The comparison of continuous and intermittent training impact on glucose-4 transporter protein level and insulin sensitivity in diabetic rats

Introduction: Diabetes mellitus (DM) is now seen as a worldwide epidemic disease with high prevalence. Exercise training (ET) is known to promote beneficial changes in diabetic patients. The aim of this study was to compare the impact of continuous and intermittent aerobic training on glucose-4 transporter protein (GLUT-4) levels and insulin sensitivity in diabetic rats. Materials and methods: This research was an experimental and interventional study. A number of 60 male rats weighing 180 to 310 grams and 13 weeks old were divided into six groups: healthy control, healthy continuous, healthy intermittent, diabetic control, diabetic continuous, and diabetic intermittent groups. The exercise protocol in both continuous and intermittent groups was aerobic training for six weeks which was conducted considering overload principle. The data were analyzed using one-way analysis of variance test in P< 0.05 level. Results: The results showed that despite the significant difference between insulin resistance and GLUT4 level among diabetic rats in different groups compared to control group, there was no significant difference between the impact of continuous and intermittent training on these indices (P=1.00). Conclusion: Through increasing the GLUT4 protein content, the continuous and intermittent training improved insulin resistance.


Introduction
The mellitus diabetes is a metabolic disorder that is widespread in the world and is associated with increase in blood glucose, inadequate secretion, and dysfunction of insulin (1). The type 2 diabetes is a result of pancreatic beta cells destruction resulting in insulin deficiency. The type 2 diabetes is diagnosed considering insulin resistance and relative reduction of insulin level. The chronic high blood sugar has many complications which lead to damages in various organs and disorder in their function including 10 years less lifetime (2, 3), 10 times more common amputation (4), 9% death (5), and visual impairment and blindness (6). There is a direct relationship between chronic complications of diabetes and high level of blood glucose (7). The high blood sugar causes non-enzymatic binding of glucose to proteins inside and outside the cells (8). Today, there is no doubt that improved glycemic control in diabetic patient results in decreasing incidence of chronic complications of this disease (9). The transportation of glucose into the muscle fiber is done by glucose transporter proteins (GLUTs); the GLUT4 is the most important glucose transporter isoform in skeletal muscles. The insulin and exercise stimulate fast and intense GLUT4 translocation to plasma membrane and cause glucose uptake in muscle and adipose tissue (10). The increased insulin sensitivity occurs after exercise simultaneously with accumulation of muscle glycogen stores (11). It is believed that the increased muscle glycogen stores after the exercise is due to increased GLUT4 (12,13) and increased GLUT4 protein translocation from inside the cell to plasma surface [13]. In this context, Park et al (2011) reported the increased GLUT4 in skeletal muscle of healthy and diabetic mice after 6 weeks running on a treadmill at a speed of 15 meters per minute (14). Moreover, Holten et al (2004) showed that resistance exercise with 70% to 80% of one maximum repetition led to 40% and 21% increase of GLUT4 and insulin receptors content in diabetic patients' muscles (15). Also, Christine (2002) showed that 7 week running on a treadmill at a speed of 22 meters per minute increases the GLUT4 level and improves insulin resistance in obese mice (16). In contrast, Rudnick (1990) showed that there was no change in GLUT4 content of soleus muscle in three weeks of running on treadmills (17). Moreover, Vannucci et al (1998) reported that the GLUT4 level is decreased due to exercise-induced insulin reduction (18). Finally, Ivy (2004) showed that exercise improves insulin sensitivity in independent ways from GLUT4 in diabetic rats (19). According to studies conducted in this field and different results in previous studies, this study aims to investigate the impact of two aerobic (continuous and intermittent) exercises on insulin sensitivity and GLUT4 indices in soleus muscle of diabetic rats and answer this question: which aerobic exercise (continuous and intermittent) protocol is more useful for diabetic rats?

Materials and methods
A total of 60 male rats weighting between 180 to 310 grams and 13-week old were bought from Laboratory Animals Breeding Centre. These animals were kept in a place with 12-hour light-dark cycle, 21± 2 ° C temperature, and a relative humidity of 45 to 55 percent (20). During the study, the animals were given enough food and water. To avoid stress and physiological conditions, the sample was stored for 2 weeks under the new conditions. The subjects were kept for 2 weeks in new conditions to avoid stress and change in physiological conditions. In the second week, the animals were trained to work on the treadmill. The training program was conducted for 5 days (at a speed of 10 meters per minute, once a day for 10 minutes) (21). After a period of adaptation to laboratory, 30 rats (except healthy control, healthy intermittent, and healthy continuous groups) got diabetic using Streptozotocin (STZ) drug with a dose of 50 mg per kg of body weight, solved in citrate buffer 1.0 Molar and acidity 4.5. After a week, the blood samples were taken with a small wound on the tail; the blood glucose levels were measured with a glucometer. After disclosure of diabetes (glucose more than 300 mg per deciliter) in animals (22). The rats were randomly divided into six healthy control, diabetic control, healthy intermittent, diabetic intermittent, healthy continuous, diabetic continuous (each group of 10 rats) groups based on the type of training protocol. The exercise protocol of both continuous and intermittent groups was six weeks of aerobic exercise which was performed with progressive intensity and duration and considering gradual overload principle (Table 1). Throughout the training period, the slope of treadmill was considered at zero degree. The interval of rest between training sessions in intermittent group was considered to be one to one-quarter. The exercise groups ran at the beginning of each training session for 3 minutes at a speed of 7 meters per minute for warming up. The treadmill speed was increased 2 meters per minute to achieve the desirable speed. The speed of treadmill was reduced reversely to initial speed at the end of each exercise session for cooling down. To avoid possible effects of electric shock (1 to 3 volts) on study results, the animals were trained through audio stimulation to avoid approaching and resting at the end section of device (23). Table 1. The process of performing training protocols (24).
week Continuous training Intermittent training 1 14 minutes at a speed of 12 meters per minute 2 repeats for 7 minutes and 12 meters per minute 2 19 minutes at a speed of 12 meters per minute 2 repeats for 9.5 minutes and 12 meters per minute  However, 48 hours after last exercise session followed by 12 hours fasting, the rats were anesthetized with inhaling ether solution in inside of glass chamber. Cutting the skin of abdomen and chest area and opening abdominal cavity, about 10 ml of blood was taken directly from the heart and was transferred to test tube. The collected samples were immediately centrifuged (at 3000 rpm for 10 min) and were used to assess the insulin level. After dissection, their soleus muscle was also taken by surgery knife. It was washed, homogenized, and frozen in liquid nitrogen and was kept at -80 ° C for subsequent analyses (25). In the present study, the commercial kit of Sweden Mercodia Company with sensitivity of 0.015 micrograms per liter and commercial kit of China-American Cosabio Company with sensitivity of 0.04 ng per ml were used to measure insulin level and a GLUT4, respectively. The samples were analyzed using Sandwich ELISA method. The insulin resistance is opposite to insulin sensitivity. It is not possible to calculate insulin sensitivity by calculating the number of insulin receptors on the cell surface. Therefore, the HOMA-IR formula (26) was used to calculate insulin resistance changes and insulin sensitivity changes. HOMA-IR= fasting insulin (Micro unit/ml) × fasting glucose (mg / dl) ÷ 405 The data were analyzed using Statistical Package for Social Sciences (SPSS), version 16. First, the Shapiro-Wilk Test was used to determine the distribution of data in all groups. According to results of this test (Table 2), the one-way analysis of variance (ANOVA) was used to determine between-groups changes of dependent variables at P<0.05 level. Also, the Tukey test was used to determine the difference between pairs.

Results
The results showed a significant difference in insulin resistance levels among different groups (P=0.001, F5, 37= 10.80). Comparing the pairs in Tukey test showed that insulin resistance in diabetic control mice is significantly higher than that of healthy control group (P=0.001). However, there was no significant difference between insulin resistance of diabetic continuous training group and healthy continuous exercise group (P=0.99) and diabetic intermittent training group and healthy intermittent training group (p=0.96). This means that continuous and intermittent training reduce insulin resistance level in diabetic mice compared to diabetic control group (Figure 1).Also, the continuous (P=1.00) and intermittent (P=0.96) training had not significant effect on insulin resistance level of healthy mice compared to healthy control group; there was no significant difference between the impact of both continuous and intermittent training on insulin resistance (P=1.00). The results showed a significant difference in GLUT4 of soleus muscle in different research groups (P=0.001, F5, 37= 20.14). Comparing the pairs in Tukey test showed that GLUT4 of soleus muscle in diabetic control mice is significantly lower than healthy control group (P=0.006). Although the results showed that the continuous (P=0.001) and intermittent (P=0.004) training increaseGLUT4 of soleus muscle in healthy mice compared to healthy control group, there was no significant difference between the effects of these two type of training (P=0.94). In addition, GLUT4 of soleus muscle in continuous training group was significantly higher than diabetic continuous training group (P=0.001); also, GLUT4 of soleus muscle in intermittent training group was significantly higher than diabetic intermittent training group (P=0.002) (Figure 2). There was no significant difference between GLUT4 of soleus muscle in diabetic continuous training group (P=0.81), diabetic intermittent training group (P=0.99), and healthy control group.

Discussion
The insulin resistance of diabetic control mice was higher than that of healthy control group. However, there was no significant difference between insulin resistance of diabetic continuous training group and that of continuous training group, and between diabetic intermittent training group and intermittent training group. In addition, the results showed that GLUT4 of soleus muscle in diabetic control group was lower than that of healthy control group. Although the continuous and intermittent training increased GLUT4 of soleus muscle in healthy mice compared to healthy control group, there was no significant difference between these two types of training. In a study conducted on rats, the increased level of GLUT4 in plantaris muscle and no change in soleus muscle was observed after three weeks of training on a treadmill (17). In this context, Vannucci et al. suggested that due to reduced level of insulin after exercise, GLUT4 was significantly reduced in rabbits (18). In addition, Ivy (2004) stated that exercise improves insulin sensitivity, but causes no change in insulin signaling and GLUT4 transmission to membrane surface in muscles of rats. It can be found that the results of present research on GLUT4 are not consistent with above findings. This may be due to difference in training program type and studied muscle. However, the research shows that muscle fiber type is involved in type of response.  (30). The GLUT protein is a major mediator for glucose uptake from blood circulation which is expressed in skeletal muscle and adipose tissue (31). The evidence has shown that the total amount of GLUT4 protein and its displacement to muscle fiber membrane determines the amount of muscle glucose uptake in response to insulin. It is now widely accepted that this protein molecule plays a key role in whole-body insulin sensitivity and glucose tolerance (32). There are two routes for stimulating glucose uptake by muscle. During the rest, the glucose uptake by muscle depends on insulin and its main role is rebuilding muscle glycogen stores. During exercise, the muscle contraction increases blood glucose uptake to help muscle glycogenolysis. Since both routes are separate, the blood glucose uptake in active muscle is natural in patients with type 2 diabetes which their insulindependent absorption is impaired. The uptake of glucose into muscle is high even after exercise, because the routes which stimulate glucose uptake remain active for hours after exercise (33,34). Due to increased GLUT4 protein, increased displacement, and increased exposure of these transport proteins at the cell surface, the muscle contraction increases membrane permeability to glucose and improves insulin action in glucose metabolism (35). The increased insulin sensitivity after exercise occurs simultaneously with the accumulation of muscle glycogen stores (11). It is believed that increased muscle glycogen stores after exercise is due to increased GLUT4 (12,13) and increased GLUT4 protein displacement from inside the cell to plasma surface (13). The findings of some studies show that exercise (36,37) may impact on AMPK activity and AMPK impact on protein GLUT4 (37). However, it can be said that continuous and intermittent exercises increase AMPK activity and stimulate GLUT4 protein in these groups. In terms of increased insulin sensitivity as one of the findings, it can be said that some mechanisms increase insulin action after aerobic (continuous and intermittent) exercise including increased insulin receptors signaling, increased protein of glucose transporter (GLUT4), increased activity of glycogen synthase and hexokinase, decreased release and increased deletion of free fatty acids, increased release of glucose from blood into the muscle due to increased muscle capillaries, and changes in composition of muscle to increase glucose uptake (26). Given the rise in GLUT 4 protein levels in this study, the increased GLUT4 level is probably one of the reasons for improvement of insulin action. Both the insulin dependent and independent routes in diabetic patients are impaired in glucose uptake, because diabetes reduces GLUT4 level and increases resistance to insulin. In contrast, the exercise acts as an alternative to compensate low levels of these indicators. In addition, although diabetes reduces GLUT4 displacement to muscle cell membrane, the exercise compensates this transportation and displacement through stimulating insulin independent route.

Conclusion
Given that the present study showed that the continuous and intermittent training impact significantly on GLUT4 protein levels and insulin sensitivity, it seems that training may overcome the damage caused by diabetes on AMPK and increases GLUT4 protein. In general, considering the positive impact of training on GLUT4 protein levels in diabetic rats, it is suggested that the findings of this study be used in clinical trials; if the results will be positive and useful, they should be considered as a complementary way in the management of diabetes. However, due to the lack of significant difference between the effects of intermittent and continuous training, the diabetics may perform any of these training to achieve positive results.