ABSTRACT
Background and objectives: Programmed autophagy is a genetically and evolutionarily conserved process that destroys long-lived cellular proteins and organelles. This study aimed to investigate effects of continuous and interval exercise training with or without atorvastatin supplementation on Beclin1, LC3-I and LC3-П expression in old rats with type 2 diabetes.
Methods: Sixty three male Wistar rats were divided into eight groups. Continuous exercise was performed at a speed of 15-29 m/min for 5-22 minutes. Interval exercise program consisted of six 2.5-minute sets that included a four-minute rest period between each set. The rats in the supplementation groups also received 20 mg/kg body weight atorvastatin daily via intraperitoneal injection. At the end of the training period, the expression of Beclin1, LC3-I and LC3-П in soleus muscle was measured by RT-PCR. One-way ANOVA was used for data analysis at statistical significance of 0.05.
Results: The results showed that both exercise trainings with or without atorvastatin significantly reduced LC3I, LC3-II and Beclin1 compared with the diabetic control group (P<0.05). In addition, the effects of the trainings and atorvastatin supplement did not differ significantly (P>0.05).
Conclusion: The results indicate that continuous and interval exercise program alone and combined with atorvastatin supplementation could significantly reduce LC3-1, LC3-II and Beclin1 level in soleus muscle of old diabetic rats.
Keywords: Interval Exercise, Autophagy, Type 2 Diabetes, Atorvastatin
 
 
INTRODUCTION
Aging is associated with biological changes in an organism, leading to a decrease
in vital and adaptive energy (1). From the age of 30 to 60 years, muscle mass decreases by about 1% per year, and this procedure is accelerated after 60 years. Decreased muscle mass can increase the risk of metabolic diseases, such as type 2 diabetes, metabolic syndrome and cardiovascular disease as well as chronic diseases of the musculoskeletal structure and cancer (2). It is estimated that the world’s older population will be doubled to 1.2 billion by 2025. In addition, the prevalence of type 2 diabetes increases with age and reaches its maximum by the age of 60-74 years (3). Diabetes mellitus is the most common endocrine disorder among the elderly (4). It is estimated that the worldwide prevalence of diabetes will increase to 366 million by 2030 (5). Type 2 diabetes mellitus is the biggest health challenge of the 21^st century (6). Skeletal muscle mass changes with the decrease in the total cross-sectional area. The proteolysis of skeletal muscle is mostly regulated via intracellular proteolytic complex systems, including the lysosomal system, activated Ca²⁺ system, cytosol and ATP-ubiquitin-dependent proteolysis (7). Diabetes reduces muscle volume and skeletal muscle mass, which consequently decrease basal metabolic rate during aging. In addition, changes in the endocrine glands, such as elevated insulin level, may lead to insulin resistance in the long run. In fact, the loss of skeletal muscle mass or muscle atrophy is due to the complex interaction of cell apoptosis, the increased production of free radicals and the activity of proteolytic systems (8).
There are several systems for the destruction of muscle proteins, the most important of which are the ubiquitin-proteasome system and the autophagy-lysosome system (9). Beclin-1 is involved in most biological processes, including stress adaptation, growth, endocytosis, immunity and aging (10). As an essential initiator of autophagy, this protein can be a key stimulator of autophagy proteins, which leads to formation of the central complex of Bcl-1, VPS34 and VPS15. Beclin1 is also a key determinant of cells exposed to autophagy or apoptosis (11).
Of three LC3 isoforms expressed in mammalian tissues (LC3-I, LC3-II, LC3-III), LC3-ll is associated with autophagy (1(2. Various stressors can strongly regulate LC3 expression and turns it into a cytosolic form. Moreover, LC3-II has been considered as the most reliable autophagy index so far (13).
Atorvastatin is a statin that stops the conversion of mevalonate to hydroxy β-methylglutaryl-CoA by inhibiting 3-hydroxymethylglutaryl coenzyme reductase, which will consequently reduce cholesterol production. Despite lowering blood cholesterol, this medicine has some antioxidant, anti-inflammatory, anti-apoptotic and tissue-protective effects in some pathological conditions (14, 15). Statins stimulate Langerhans cells to release more insulin in order to lower blood glucose. In general, this drug can inhibit diabetes-related pancreatic tissue damage and necrosis by limiting oxidative stress and pancreatic inflammation (16). However, statins have dose-dependent side effects on skeletal muscles such as congestion, muscle pain, weakness and acceleration of skeletal muscle decomposition, leading to cell death (17).
Since aging is associated with physiological changes, such as structural skeletal muscle change, it can enhance insulin resistance and diabetes. Given the limited number of studies in this regard, we aimed to investigate effects of continuous and interval aerobic exercise along with statin administration on Beclin1 and LC3 expression in old rats with type 2 diabetes.
                                                                                        
MATERIALS AND METHODS
In this experimental study, 63 old male Wistar rats (weighting 300-350 g) were selected. The animals were randomly divided into eight groups of  healthy control (CN), diabetic control (CD), diabetic + continuous exercise (CED), diabetic + interval exercise (IED), diabetic + atorvastatin (AD), diabetic + continuous exercise + atorvastatin (ACED), diabetic + interval exercise + atorvastatin (AIED) and saline (SD). Diabetes was induced in rats by intraperitoneal injection of 50 mg/kg streptozotocin. Blood samples were taken from the corners of the eye and glucose level of above 250 mg/dl confirm the induction of diabetes (18). All study procedures were carried out according to the standard guidelines of working with laboratory animals.
Before starting the main protocol, the rats became familiar with the exercise by running on a treadmill for five minutes, five sessions a week, at speed of 8-10 m/min. The exercise program consisted of two continuous and interval exercise protocols (19). Daily atorvastatin supplement (20 mg/kg) was intraperitoneally injected to the supplementation groups (18).
Forty eight hours after the last exercise session and after 10-12 hours of fasting, the rats were anesthetized by intraperitoneal injection of ketamine-xylazine, and soleus muscle tissues were separated and stored at -80 °C. Then, the samples were sent to the laboratory for measuring LC3I, LC3-II and Beclin1 expression via RT-PCR using specific primers (Table 1).
Table 1. The primers sequence for studied genes

 
Quantification of the values of target gene expression was performed using the 2^-CTΔΔ formula.
After confirming the normality of data distribution using the Shapiro–Wilk test, descriptive statistics including mean and standard deviation were used to describe the data. Inferential statistics, one-way ANOVA and Tukey post hoc test were used for data analysis. All statistical analyses were performed in SPSS 20 and at significance of 0.05.
 
RESULTS
Based on the results of ANOVA, LC3-I expression was significantly lower in AD, CED, IED, ACED and AIED groups than in the CD group (p=0.001) (Figure 1).
 
 As shown in figure 2, LC3-II expression differed significantly between the study groups (p<0.001). The LC3-II expression level in groups AD, CED, IED, ACED and AIED was significantly lower than in the CD group (p=0.001).