4.1 Effects of SIRT1 inhibitor and aerobic exercise on gastrocnemius histomorphology and glucose metabolism in high-fat mice
FBG level and OGTT results demonstrated a rise in body fat content and baseline glucose levels after 14 weeks of a high-fat diet. These findings imply that the glucose metabolism in mice was disturbed, resulting in severe insulin resistance and glucose metabolism disorder. However, the addition of an exercise training regimen to a high-fat diet effectively alleviated the increase in blood glucose level and body fat content. Due to the prolonged duration of the high-fat diet (14 weeks), exercise alone could not completely reverse the damaged metabolic pathway. Electron microscopy showed that abnormal vacuoles, cristae breakdown, and destruction of mitochondrial structure in high-fat mice led to abnormal energy metabolism, which further promoted fat accumulation and insulin resistance. Myelin-like lamellar structures were formed as a result of mitochondrial membrane damage in high –fat diet mice. However, the mitochondria in the HE group had self-repairing ability to a certain extent, which indicated that exercise could induce mitochondrial biosynthesis and promote energy metabolism.
The HES group had higher body fat content and insulin resistance compared to those in the HE group. The cross-sectional area and wet weight of skeletal muscle of the HES group were lower than those of the HE group. There were many vacuoles in the mitochondria and some of the mitochondrial cristae were broken. This supported the hypothesis that SIRT1 plays a regulatory role in preventing insulin resistance caused by high fat and improves muscle structure and function through exercise. Overexpression of SIRT1 in skeletal muscles increased the number of skeletal muscle nuclei and improved the repair process after muscle injury. SIRT1 can actively participate in muscle hypertrophy by up-regulating anabolic processes and down regulating catabolic mechanisms. Activation of SIRT1 by ginsenoside can significantly promote mitochondrial biogenesis and increase energy metabolism in cardiomyocytes . Low-to-medium-intensity sports can reduce the area of myocardial infarction and fibrosis by increasing the expression of SIRT1, thus, exerting the beneficial effects of sports on cardiovascular diseases . Previous studies have shown that that SIRT1 is a sensor of energy metabolism. By evaluating the serum SIRT1 levels of 80 subjects, it was found that its content was closely and positively correlated with leptin and adiponectin. SIRT1 can be used as a plasma marker in clinical applications to evaluate energy metabolism and can be applied to weight management in obese people [14, 15].
4.2 Effects of SIRT1 inhibitor and aerobic exercise on oxidative stress of gastrocnemius muscle in high-fat mice
A high-fat diet reduced the activity of antioxidant enzymes in the mouse gastrocnemius muscle and significantly aggravated mitochondrial peroxidation damage. Exercise can effectively improve the activity of antioxidant enzymes in muscles and can increase the ability of the body to resist oxidative damage. Therefore, reducing the concentration of 4-HNE and other oxidative damage byproducts has a protective effect on mitochondria. The concentration of 8-OHdG in the mitochondria of the exercise group was significantly lower than that of the SIRT1 inhibitor group. Exercise can promote the expression of SIRT1 in skeletal muscles and can increase mitochondrial biogenesis and oxidative metabolism . The improvement in mitochondrial structure and function is helpful in maintaining insulin sensitivity and normal glucose and lipid metabolism in skeletal muscles. Aerobic exercise can significantly increase the expression of GLUT4 on skeletal muscle cell membranes and promote glucose transport into the muscle tissue. This could be an important reason behind why glycogen in the gastrocnemius muscle of the HE group increased significantly, and its blood glucose level was significantly lower than that in the NH group.
The HES group showed significant reversal of the physiologic changes brought about by exercise on insulin resistance. The activities of antioxidant enzymes such as Mn-SOD and CAT in the HES group were significantly lower than those in the HE group, but there was no significant difference between NH and HES groups. The expression of GPX4 was significantly decreased in HES, which can cause severe lipid peroxidation damage. The concentration of the mitochondrial peroxidation damage marker 8-OHdG was significantly higher in the HES group than in the HE group. The increased NOX1 in HES group promotes peroxide damage and apoptosis. A decrease in the expression of GLUT4 was observed in the gastrocnemius muscle of the HES group-mice in comparison to that of the control- and HE group-mice, along with a decrease in glucose transport, which led to low muscle glycogen content and high blood glucose concentration. The PGC-1α concentration was decreased, and the structure and function of gastrocnemius mitochondria were damaged in the HES group. This further shows that SIRT1 plays a key role in maintaining mitochondrial homeostasis. Studies on nematodes and mice have shown that increasing the activity of NAD⁺ and SIRT1 can enhance the function of mitochondria, maintain metabolic functions, and prolong life span [1, 16]. In this experiment, the NAD⁺/NADH ratio of skeletal muscles increased after exercise, which further increased SIRT1 expression. This data is consistent with the results of previous studies . GPX4 specifically catalyzes lipid peroxides to inactivate them, thereby protecting cells.
4.3 The role of SIRT1/FoxO1 pathway of skeletal muscle in preventing insulin resistance by aerobic exercise
The experimental mice were fed a high-fat diet, and they adhered to an exercise regimen and received an SIRT1 inhibitor. The results showed that Selisistat had a good inhibitory effect on SIRT1, and the expression of SIRT1 in the gastrocnemius decreased significantly. The acetylation of FoxO1, which is present downstream of SIRT1, was increased significantly. Acetylation and deacetylation play important roles in glucose metabolism. NAD+-dependent SIRT1 can promote the deacetylation of key metabolic enzymes, accelerate glucose decomposition, and play an important role in the prevention and treatment of insulin resistance [17, 18]. The results showed that exercise could improve insulin resistance in mice by promoting NAD+ expression, activating SIRT1, and reducing FoxO1 acetylation. Exercise can promote the expression of PGC1-α in skeletal muscles, while SIRT1 inhibitors can reduce PGC1-α. Research has shown that SIRT1 may directly affect PGC1-α, and it may also act on PGC1-α through FoxO1.
Active substances such as resveratrol, apple polyphenol extract , ginsenoside, astaxanthin , and vitamin D can promote the expression of SIRT1 and activate AMPK, FoxO1, PGC1-α , and Nrf2 downstream of SIRT1. These substances can also decrease the expression of SIRT1. The activation of the SIRT1/FoxO1 pathway plays a role in the improvement of brain function and in the treatment of depression and Parkinson's disease (PD). Serum SIRT1 levels in patients with PD and healthy controls were measured using ELISA, and resulting SIRT1 levels in patients with advanced PD decreased in relation to disease severity and cognitive dysfunction . NAD+, miR-34a , and miR204  are the key regulatory factors upstream of SIRT1. The influence of exercise on these regulatory factors in the prevention and treatment of metabolic diseases, depression, and PD needs to be studied further [27, 28].
In conclusion, SIRT1 inhibitor significantly reversed the physiologic adaptations brought about through exercise in mice with insulin resistance induced by a high-fat diet. Addition of the SIRT1 inhibitor resulted in increased body fat content, higher FBG level, and oxidative damage along with the destruction of structural mitochondrial tissue. Glycogen stores depleted, acetylation of FoxO1 increased, and antioxidant enzymatic activity decreased in the SIRT1 inhibitor group. These results support the claim that exercise improves insulin resistance in the skeletal muscle of mice that are fed a high-fat diet through the deacetylation of FoxO1 by SIRT1. The SIRT1/FoxO1 regulatory pathway is crucial in improving insulin resistance and preventing peroxidation damage in skeletal muscles of mice during aerobic exercise.