INTRODUCTION
Sarcopenia is a gradual decrease in muscle mass and strength with age [1,2]. Sarcopenia lowers the quality of life of individuals by reducing their physical function and the ability to recover from muscle damage [3,4]. Sarcopenia has been extensively studied in older adults because it is part of the aging process; however, recent studies have demonstrated that it is also prevalent in younger adults with inflammatory diseases [2,5,6]. Aging causes sarcopenia due to decreased levels of growth hormones and testosterone, reduced neuromuscular function, diminished protein synthesis rate, lack of exercise, and nutritional imbalances [2]. However, sarcopenia in young individuals is different from age-induced sarcopenia because it is caused by a lack of physical activity (PA), malnutrition, chronic stress, lack of sleep, and chronic diseases [7,8]. Obesity promotes the secretion of inflammatory cytokines (e.g., tumor necrosis factor [TNF]-α, interleukin [IL]-6) from body fat, activating the mechanistic inflammatory signaling, activin, and metabolic syndrome pathways. This increases muscle protein breakdown and inhibits muscle synthesis, leading to sarcopenia [9-11]. Persistent obesity causes metabolic diseases by activating a vicious cycle of inflammatory cytokine [12,13] secretion and muscle breakdown stimulation [14-16], leading to weight gain in the form of fat [9,17]. Therefore, the prevention or treatment of sarcopenia in young individuals must include not only increased activity levels but also obesity treatment. Treatments for sarcopenia include physical exercise, high-protein diet, and drug intervention [18,19]. Among these, exercise therapy has been demonstrated to be most effective in alleviating sarcopenia in clinical studies [20-22]. However, South Korean adolescents and young adults have lower PA levels than those in other developed countries. Physical activity has decreased further owing to the increased use of smartphones and digital devices, academic stress, and restrictions on outdoor activities during the COVID-19 pandemic. The 2021 Student Physical Activity Assessment System results revealed that only 23.4% of male and 8.8% of female middle and high school students engaged in PA for more than 60 min a day. In addition, the obesity rate has increased, from 13.8% in 2019 to 17.5% in 2021 in male students, and from 8.1% to 9.1% in female students over the same period. The rate of PA among young individuals decreased from 58.3% in 2015 to 47.9% in 2021, a decrease of more than 10% over 6 years (Korea Disease Control and Prevention Agency, 2022 National Health and Nutrition Survey). Considering this realistic situation, increasing PA or providing nutritional prescriptions for the treatment (prevention) of sarcopenia in young patients with obesity is ineffective.
Lithium is a drug used in the treatment of mental disorders, such as bipolar disorder, and plays an important role in neuropsychiatry [23]. Recently, interest in the anti-inflammatory effects of lithium on tissues has increased [24,25]. Obesity-induced chronic inflammation causes muscle fiber damage, mitochondrial dysfunction, and muscle protein synthesis disorders [26-27]. Therefore, the reduction of chronic inflammation by lithium may be effective in preventing and treating sarcopenia in patients with obesity. However, a few studies have investigated the suppressive effect of lithium on sarcopenia in young patients with obesity. Aerobic exercise plays an important role in preventing and treating sarcopenia [28-30]. Endurance exercise exerts a positive effect on the treatment of sarcopenia by maintaining and improving muscle function, improving blood circulation and insulin sensitivity, reducing body fat, and improving immune and metabolic functions [18,31-33]. Most studies conducted on young obese individuals have aimed to reduce body fat. Relatively little information is available regarding sarcopenia in these individuals.
We have previously reported that lithium and low-intensity endurance exercise improved insulin resistance in obese mice. Based on this previous study, we investigated the effects of low-intensity endurance exercise and low-dose lithium treatment on muscle atrophy factors in the skeletal muscles of high-fat diet (HFD)-induced obese rats in the present study. The results of this study are expected to be used as basic scientific data to help prevent and treat sarcopenia in young obese individuals.
METHODS
Experimental rats and experimental procedure
Twenty 6-week-old male Wistar rats were purchased and underwent a 1-week environmental adaptation period. Obesity was induced by administering an HFD (60% calories from fat diet; Harlan Teklad, USA) for 8 weeks. After the obesity induction period, the rats were randomly assigned to four groups as follows: control group (CON), exercise group (Ex), lithium administration group (Li), and lithium + exercise training group (Lex). Lithium administration or exercise was performed for 8 weeks. An HFD was maintained during the 8-week treatment. The Li group was orally administered lithium chloride (LiCl, L4408, Sigma-Aldrich, USA) in 3 mL saline once a day at the same time. The other groups were orally administered the same amount of saline to provide the same stress as oral administration. Food and water were allowed ad libitum during the experimental period. The temperature of the breeding room was maintained at 21℃. The light and dark periods were controlled for 12 h each, and the dark period was controlled from 7:00 AM to 7:00 PM. During the experimental period, body weight and feed intake were measured at the same time every 3 days. This study was approved by the Animal Research Committee of Keimyung University (KM2020-013).
ㆍControl group (CON): saline intubation, n = 5
ㆍ Exercise group (Ex): treadmill, 17 m/min for 30 min, slope 0%, 5 days/week, n = 5
ㆍ Li administration group (Li): 10 mg/kg LiCl, intubation, 5 days/week, n = 5
ㆍ Lithium + exercise training group (LEx): treadmill exercise after lithium administration, n = 5
Lithium administration and exercise training
According to the results of Jung et al. [34], 10 mg/kg LiCl (L4408, Sigma-Aldrich) was dissolved in 300 mL of saline and administered orally once a day to rats, 5 days a week at the same time (10:00 AM). The exercise and lithium combination groups were administered Li before exercise. Endurance exercise was performed 5 days a week using an electric treadmill for laboratory animals (FT-200, Techman Soft.). Exercise intensity was set by modifying the methods described by Wang et al. [35] and Kemi et al. [36]. The exercise intensity was initially 10 m/min for 10 min, and the exercise time and speed were gradually increased over 2 weeks. From the 3rd week, the rats were instructed to exercise at a target intensity of 17 m/min for 30 min.
Sample preparation
After 8 weeks of treatment, the rats were fasted for 12 h and anesthetized with sodium pentobarbital (5 mg/100 g body weight). Body composition was measured using dual-energy X-ray absorptiometry (DEXA) and tissues were extracted. The extracted liver and kidney tissues were fixed with 4% paraformaldehyde, and hematoxylin and eosin (H&E) staining was performed. Skeletal muscles (soleus and tibialis anterior muscles) were extracted, pressed frozen, and stored at −80℃ until analysis. After muscle extraction, the abdominal cavity was opened, 5 mL of blood was collected from the abdominal artery, and treated with 50 μL of heparin to prevent clotting. The blood was centrifuged (1500 × g, 15 min) to extract only the plasma and stored at −80°C until analysis. Visceral fat pads (epididymal, mesenteric, and retroperitoneal) were removed and weighed.
Analysis
Plasma analysis: Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity were measured using an enzyme-linked immunosorbent assay kit (Cayman Technology, USA).
H&E staining. The liver and kidneys were fixed with 4% paraformaldehyde and embedded in paraffin blocks. They were cut into 4-μm thick sections and attached to glass slides. The attached tissues were deparaffinized using xylene (5 min × 3 times), rehydrated (100% ethanol for 3 min, 90% ethanol for 3 min, and 80% ethanol for 3 min), and stained with H&E. The slides were converted into virtual slide files using an Aperio Scanscope XT (Aperio, CA92081, USA).
Western blotting: The tibialis anterior muscle was homogenized with radio-immunoprecipitation assay buffer (containing a protease inhibitor, 4℃). The homogenized sample was frozen, thawed thrice, and centrifuged at 3,500 rpm for 15 min. The proteins were quantified using the Bradford method, dissolved in the Laemmli buffer, distributed on a sodium dodecyl sulfate (SDS)-polyacrylamide gel, and subjected to electrophoresis. The following antibodies were used for immunoblotting: anti-phospho-mTOR (Cell signaling, #5536), anti-mTOR (Cell signaling, #2972), anti-phospho-FOXO1 (Cell signaling, #9464), anti-FoxO1 (Cell signaling, #2880), Atrogin-1 (Santa Cruz Biotechnology, sc-166806), MuRF-1 (Santa Cruz Biotechnology, sc-398608), anti-GAPDH (Cell signaling, USA, #2118). Each secondary antibody was visualized using enhanced chemiluminescence (ECL) and quantified by densitometry (Sigma-Plot 8.0 system).
Statistical analysis
The results of each measurement were calculated as the mean and standard error (SE), and statistical analysis was performed using the SigmaPlot 12.0 statistical package. One-way analysis of variance was performed to verify the differences between groups, and Tukey’s post-hoc test was used. The statistical significance level was set at α = 0.05.
RESULTS
Food consumption and body composition
No significant differences were noted in food intake among the four groups at 8 weeks (Figure 1a). However, after 5 weeks of treatment, the body weights of rats in the Li and LEx groups were significantly lower than those in the CON and Ex groups (Figure 1b, p < 0.05). After 8 weeks of treatment, the lean body mass of rats in the LEx group was significantly higher than that in the CON group in body composition measurements using DEXA (Figure 1c, p < 0.05). However, the distribution and amount of body fat (retroperitoneal, epididymal, and mesenteric fat pads) did not differ between the groups (Figure 1d-f).
Toxicity
The plasma ALT and AST concentrations were measured to evaluate the toxicity of lithium, and concentrations in all four groups were within the normal range, with no significant differences between the groups (Table 1). In addition, no specific cell morphology was observed in the kidney or liver tissues (Figure 2a, b).
Molecular analysis
The effects of endurance exercise and lithium on muscle synthesis and atrophy mechanisms in skeletal muscles were analyzed by measuring the expression of proteins implicated in muscle synthesis and degradation factors. The mechanistic target of rapamycin (mTOR) is an important signaling pathway that regulates cell growth, proliferation, metabolism, and survival and promotes protein synthesis to assist in cell growth and muscle development. No changes in the mTOR activity were noted following exercise or Li treatment (Figure 3b).
Forkhead box O1 (FOXO1) is a transcription factor involved in several biological processes such as cell survival, metabolism, oxidative stress response, aging, and immune regulation. It promotes protein degradation in muscles and affects muscle mass loss. Moreover, it exacerbates insulin resistance in obese individuals and causes adipose tissue dysfunction. It aggravates obesity-related inflammation by increasing the expression of inflammatory cytokines. We found that the expression of phospho-FOXO1 was significantly increased in the Li and LEx groups compared to that in the CON and Ex groups (Figure 3c, p < 0.001). FOXO1 is inactivated when phosphorylated, and an increase in the expression of phospho-FOXO1 indicated its inactivation.
The expression of Atrogin-1 was significantly lower in the Li and LEx groups than in the CON group (Figure 3d, p < 0.001), and that of MuRF-1 was significantly lower in the Ex, Li, and LEx groups than in the CON group (Figure 3e, p < 0.001). Muscle proteins are degraded by the ubiquitin-proteasome system (UPS) and autophagy. Autophagy is a process that degrades and recycles damaged organelles and proteins within cells. The UPS is the primary pathway that degrades damaged or no longer-needed proteins within cells. Atrogin-1 and MuRF-1 are muscle-specific E3 ligases in the UPS that recognize degraded proteins and conjugate ubiquitin to them and are overexpressed during muscle atrophy.
TNF-α is an inflammatory cytokine that plays an important role in muscle atrophy (wasting) by promoting catabolic processes and inhibiting anabolic pathways in skeletal muscles. As white adipose tissue (WAT) increases during obesity, physical and endoplasmic reticulum stress occurs, and the secretion of the inflammatory cytokine (TNFα) along with free fatty acids increases. Consequently, the expression of TNFα in the Ex, Li, and LEx groups was significantly lower than in the CON group (Figure 3f, p < 0.05).
DISCUSSION
Skeletal muscles, the largest organ in the body, account for approximately 40% of the body weight of healthy women and men. A sedentary lifestyle causes the accumulation of intra-abdominal fat, which is accompanied by the infiltration of pro-inflammatory immune cells into the adipose tissue, leading to an increased release of adipokines and the development of a low-grade systemic inflammatory (LGSI) state [37,38]. LGSI is intricately associated with the development of insulin resistance, atherosclerosis, neurodegeneration, and tumor growth [39,40]. In particular, LGSI significantly contributes to muscle atrophy, which disrupts the balance between the anabolic and catabolic actions that build muscles [41,42]. The muscle atrophy mechanism of LGSI is not yet clear; however, it includes increased levels of inflammatory cytokines, oxidative stress, insulin resistance, and increased NF-κB activation [6,43,44]. Elevated levels of inflammatory cytokines (TNF-α, IL-6, and C-reactive protein [CRP]) decrease muscle mass, strength, and muscle function by increasing protein degradation through pathways such as the UPS and lysosomal degradation while impairing protein synthesis [45]. Previous studies have demonstrated that Li exerts its anti-inflammatory effects via several pathways. Representative mechanisms of lithium-induced anti-inflammatory action include a reduction in the levels of inflammatory cytokines, inhibition of glycogen synthase kinase-3β (GSK-3β) activity, regulation of the Toll-like receptor (TLR) pathway, and reduction in oxidative stress [46,47]. In this study, we analyzed the change in the expression of TNFα, an inflammatory cytokine; lithium treatment for 8 weeks significantly reduced its expression in skeletal muscles. In addition, the expression of Atrogin-1 and MuRF-1, which are major components of the UPS, was significantly decreased in the skeletal muscles of rats in the lithium-treated group. We analyzed the activity of FOXO-1, an upstream regulatory factor, to elucidate the regulatory mechanisms of these factors. FOXO1 is a transcription factor of the FOXO family that regulates different cellular processes, including metabolism, apoptosis, oxidative stress resistance, and the immune response. FOXO1 plays an important role in muscle atrophy by regulating the expression of genes involved in protein degradation and inhibiting protein synthesis. FOXO1 increases protein degradation in skeletal muscles by upregulating the expression of Atrogin-1 and MuRF-1. Altogether, the activity of FOXO-1 was significantly reduced in the Li-treated group.
Endurance exercise (aerobic exercise) plays an important role in preventing and improving muscle atrophy [48,49]. Muscle atrophy primarily occurs when muscle protein breakdown exceeds synthesis. Endurance exercise has been reported to prevent muscle atrophy by suppressing inflammation, improving mitochondrial function, and activating satellite serine, which plays a crucial role in muscle regeneration [33,50]. Low-intensity endurance exercise did not affect the mTOR signaling system, a muscle synthesis pathway in skeletal muscles of obese rats; however, it significantly reduced the expression of TNFα and the activity of FOXO1, a muscle atrophy signaling system.
This study investigated the effects of low-intensity endurance exercise and low-dose lithium treatment on muscle atrophy in rats with HFD-induced obesity. Low-intensity endurance exercise and low-dose lithium alone did not affect muscle atrophy. However, the combined treatment inhibited the FOXO1 signaling pathway, a muscle atrophy factor in skeletal muscles, and significantly increased the lean body mass. Considering these results, it is believed that the synergistic effect of combination treatment reduces obesity-induced muscle atrophy. However, this study was conducted in HFD-induced obese rats; therefore, its application is limited. In addition, the study did not measure direct morphological changes in skeletal muscles, other than changes in lean body mass. If this point is clarified in future research, the results of this study will become more valuable.
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