Highlights
Fatty liver may be associated with autophagy. Supplementation with exercise increases the synergistic effects of regulating and improving the disorder. In this experimental study, some indicators of autophagy under the influence of alpha lipoic acid and strength training were investigated. Improvement of inflammatory, oxidative and autophagy factors was a common result of supplementation and exercise.
Introduction
One of the complications of diabetes is a decrease in muscle mass in the lower extremities
(1). In old age, the breakdown of muscle protein reduces the volume of muscle mass. Lysosomal autophagy and proteasomal ubiquitin systems are activated during muscle atrophy, which ultimately increases insulin resistance. Autophagy is a self-eating system by which damaged organelles and cellular byproducts are degraded in the lysosome to help maintain cellular homeostasis
(2). Autophagy is the major intracellular degradation system and there are three classes: macroautophagy, microautophagy, and chaperone-mediated autophagy
(3). Autophagic substrates are nonselective encapsulated and conjugated by the lipid and active form of chain LC3-II and p62
(2). Beclin-1 and LC3-1 are the main regulators of the autophagy pathway. Physical activity and mobility can reduce the process of autophagy. But diabetes increases oxidative stress and physiological changes such as aging, apoptosis, cell growth, and immune response due to autophagy
(4). However, a moderate-intensity exercise in male mice improved muscle atrophy and inhibited the autophagy system
(1). Also, contradictory findings have been observed acute aerobic exercise is likely to increase the autophagic responses in skeletal muscle
(2). Researchers observed that muscle autophagy may be concomitantly altered along with mitochondrial adaptations over the course of chronic muscle activity. Changes in mtor, Beclin 1, LC3-1, and P62 central autophagy genes play a very important role in sarcopenia due to type 2 diabetes in the elderly
(5). The first selective adapter for autophagy in mammals is P62, which is also a scaffold and stress-induced protein
(6). Multiple P62 domains and their transcription are modulated by oxidative stress
(7). With the onset of autophagy, P62 expression decreases
(8), but if P62 is overexpressed, the protein accumulates too much and has a protective effect on cell survival. If P62 is removed, the formation of LC3II, exosomes, and autophagosomes is disrupted, which increases cell damage
(9). Researchers have shown that low levels of P62 cause ubiquitin-positive masses to form in autophagic mice, so the presence of P62 is essential for the accumulation of these proteins
(10). Atg dependent genes encode specific proteins that regulate autophagy. Among the ATGs present, Atg8 or LC3 is a modulatory protein essential for autophagy, biogenesis, and maturation of autophagosomes. When LC3 is degraded in autophagy it is a sign of autophagy substrate. LC3 also regulates the phagocytic process that is LC3-dependent
(11). Induction of autophagy increases LC3 levels by a large amount in autophagosomes. Some researchers concluded that continuous and intermittent aerobic exercise combined with atorvastatin supplementation played a significant role in it has decrease in autophagy indicators and can reduce that mechanism in type 2 elderly diabetic rats
(12). Also, the effect of 6 weeks of intermittent exercise with selenium nanoparticles on Bcl2 and LC3 gene expression in tumor tissue of female mice showed, LC3 increased in the exercise and the supplement + exercise groups. Consumption of selenium nanoparticles along with intermittent exercise had a synergistic effect against tumor growth, which is probably due to changes in Bcl2 and LC3 gene expression in tumor tissue
(13). Do Keun et al. (2017) studied the effect of exercise on skeletal muscle autophagy in obese rats. The results showed that endurance training had a significant effect on insulin resistance. But no significant difference was observed in Beclin1, P62, Lc3-I, LC3-II as indicators of autophagy in soleus muscle
(14). Several studies have shown that methods of training and supplements are effective in the progressive effects of autophagy
(15). Also, the basal autophagy flux and autophagy protein expression have been reported to be increased after 4 weeks of voluntary running
(16). Some key markers important in examining autophagic flux including the LC3-II/LC3-I ratio, LC3-II, and p62 have been reported to be upregulated
(3). Therefore, the aim of this study was to investigate the effects of resistance training with alpha-lipoic acid consumption on LC3-1 and P62 gene expression on soleus muscle tissue of older mice with type 2diabetes and fatty liver.
Materials and Methods
The experiment was performed on male LC3-1 and P62 gene expression mice. The statistical population was 100 old male Wistar rats that had not been studied until the implementation of the training protocol. Rats were purchased from the Pasteur Institute and transferred to the animal room of the university laboratory. Among them, 35 rats were randomly selected as subjects. In this study, rats were kept in separate polycarbonate cages measuring 20×27×47 cm. The ambient temperature was set at 22±1.4°C, the light cycle was set at 12:12 pm and humidity was set at 55% ± 0.6. Rats were fed with foods produced by the Pars-Iran Company Animal Feed Production Center. Also, the water required for each animal was provided in 500 ml bottles for laboratory animals. All stages of the research were carried out in accordance with the ethical principles of working with animals. After a week of familiarity with the laboratory environment, the rats first became diabetic, and then their liver became oily and was randomly divided into 5 groups: healthy control, diabetic, diabetic + resistance training, Diabetic + supplement, Diabetic + resistance training + supplement.
For inducing type 2 diabetes in the study sample, after 12 hours of fasting, a solution of nicotine amide dissolved in normal saline at a dose of 120 mg/kg. After 15 minutes, streptozotocin was used 0.1 M citrate buffer solution was injected intraperitoneally at a dose of 65 mg/kg. By examining blood samples of the eye and fasting glucose above 426 mg/dl, we confirmed that the rats were diabetic
(18). To creating fatty liver, the healthy control group was fed a standard diet of rodents and the other groups were fed a high-fat diet for 10 weeks
(19). Serum cholesterol concentration was measured as one of the indicators of the fatty liver at the end of the study. Alpha-lipoic acid supplement, at a dose of 50 mg/kg as a solution of dimethyl sulfoxide, 3 times a week by intraperitoneal injection was given to rats in the training + groups used. Rats in the training group, the healthy control group, and the patient control group received 5% normal saline solution. Because 4 groups are equally affected by the physiological effects of the intraperitoneal injection. In this study, all ethical points of working with animals based on the principles of care and use of laboratory animals approved by the Ethics Committee of the Faculty of Medical Sciences of Islamic Azad University, Varamin Pishva Branch was observed.
The normality of the data was assessed using the Shapiro-Wilk test and the homogeneity of variances was assessed by the Leven test. One-way analysis of variance was used to examine the differences between groups. Tukey post hoc analysis for multiple comparisons was used to determine any significant changes between groups (P < 0.05, SPSS statistical software, version 21).
Result