Westernized eating habits and lack of exercise have increased the prevalence of various diseases. Among these, Type 2 diabetes mellitus (T2D) is characterized by the failure of normal glucose absorption and increased blood glucose levels¹. At present, there are 400 million patients worldwide with T2D; in 20 years, this number is expected to increase to more than 600 million². The pathophysiological features of T2D include chronic systemic inflammation, oxidative stress, endoplasmic reticulum stress, and apoptosis^{2, 3}. Recent studies have reported that autophagic dysfunction is involved in the pathogenesis of T2D, and studies have shown a trend toward increasing insulin resistance and autophagy^{2, 4-7}.

Autophagy maintains intracellular homeostasis through protein turnover and is a cell survival mechanism activated by starvation, oxygen-free radicals, and hypoxia⁸. The process of autophagy is initiated through the pathway of nucleation-elongation/enclosure-degradation; autophagosomes are formed, which then fuse with lysosomes, resulting in disintegration of abnormal proteins and cell organelles. Studies by Koga et al.⁹, Kosacka et al.¹⁰ and Papáčková et al.¹¹ showed that an increase in insulin resistance interferes with the fusion of autophagosomes and lysosomes, resulting in defective intracellular autophagy. Liu et al.¹² studied a mouse model with T2D induced by a high-fat diet, and reported a decrease in the microtubule-associated protein-1 light chain-3 (LC3-II/LC3-I) ratio, a primary marker of autophagic activity, an increase in p62, and a decrease in vacuolar protein sorting 34 (PS34), autophagy-related gene 12 (ATG12), and GABA receptor-associated protein-like 1 gene (GABARAPL1), which are involved in autophagy.

Autophagy in the liver, muscle, heart, pancreas, and skeletal muscle plays an important role in glucose metabolism and utilizes about 80% of the glucose that mediates insulin action^{2, 13-16}. Thus, intramuscular insulin resistance is a major cause of glucose intolerance and T2D. Shi et al.¹⁵ reported reduced insulin resistance associated with autophagic activity in skeletal muscle upon administration of dihydromyricetin, an antioxidant substance, to wild-type experimental animals for 10 weeks. On the basis of their study findings, the researchers applied the same treatment in a T2D experimental animal model induced by a high fat diet, and found that autophagy was induced in skeletal muscle by AMP-activated protein kinase (AMPK), which is the primary mechanism of autophagy. Furthermore, an improvement in T2D with an increase in insulin receptor substrate-1 (IRS-1) has been reported¹⁵. In addition, Zhou et al.¹⁶ reported decreased insulin resistance in muscle cells, with improvement in autolysosome activity, which is responsible for disintegration of autogranular bodies, as a result of intermittently administering osteocalcin, an osteogenic hormone, to a T2D mouse model induced by a high fat diet.

Exercise is effective in preventing components of metabolic syndrome, including hypertension, hyperlipidemia, and obesity¹⁷, and maintains and improve the qualitative and functional status of skeletal muscles. Repetitive muscle contraction through exercise reportedly activates autophagy as part of the mechanism of homeostasis in skeletal muscle^18-20. According to Lira et al.²¹, the overall improvement in physical status induced by voluntary endurance exercise for 4 weeks is the result of an increase in the basal level of autophagy in skeletal muscle. Grumati et al.²² and He et al.²³ reported an increase in autophagic flux based on an increase in the conversion rate of LC3II/LC3I induced by exercise, and a decrease in p62, an autophagic adapter protein. Furthermore, Masiero et al.¹⁸ reported a correlation between autophagy and endurance exercise ability, based on the finding that a mouse model lacking the ATG7 gene in skeletal muscle was incapable of normal endurance exercise.

Accordingly, improvement in autophagy through exercise can be an effective noninvasive intervention to prevent progression of T2D. Therefore, the purpose of this study was to investigate changes in blood glucose levels and insulin resistance during an 10 weeks treadmill exercise in a T2D animal model induced by a highfat diet, and to confirm the change of Beclin-1, which is involved in the autophagic initiation process, and LC3-II/I, p62, and LAMP-2, which are involved in the disintegration phase.

Reduced activity and excessive dietary intake in association with improved living standards have contributed to increased obesity²⁷ and obesity-related disease. Fat accumulation due to ingestion of a high-fat diet has an effect on energy metabolism and function²⁸. Excessive fat accumulation is called obesity and causes dyslipidemia. Atherosclerosis and myocardial infarction are associated with obesity and dyslipidemia²⁹. With an increase in obesity, the risk of related disease is also increasing. Exercise and dieting can reduce this risk.

Exercise has long been suggested as a good way to treat and prevent obesity. In particular, endurance exercise is effective in reducing total body fat, reducing visceral fat, and improving insulin resistance by enhancing glucose metabolism³⁰. According to a study by Delghingaro-Augusto et al.³¹, rats with obesity induced by a high-fat diet tended to have decreased body weight and body fat after spontaneous wheel exercise for 6 weeks. In the case of autophagy, Levine et al.³² reported that exercise stimulates autoregulation and alleviates neurodegenerative diseases such as Alzheimer's disease and diabetes.

Autophagy is very important because it affects the removal of abnormal proteins as well as infectious agents such as bacteria and viruses³³. Therefore, the purpose of this study was to investigate the general characteristics, changes in blood markers, and the expression level of proteins related to autophagy in the soleus muscle after 10 weeks of endurance exercise in rats with obesity induced by a 60% high-fat diet.

Weight was increased in both HF-SED and HF-TE groups in comparison with the ND-SED group, and showed a noticeable decrease in the HF-TE group. In addition, triglyceride, total cholesterol, LDL-cholesterol, insulin, glucose, and HOMA-IR values increased in the HF-SED group. In other words, increased body weight was reduced through endurance exercise despite a high-fat diet. According to McTiernan et al.³⁴, endurance exercise activates fat-splitting enzymes, resulting in a decrease in body weight, abdominal fat percentage, and body fat. Additionally, western blotting was conducted to analyze autophagic flux and how a high-fat diet affects the process of autophagy. Tissue in the soleus muscle was used for analysis. Autophagic flux refers to a series of processes in which degradation occurs from the initiation of autophagy³⁵.

Beclin-1 is a protein that affects the initiation stage of autophagosome formation. There was no significant difference in the level of Beclin-1 expression in the ND-SED, HF-SED, and HF-TE groups. These results suggest that high-fat diets did not affect the initiation of autophagy.

The p62 (sequestosome 1) protein plays a role as an adapter to promote autophagy through association with ubiquitinated proteins^{36, 37}. Thus, p62 is increased when the autophagy process becomes active³⁸. However, in this study, the expression of p62 was expected to increase in the HF-SED group with a high-fat diet, but the difference was not statistically significant. Previous studies have shown that p62 increased in the fasting group, but not in the fasting group. This is consistent with the findings in this study³⁹.

The LC3 protein is a key marker of autophagy, which is involved in the formation of autophagosomes by binding to phosphatidylethanolamine⁴⁰. Penfei et al.13 showed that the levels of streptozotocin-induced autophagy-related factors such as Beclin-1 and LC3 in diabetic rats did not change statistically when compared to those in the ND-SED group. These results show that there is no change in the process of autophagy.

Lysosome-associated membrane protein-2 (Lamp-2) protein is important in the fusion of autophagosomes and lysosomes and requires enzymes such as cathepsins B, D, and L, which are lysosome resolvents. The expression level of Lamp-2 protein was not significantly different in this study. In other words, this study showed that a high-fat diet and exercise did not affect the autophagy process.

The soleus muscle was exercised on the treadmill, and was analyzed in this study. However, there was no significant difference in the results of western blot analysis. For this reason, Jamart et al.³⁹ reported that LC3- II b expression was increased when fasting and exercise were performed simultaneously. The authors reported that autophagy was not activated because autophagy had already adapted to hyperglycemia in the muscles of the high-fat diet group, with differences in muscle mass in each group7. Therefore, chronic hyperglycemia in which fat accumulates due to long-term high-fat diets adversely affects not only skeletal muscle but also autophagy in the liver and adipose tissue.

Although the reason is unclear, the method and intensity setting of the exercise performed in this study may be one of causes. Treadmill exercise can be an effective way to reduce weight, but may be less effective than resistance exercise to improve muscle performance. There was no difference in soleus muscle weight between the groups. In addition, we tried to apply moderate-intensity exercise as suggested by Kim et al. (2003)²⁴ and Jee et al. (2008)²⁵, but exercise was not performed normally because the HF-SED group was overweight. Therefore, inevitably, low-intensity exercise was performed. For this reason, exercise may not have had much effect on the process of autophagy.

In addition, the muscle fiber distribution in the soleus muscle might have affected the process of autophagy. Penfei et al.¹³ reported that the gastrocnemius muscle responds more sensitively to autophagy than the soleus muscle. This may be due to the control of protein synthesis and differences between slow-twitch and fasttwitch muscle fibers. In other words, the gastrocnemius muscle compared with the soleus muscle is more susceptible to external changes such as starvation, resulting in protein turnover⁴¹. Autophagy tends to be activated in the starvation state. However, a hyperlipidemic state, in which glucose is elevated by a long-term high-fat diet, results in an excess of nutrients rather than a starvation state.

This study showed statistically significant increases in body weight, blood chemistry levels (total cholesterol, LDL-cholesterol, HDL-cholesterol, glucose), and the HOMA-IR index in the high-fat diet group. However, endurance exercise using a treadmill led to improved body weight, insulin resistance, and blood lipid levels. Western blotting was performed to examine flux in the soleus, but there was no significant difference between the groups in any part of the autophagic process. These results indicate that aerobic exercise can be effective in the treatment of obesity, but does not seem to affect autophagy. However, studies should be performed based on the characteristics of different muscle fibers.

This study investigated the effects of treadmill exercise on body weight and blood biochemical indicators, and analyzed HOMA-IR indices and autophagy-related factors in obese rats. Treadmill exercise led to improvement in body weight, body fat, and biochemical indicators of obesity and Type 2 diabetes, but did not affect autophagy in the soleus muscle.