Long-term effects of moderate physical exercise during early childhood on insulin sensitivity in rats during adulthood

É de grande importância a investigação dos efeitos do exercício para a saúde metabólica quando realizado no início da vida, e quais seriam as respostas durante a fase adulta. O objetivo desse estudo é analisar os efeitos do exercício físico de intensidade moderada durante a infância na adiposidade e sensibilidade à insulina de ratos durante a fase adulta. Vinte ratos Wistar desmamados de 28 dias de vida foram divididos em grupos Controle e Treinado. O grupo Controle permaneceu sedentário, enquanto o grupo Treinado realizou exercício de natação a 80% do limiar anaeróbio (determinado pelo teste de lactato mínimo) por 1hora/dia, 5dias/semana do 28º até o 90º dia de vida. No fim do experimento, a massa corporal, adiposidade, sensibilidade à insulina, glicemia, insulinemia, insulina pancreática, atividade lipogênica do tecido adiposo mesentérico, atividade lipogênica e estoques de glicogênio no músculo gastrocnêmio e captação de glicose pelo músculo sóleo foram analizadas. O grupo Treinado apresentou menor ganho de massa corporal, acúmulo de gordura, síntese de lipídios, glicemia, insulinemia e insulina pancreática. Kitt, HOMA-IR, estoques de glicogênio no gastrocnêmio e captação de glicose pelo sóleo foram maiores nesse grupo. Portanto, concluímos que o exercício físico moderado realizado durante a infância pode contribuir para a redução da adiposidade corporal e resistência à insulina durante a fase adulta em ratos Wistar.


Introduction
Long-term effects of moderate physical exercise during early childhood on insulin sensitivity in rats during adulthood PALAVRAS-CHAVE: Moderate Exercise; Weaned Rats; Insulin Resistance; Overweight; Obesity; Diabetes.
Th e prevalence of overweight and obesity has been growing over the years, and it is estimated that in 2030 more than half the world's population will be obese 1 . Obesity is considered one of the biggest public health problems in the world, having as main feature high levels of fasting insulin in response to glucose consumption, phenomenon known as insulin resistance (IR) 2 . Th erefore, a number of studies demonstrate that its development is infl uenced by behavioral, genetic and environmental factors 3 .
In this context, regular physical exercise is considered an important strategy for the treatment and prevention of obesity and IR, promoting reduction in fat mass and increased energy expenditure 4 . Th ese benefi ts provided by the exercise occur by its ability to modulate various proteins involved in the insulin signaling pathway in key tissues of glucose homeostasis control, such as skeletal muscle, liver and hypothalamus 5 .
Although the beginning of physical exercise is proposed from youth 6 , it is still unclear what would be the metabolic benefi ts in adulthood from this practice. In this sense, few studies using rodents have shown that voluntary practice exercise after weaning promotes a number of benefi ts related to metabolism, such as lower levels of circulating leptin 7 , reduction of body fat and increase in the amount of brown adipose tissue 8 . Also, the increase of leptin sensitivity and resistance to the development of obesity can remain for weeks, even after the cessation of exercise practice 9 . However, these studies did not investigate the eff ects of systematic practice exercises, controlling the training variables such as individual volume and intensity of eff ort.
In this sense, it is of great importance to investigate the eff ects of exercise for metabolic health when it is performed in early life, and what the adulthood response would be. Studies on this fi eld can provide Method Animals a better knowledge about the exercise benefi ts in early life and encourage the controlled training programs participation. Th is strategy can be an important way to combat obesity and IR, since it is proposed that physically active subjects at early stages of life tend to maintain that level of activity when adults 10 . Th erefore, the present study aimed to analyze the long-term eff ects of a moderate intensity physical exercise protocol during childhood on the adipose tissue and insulin sensitivity of rats during adulthood.
Twenty weaned Wistar rats were used at 21 days of age at the beginning of the experiment. Th ey came from central vivarium of the State University of São Paulo (UNESP, Botucatu/SP -Brazil). Th e animals were allocated in polyethylene cages, measuring 37 x 31 x 16cm (fi ve mice per cage) and maintained at a temperature of 21°C. Th e rats remained in a room with a photoperiod of 12h light/dark and were fed, ad libitum, with balanced standard Purina food and water. From 28 to 90 days of age, the animals were divided into two groups: Control (n=10) that remained sedentary throughout the experiment and Trained (n=10) that were subject to a swimming training protocol over the entire experiment. In order to establish an initial homogeneity between groups, diff erent variables such as weight, glycemia, insulinemia, triglycerides concentrations, and lipogenic activity were measured at the beginning of the experiment (28 days). All animal experiments were performed in accordance with the Brazilian law for scientifi c use of animals (law 11.794, October 8, 2008). Th e protocols were approved by the Ethics Committee on Animal Use (CEUA) from the Institute of Biosciences of UNESP -Rio Claro (Protocol: 4573/2010).

Training protocol
From the weaning age to adulthood (28 to 90 days of age), the trained animals were submitted to swimming exercise in the same controlled individual tanks 1 hour per day, fi ve days per week, supporting a overload of lead tied to the thorax, equivalent to 80% of individual AT, previously identifi ed by the result of LMT.

Adaptation to the water environment
All trained animals were adapted to the water environment that consisted of keeping the animals in shallow water for 3 days for 10, 20 and 30 min/day respectively at a temperature of 31±1°C to minimize the stress experienced.

Determination of anaerobic threshold through lactate minimum test
Th e moderate physical exercise was determined according to Moura et al. 11 . In summary, the lactate minimum test (LMT) was performed at 28, 50 and 70 days of age for determination of the anaerobic threshold in the trained animals, as proposed for humans 12 and modifi ed for rats 11 . For a better load control, this test was performed at diff erent times throughout the experiment (28, 50 and 70 days old). For the hyperlactacidemia induction, the animals were placed individually in tanks (100x80x80cm) containing water at 31±1°C, supporting overloads according to body weight (20% at 28 days, 13% at 50 and 70 days old) and swam for 30 seconds. After this sub-maximal eff ort, the rats rested for 30 seconds. After that, they were then allowed to swim again with the same overloads until exhaustion. After a resting period (12min at 28 days of age and 9min at 50 and 70 days of age), blood samples were collected by cutting the end of the tail to determine lactate concentration, and the animals began a swimming exercise with intensities progressively higher 11 . Th e determination of blood lactate concentrations was performed by the enzymatic method 13 .

Insulin tolerance test (ITT)
At the beginning and at the end of the experiment (28 and 90 days of age), rats were submitted to an insulin tolerance test (ITT). Th e test was performed with fed and rested animals for 24 hours. Th e fi rst blood sample was taken from the tail vein (time 0). Next, an insulin solution dose of 1U/Kg of body weight was injected intraperitoneally. Blood samples were collected after 4, 8, 12 and 16 minutes post insulin injection with heparinized and calibrated capillaries (25μL) in order to determine the glucose concentrations through the glucose oxidase method (commercial kits -Laborlab®). A single cut on the end of the tail was enough to collect all blood samples. Th e results were analysed by calculating the removal rate of serum glucose (Kitt) and applying the formula (0.0693/t1/2) x 100 and expressed in %/min 14 . Th e removal of blood glucose (t1/2) was calculated by the analysis curve of the minimum squares of the levels of blood glucose in the decay times after administration of insulin 15 using Microsoft Excel ® 2013 software.

Blood and Pancreas
Seventy two hours after the last exercise session and forty eight hours after the last "in vivo" evaluation, fed animals were sacrifi ced with CO 2 anaesthesia, and the blood was collected for serum separation and measurement of glucose (Laborlab®) and insulin by radioimmunoassay 16 for determining the HOMA index (Homeostasis Model Assessment) according to Matthews et al. 17 . The entire pancreas was excised, weighed and macerated to determine insulin concentration also through radioimmunoassay 16,18 .

Gastrocnemius muscle
The gastrocnemius muscle was excised for determination of the intramuscular triglycerides concentration (using commercial kit Laborlab®) at the beginning and at the end of the experiment (28 and 90 days of age) by enzymatic-spectrophotometric method according to the manufacturer's recommendation. A separate batch of animals (n=6 animals/group) was sacrifi ced 60 minutes after receiving intraperitoneal administration of tritiated water (3mCi 3H 2 O). Aliquots of gastrocnemius muscle tissue were extracted and weighed to determine the rate of lipogenesis, as previously described 19 . Th e rate of lipid synthesis was expressed as micromol (μmol) of 3H 2 O incorporated into lipids per hour per gram of tissue. Furthermore, at 90 days of age the concentration of gastrocnemius muscle glycogen was investigated 20,21 .

Soleus muscle analyses
After sacrifice, the soleus was removed and slices weighing between 25 and 35mg were subjected to incubation previously described 22 , modifi ed for the test of glucose uptake. In summary, the slices were placed in Krebs-Ringer bicarbonate buff er, supplemented with glucose (5,5 mM), containing [U -14C] glucose (0,5 mCi/mL), [3H+] 2-deoxiglicose (0,5 15 μCi/mL) and insulin (100 μU/mL) in vial glass for 90 minutes, and continuous gassing with O 2 / CO 2 (95%/5%) under constant stirring in a water bath (37°C). Th e released CO 2 was captured by hyamine 10x, placed in glass apparatus inserted in the above mentioned vial. Glucose uptake was assessed using the 2-deoxyglucose (2-DG) as marker, and the incorporation of 14C in the glycogen (synthesis) by measuring the radioactivity of the 3H of 2-DG and glucose 14C respectively. Th e measurement of the amount of oxidized glucose was performed by radioactivity of 14C in hyamine. The radioactive lactate released into the incubation medium was determined by separation of metabolites in the ion exchange column (Dowex-2, Sigma).

Adipose tissue analyses
At the beginning and at the end of the experiment (28 and 90 days of age), the adipose tissue from diff erent regions (subcutaneous, mesenteric, retroperitoneal and epididymal) was removed according to the description of Cinti 23 for total weighing and determination of the triglyceride concentrations through enzymatic-spectrophotometric by glycerol phosphate (using commercial kit -Laborlab®). A batch of animals (n=6 animals/group) was separated and the aliquots of mesenteric adipose tissue were extracted and weighed 60 minutes after receiving intraperitoneal administration of tritiated water (3mCi 3H2O) to determine the rate of lipogenesis as previously described 19 . Th e amount of tritium incorporated was assessed in the adipose tissue 60min after 3H 2 O administration. Th e rate of lipid synthesis was expressed as micromol of 3H2O incorporated into lipids per hour, per gram of tissue.

Statistics
Results were expressed as mean ± standard deviation. Th e student t-test for independent samples was used to compare the diff erence in means between the beginning and the end of the experiment for the trained and the control groups. Th e predetermined level of signifi cance was p<0.05.
At baseline, there was no difference in the physiological profi le of both groups (TABLE 1).
At the end of the experiment, the rats from the trained group had a signifi cantly lower body weight gain and a higher insulin sensitivity when they were compared to the animals from the control group (FIGURES 1A and 1B). Moreover, the analysis Physiological profi le at the beginning of the experiment. TABLE 1 -of adipose tissue showed a signifi cant reduction in fat mass in all regions analysed: mesenteric, subcutaneous, retroperitoneal and epididymal (FIGURES 2A-D). Th e analysis of lipogenic activity in the mesenteric region showed that the animals submitted to exercise had lower lipid synthesis (FIGURE 2E).

Control Trained
In vivo

Results
Body weight (A) and serum glucose removal rate (Kitt) (B) in the control and trained groups at the end of the experiment (90 days of age). * p<0.05.   The blood collected to determine the serum glucose and insulin showed that physical training reduced glucose and insulin levels, even at the serum and pancreatic levels, resulting in an increase in the HOMA index (FIGURES 3A-D).
Th e lipidic and glucose profi le in the gastrocnemius muscle was determined. Although physical training was not eff ective in changing the lipogenic activity (tritium incorporated) or triglyceride concentrations, it was possible to observe an increase of glycogen storage in this muscle (FIGURES 4A-C). Furthermore, the glucose uptake by the soleus muscle was increased in the trained animals as compared to the untrained group (FIGURE 4D).

Discussion
Physical inactivity is considered one of the main causes of obesity. Since this disease is growing alarmingly, studies related to the prevention and / or treatment are of paramount importance. Th e present study aimed to investigate the eff ects of moderate exercise in adult mice when it was started in the childhood, on parameters related to obesity and IR. Th us, we observed positive changes in body composition of exercised animals compared to sedentary, such as less weight and amount of fat tissues, and a better response to insulin.
In this study, the physical training was able to reduce the body weight gain (FIGURE 1A) and, consequently, the fats stocks in all regions analyzed (mesenteric, epididymal, subcutaneous and retroperitoneal) (FIGURE 2A-D), and through a more sensitive analysis of fat synthesis in the mesenteric region, the lipogenic activity, we observed that the animals submitted to exercise had lower lipid synthesis (FIGURE 2E). Th e smaller gain in fat mass by trained group can be attributed to the fact that the exercise can activate the hormone sensitive lipase (HSL), increasing lipolysis and thus providing energy substrate for the activity performed 24 . Another factor that may have contributed to lower fat mass accumulation was the decrease in insulin concentrations, hormone that directly participates of lipogenesis 25 . According to the our fi ndings, the fat mass reduction contributed to improvement in carbohydrate metabolism and generated favorable environment for better insulin sensitivity to peripheral tissues as shown by ITT (FIGURE 1B) and analysis of the HOMA index (FIGURE 3D).
The IR, when untreated, generates a hyperinsulinemia state and thus can trigger the failure of the pancreatic beta cells 26 . At the end of this experiment, when blood glucose, serum insulin, pancreatic insulin and HOMA index were analyzed, it was seen that the sedentary lifestyle makes that a state similar to IR and prediabetes was developed in the control group (FIGURE 3A-D). It is already established in the literature that the aging associated to physical inactivity can collaborate in the development of IR, providing greater gain of fat mass and lean mass reduction 27 . Th us, the physical inactivity in childhood, ally with the natural aging process, can be a determining factor for metabolic health. Therefore, regular physical exercise has been widely indicated for the prevention and/or   28,29 , since the practice of exercises besides to increasing energy expenditure, elevate interleukin-6 (IL-6) levels, that when coming from the muscle contraction is able to minimize the infl ammatory disorders caused by adipose tissue excess, from the peripheral levels until the central 30 . In addition, physical exercise contributes to better signaling of proteins involved in the metabolism of carbohydrates and lipids, moreover muscle contraction also has a positive eff ect on glucose uptake independently of insulin 31,32 .
In this study, when the gastrocnemius muscle was analyzed, no change in lipogenic activity and triglyceride concentration was found in the T group (FIGURE 4A-B). It allows us to conclude that the action of insulin has been improved in this tissue, since even with a lower concentration of this hormone, the group maintained its lipogenic activity and its lipid reserves unchanged. However, the body weight of trained animals and all portions of fats were lower (FIGURE 1A and 2A-D). Th us, we believe that there is also a greater capacity for mobilization and oxidation of fatty acids, which refl ects an improved overall metabolic health, since there is evidences that muscle ability to oxidize fatty acids is an important regulator of insulin sensitivity, and this capacity is impaired by physical inactivity 33 .
During long physical exercise session, glycogen is the main muscle energy reserves 34 , providing glucose to the cells. Th e energy from glucose is an energy provided quickly, so its use is directly proportional to the intensity of the exercise 35 . Th e depletion of glycogen is widely used by athletes, once that after their depletion a state called glycogen overcompensation is installed, allowing the athlete to higher accumulation over basal levels, thus providing a better performance in tests using this energetic substrate 36 . In the present study, animals were exercised for 1 hour per day in moderate intensity. As expected, an increase in muscle glycogen levels in trained animals was observed (FIGURE 4C), as well as a greater glucose uptake by the soleus muscle (FIGURE 4D). Right after the end of the exercise protocol, muscle slices were incubated with insulin and glucose at baseline levels, revealing that even without a higher availability of glucose after depletion, there is a greater uptake of this substrate as a consequence of exercise.
Th e knowledge of the exercise intensity to be worked also shown to be extremely important. Th ere are several studies that showed diff erent responses to different exercise intensities [37][38][39] , therefore when compared to other variables such as weekly frequency and session volume, the intensity seems to be more relevant to the improvement of IR 40 . In this context, other studies also carried out aiming to investigate the benefi ts off ered by exercise when started soon after weaning rodents [7][8][9] . However, these studies only provided to the animals access to the running wheel, and the eff ects observed by the authors related only to the spontaneous practice of exercises. Contrary to that found in our study, Acosta and colleagues found no signifi cant reduction in body mass of animals exposed to the running wheel 7 . In another study, Schroeder and colleagues found no diff erence in the amount of retroperitoneal and epididymal fat in rats 8 . Th us, our study shows up as the fi rst in the literature to demonstrate that moderate physical exercise performed since childhood, with controlled exercise intensity, can promote consistent improvement in body fat during adulthood, with this improvement refl ecting in a better glycemic control and less production of insulin by the pancreas.
In summary, we found that moderate physical exercise performed during childhood can be an eff ective strategy to prevent body fat accummulation and insulin resistance during adulthood life.
Authors declare no confl ict of interest.