat production in obese exercised mice ( P < 0.05). The increased energy expenditure in the obese exercised animals occurred independently of changes in spontaneous activity. Thus, this is the first study demonstrating that acute physical exercise can increase leptin‐induced hypothalamic ERK1/2 phosphorylation and energy expenditure of obese mice1201697704CNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulo306535/2017‐32016/18488‐8OCR

Physical training reverses changes in hepatic mitochondrial diameter of Alloxan-induced diabetic rats

<div><p>ABSTRACT Objective To investigate the effects of physical training on metabolic and morphological parameters of diabetic rats. Methods Wistar rats were randomized into four groups: sedentary control, trained control, sedentary diabetic and trained diabetic. Diabetes mellitus was induced by Alloxan (35mg/kg) administration for sedentary diabetic and Trained Diabetic Groups. The exercise protocol consisted of swimming with a load of 2.5% of body weight for 60 minutes per day (5 days per week) for the trained control and Trained Diabetic Groups, during 6 weeks. At the end of the experiment, the rats were sacrificed and blood was collected for determinations of serum glucose, insulin, albumin and total protein. Liver samples were extracted for measurements of glycogen, protein, DNA and mitochondrial diameter determination. Results The sedentary diabetic animals presented decreased body weight, blood insulin, and hepatic glycogen, as well as increased glycemia and mitochondrial diameter. The physical training protocol in diabetic animals was efficient to recovery body weight and liver glycogen, and to decrease the hepatic mitochondrial diameter. Conclusion Physical training ameliorated hepatic metabolism and promoted important morphologic adaptations as mitochondrial diameter in liver of the diabetic rats.</p></div