Ganoderic Acid A Enhances Glycometabolism in Mouse Gastrocnemius Muscle During Exercise‐Induced Fatigue via AMPK/PGC‐1α Pathway Activation

ABSTRACT

Exercise‐induced fatigue (EIF) is closely associated with impaired glycometabolism in skeletal muscle. This study investigated the protective effects of Ganoderic acid A (GAA) on glycometabolism in EIF mice and explored its underlying mechanisms. 60 KM mice were divided into five groups: a blank control (BC), a model control (MC), and three GAA‐treated groups (20, 40, and 60 mg/kg/d). After a 7‐week intervention, exhaustive treadmill tests and biochemical analyses were conducted to assess fatigue resistance, metabolic parameters, and molecular pathways. GAA administration significantly prolonged the exhaustive running time (p < 0.01), reduced serum levels of blood urea nitrogen (BUN), creatine kinase (CK), lactate dehydrogenase (LDH), and lactate (LD) (p < 0.05), and increased glycogen content in the liver and gastrocnemius muscle. Mechanistically, GAA increased AMPK phosphorylation, suggesting activation of the AMPK pathway, upregulated PGC‐1α and GLUT4 expression, and enhanced succinate dehydrogenase (SDH) and Ca²⁺‐Mg²⁺‐ATPase activities. The results demonstrate that GAA alleviates EIF by enhancing energy metabolism through the AMPK/PGC‐1α/GLUT4 pathway. These findings highlight GAA as a promising natural supplement for combating exercise‐induced fatigue by improving glycometabolism.

This study investigated the effects of GAA, an active ingredient in Ganoderma lucidum, on exercise‐induced fatigue. GAA effectively enhances exercise performance by improving endurance duration while simultaneously reducing metabolic byproducts and muscle fiber damage. Furthermore, GAA increases glycogen storage and enhances energy metabolism. Additionally, GAA stimulates AMPK and PGC1‐α, which induces GLUT4 expression, thereby improving glucose uptake and mitochondrial function. These findings suggest that GAA alleviates Exercise‐induced fatigue by improving energy metabolism via the AMPK/PGC‐1α/GLUT4 pathway, highlighting its potential as a natural anti‐fatigue supplement.

1. Introduction

Exercise‐induced fatigue is a non‐pathological condition characterized by a reduction in muscle performance resulting from excessive physical activity. This type of fatigue significantly impacts the daily lives of individuals, particularly athletes, military personnel, and manual laborers. It is closely linked to muscular fatigue in the skeletal muscles and an energy imbalance within the body (Hargreaves and Spriet 2020; Hood et al. ²⁰¹⁹; Smith and Newham ²⁰⁰⁷). The primary causes of exercise‐induced fatigue include a deficiency of energy substrates and the accumulation of secondary metabolites (Zhu et al. 2021; Xie et al. ²⁰²⁰; Morales‐Alamo et al. ²⁰¹⁵). The substantial consumption of glucose and glycogen during high‐intensity exercise leads to an inadequate supply of adenosine triphosphate (ATP), thereby impeding the muscle's ability to maintain a continuous contraction state and ultimately resulting in a decline in athletic performance (Hood et al. 2019). Concurrently, the increase in anaerobic metabolism, where glycogen or glucose is converted through the glycolysis pathway, results in the accumulation of lactic acid and other metabolites, which alters the body's microenvironment. This process causes intracellular acidosis within muscle cells, adversely affecting muscle contraction and exacerbating muscle fatigue (Burnley and Jones 2018; Verschueren et al. ²⁰²¹; Westerblad et al. ²⁰⁰²). Therefore, enhancing glucose uptake and increasing glycogen synthesis to reduce the accumulation of secondary metabolites presents an effective strategy for alleviating exercise‐induced fatigue.

Skeletal muscle serves as the primary organ for body movement, converting chemical energy into mechanical energy through contraction and relaxation, thereby facilitating skeletal movement and maintaining body posture (Qu et al. 2021). The gastrocnemius muscle, being a bi‐articular muscle, impacts both the ankle and knee joints and plays an important role in supporting and mobilizing the body during physical activity. Its functionality is influenced by various factors including glucose uptake and mitochondrial function (Yu et al. 2023; Chen et al. ²⁰¹⁷). ATP supply becomes inadequate, resulting in an elevated concentration of adenosine monophosphate (AMP) and a subsequent decrease in the AMP/ATP ratio. (Ke et al. 2018; Viollet et al. ²⁰⁰⁹). AMPK acts as a master regulator of energy metabolism capable of activating downstream PGC‐1α through phosphorylation and deacetylation (Daval et al. 2006; Fang et al. ²⁰²²; Wen et al. ²⁰²¹). The activated AMPK/PGC‐1α pathway regulates ATP levels in the gastrocnemius muscle and influences ATP biosynthesis in mitochondria (Herzig and Shaw 2018). Furthermore, AMPK can activate GLUT4, thereby facilitating the transport of extracellular glucose into cells. This process enhances the oxidative metabolism of glucose, leading to increased ATP production and improved glucose uptake by the gastrocnemius muscle (Qualls‐Histed et al. ²⁰²³; Yap et al. ²⁰²⁰). Some studies have indicated that dietary supplementation with natural substances, such as Rhodiola rosea (Lee et al. 2009), Antrodia camphorata (Liu et al. 2017) and Astragalus membranaceus (Li et al. 2014) can enhance glycogen storage in the body, help stabilize the intracellular AMP/ATP ratio, and mitigate exercise‐induced fatigue.

Traditional Chinese medicines (TCMs) have garnered significant attention for their efficacy in alleviating exercise‐induced fatigue, particularly due to their favorable therapeutic effects and minimal side effects. Ganoderma lucidum, a renowned medicinal fungus, has been utilized in China for millennia. In the earliest monograph on traditional Chinese medicine, the Shennong's Classic of Materia Medica (Shennong Bencao Jing), Ganoderma lucidum was categorized as a top‐class medicine (Hu et al. 2017). Ganoderic acid A, a triterpenoid found in Ganoderma lucidum, exhibits significant pharmacological activities including anti‐inflammatory, antioxidant, antidepressant, and anticancer effects (Ma et al. 2021; Bao et al. ²⁰²¹; Jia et al. ²⁰²³). Research indicates that a combination of ganoderic acid A, B, and C₂ enhances mitochondrial function and increases glycogen content, thus alleviating chemotherapy‐induced fatigue (CRF) in murine models with colon tumors (Abulizi et al. 2021). Although the anti‐fatigue properties of ganoderic acid have been preliminarily established, the exact mechanisms by which a single dose of GAA mitigates exercise‐induced EIF, especially in relation to glucometabolic regulation of skeletal muscle, remain to be fully elucidated. While prior studies have largely concentrated on multi‐component herbal extracts, such as Rhodiola rosea and Astragalus membranaceus, there exists a notable gap in knowledge regarding the therapeutic potential of GAA—a bioactive triterpenoid derived from Ganoderma lucidum—in modulating glucose metabolic pathways within skeletal muscle tissue under EIF conditions. Current literature lacks substantial exploration of this purified compound's capacity to influence critical aspects of muscle energy homeostasis, including glycogen metabolism, glucose uptake mechanisms, and mitochondrial bioenergetics, despite its documented pharmacological activities in other physiological contexts. This dearth of research highlights the necessity for a systematic investigation into GAA molecular targets and regulatory effects on skeletal muscle carbohydrate metabolism during exercise induced fatigue. Accordingly, the present study utilized a mouse model of exercise‐induced fatigue and administered varying doses of GAA to investigate its potential anti‐fatigue mechanisms. This research aims to provide novel ideas and methods for the prevention and treatment of exercise‐induced fatigue.

2. Materials and Methods

2.1. Chemical Reagents

Ganoderic acid A was purchased from Shanghai Yuanye Bio‐technology Co. By employing commercial kits (Nanjing Jiancheng Bioengineering Institute), Urea Assay Kit (C013‐2‐1), Lactate dehydrogenase assay kit (A020‐1‐2), Lactic Acid assay kit (A019‐2‐1) and Creatine kinase assay kit (A032‐1‐1), concentrations were determined. The glycogen in the liver and gastrocnemius muscle, Adenosinetriphosphatase contents and SDH were determined using a glycogen assay kit (LDQB‐E‐1277), an Adenosinetriphosphatase assay kit (LDQB‐E‐1024) and Succinate Dehydrogenase assay kit (LDQB‐E‐1177) from Quanzhou Ledda Qibo Biotechnology Co. Hematoxylin, eosin, and neutral gum were acquired from Beijing Suobao Technology Co. The BCA Protein Assay Kit was obtained from Shanghai Epizyme Biomedical Technology Co. Additionally, 5‐AMP activated protein kinase (AMPK) was purchased from Wuhan Feiyue Biotechnology Co. Phosphorylated 5′‐AMP‐activated protein kinase (p‐AMPK) was purchased from Shanghai Beyotime Biotechnology Co. Glucose transporter 4 (GLUT4) and peroxisome proliferator‐activated receptor γ coactivator‐1 (PGC‐1α) were purchased from Wuhan Sanying Biotechnology Co. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), HRP goat anti‐rabbit IgG, HRP goat anti‐mouse IgG were purchased from Affinity Biosciences. Polyvinylidene fluorides (PVDF) membrane were purchased from Merck Millipore, USA. 5% BSA blocking buffer was purchased from Wuhan Carnoss Technology Co. RIPA lysis buffer, Tris Buffered Saline, and ECL chemiluminescence solution were purchased from Beijing Lanjieke Technology Co.

2.2. Animals and Experimental Design

The animal care and procedures received approval from the Ethics Committee of Guangxi Normal University (Ethics review acceptance code: 202412–006). Sixty male KM mice (4‐week‐old; body weight 13–17 g) were purchased from Hunan SJA Laboratory Animal Co. The KM strain, an outbred model commonly used in regional pharmacological studies, was selected to introduce genetic diversity that may better reflect population heterogeneity. Animals were housed in a controlled environment maintained at a temperature of 22°C ± 1°C and a humidity level of 55%–60%. A 12‐h light/dark cycle was implemented, with lights on from 7:00 AM to 7:00 PM, and the animals had free access to regular food and water. Following a 1 week acclimatization period, the mice were randomly assigned to 5 groups (n = 12) using a random number table:

Blank control group (BC): Gavage with 0.9% saline solution.

Model control group (MC): Gavage with 0.9% saline solution.

Low‐dose GAA group (L‐GAA): Gavage with GAA 20 mg/kg·BW/d.

Middle‐dose GAA group (M‐GAA): Gavage with GAA 40 mg/kg·BW/d.

High‐dose GAA group (H‐GAA): Gavage with GAA 60 mg/kg·BW/d.

The selection of GAA doses (20, 40, and 60 mg/kg/d) was based on prior efficacy studies of Ganoderma triterpenoids in anti‐fatigue and related models (Abulizi et al. 2021), aiming to cover a range from low to high potency to effectively evaluate the dose–response relationship. The intervention groups were gavaged with different doses of GAA solution (prepared with GAA solution dissolved in 0.9% normal saline containing 0.5% CMC sodium). After 56 consecutive days of daily drug intervention, groups (MC) to (H‐GAA) underwent an exhaustive treadmill test, while the BC group served as a non‐running control. Following the final test (within 5 min after the mice were physically exhausted), all mice were sacrificed, and blood, skeletal muscle, liver, heart and kidney were collected. Blood samples were centrifuged at 3000 rpm for 15 min at 4°C to separate serum, and all samples were stored at −80°C for subsequent detection.

2.3. Exhaustive Treadmill Test

Increased exhaustive running time indicates enhanced exercise endurance. In the exhaustive running test, prolonged running duration reflects improved exercise capacity and reduced fatigue (Ji et al. 2024; Liu et al. ²⁰²²). In this experiment, the exhaustive treadmill test was conducted using an animal treadmill (Beijing Zhong Shi Di Chuang Science and Technology Development Co.). The exercise program was based on the method of Chen (Chen et al. 2021) et al. with minor modifications. Prior to the formal experiment, the mice underwent a one‐week acclimation period, during which they ran at a speed of 10 m/min for 30 min. The incline of the treadmill was fixed at 10°C throughout the testing period. The mice began running at 10 m/min, with the speed increasing by 2 m/min every 5 min until reaching 20 m/min. This speed was maintained for 10 min, after which it was increased to 25 m/min until exhaustion, at which point the exhaustion time was recorded. The state of exhaustion was defined as the inability of the mouse to return to the treadmill belt despite gentle mechanical stimulation (nudging) for more than 10 s. This quantifiable endpoint was typically preceded by observable signs of severe fatigue, such as the animal consistently remaining in the posterior third of the treadmill and exhibiting significant difficulty in maintaining running motion. To minimize bias, the exhaustive treadmill test and subsequent sample collection and analysis were performed by investigators who were blinded to the group assignments throughout the experiment.

2.4. Biochemical Serum and Tissue Analysis

Mice were not fasted prior to blood sampling. Blood collection was performed immediately after the exhaustive treadmill test (upon exhaustion, within 5 min) for MC and GAA‐treated groups or directly from the cage for the BC group. It is important to note that the handling and brief restraint required for blood sampling can induce acute stress in mice, which may elevate serum lactate levels above true resting baselines. Serum concentrations of BUN were measured enzymatically using the urease method. Serum LDH, CK, and LD levels, hepatic and muscular glycogen content, and gastrocnemius energy metabolism markers including SDH and Ca²⁺‐Mg²⁺‐ATPase activities were determined by colorimetric assays according to the manufacturer's protocols. The activity of Ca²⁺‐Mg²⁺‐ATPase in the gastrocnemius muscle was assessed as follows. Muscle samples were first homogenized in ice‐cold buffer containing 50 mM Tris–HCl (pH 7.4), 1 mM EDTA, and 0.25 M sucrose. The homogenate was then centrifuged at 12,000 × g for 10 min at 4°C, and the resulting supernatant was used as an enzyme source. The reaction mixture for the determination of Ca²⁺‐Mg²⁺‐ATPase activity consisted of 50 mM Tris–HCl (pH 7.4), 5 mM MgCl₂, 1 mM CaCl₂, 5 mM ATP, and enzyme samples. After incubation at 37°C for 30 min, the reaction was terminated by the addition of trichloroacetic acid. The released inorganic phosphate (Pi) was then quantified by the ammonium molybdate malachite green method. The absorbance of the resulting complex was measured at 700 nm. The Ca²⁺‐Mg²⁺‐ATPase activity was calculated as the amount of Pi released per unit of protein per unit time (μmol Pi/mg protein/h), and the protein concentration was determined by Bradford method. All assays were performed in strict accordance with the commercial kit instructions provided by the manufacturer.

2.5. Histopathological Examination

The gastrocnemius muscle was fixed in a 10% neutral buffer formalin solution for 24 h. To ensure consistency, tissue samples were collected from the mid‐belly region of the gastrocnemius muscle. Subsequently, the tissue was dehydrated using ethanol, clarified in xylene and embedded in paraffin. A 4 μm section was then sliced from the paraffin‐embedded tissue using a microtome (Lecia Biosystems, Germany). Tissue sections were stained with hematoxylin–eosin and mounted with neutral gum. Microphotographs were captured using a microscope (Olympus, Japan) at 100× magnification from randomly selected fields of view to document the morphological changes in skeletal muscle tissue.

2.6. Western Blot Analysis

To extract protein from the gastrocnemius muscle, RIPA lysis buffer was applied on ice. Following centrifugation, the supernatant was collected, and protein concentration was quantified using the Bicinchoninic Acid (BCA) method. Proteins including GAPDH, AMPK, p‐AMPK, PGC1‐α and GLUT4 were separated using an SDS‐PAGE gel electrophoresis system (Shanghai Tanon Technology Co.). The protein was transferred onto a polyvinylidene fluoride (PVDF) membrane by an electro‐transfer membrane system (Shanghai Tanon Technology Co.) and blocked in 5% BSA for 1 h at room temperature. The PVDF membrane was then incubated overnight at 4°C with antibodies against GAPDH (1:5000), AMPK (1:1000), p‐AMPK (1:1000), PGC1‐α (1:1000), and GLUT4 (1:1000). After washing with 1 × TBST, HRP Goat Anti‐Rabbit IgG (1:5000) and HRP Goat Anti‐Mouse IgG (1:5000) were applied for 1 h at 25°C. Detection was performed using ECL chemiluminescence solution in a chemiluminescent imaging system (Shanghai Tanon Technology Co.), after a final wash with 1 × TBST. The relative protein expression was normalized to GAPDH using Image J software.

2.7. Statistical Analysis

Data collection and analysis were performed by researchers blinded to the experimental groups. The experimental data were analyzed statistically using SPSS 27.0 (IBM corporation, Armonk, NY, USA). One‐way ANOVA followed by Tukey's post hoc test was used to assess differences among groups. Data are expressed as mean ± standard deviation (mean ± SD). Results with * p < 0.05, ** and p < 0.01 were deemed statistically significant. Graphs were generated using GraphPad Prism version 9.00 (GraphPad software, San Diego, CA, USA). The sample size (n = 12 per group) provided adequate statistical power to detect significant differences despite the genetic variability inherent in the outbred KM model, and the observed treatment effects were consistent within groups.

3. Results

3.1. Effects of GAA on Body Weight and Organ Index in Mice

Body weights were monitored weekly throughout the experimental duration. No statistically significant intergroup differences in body weight were observed following 56 days of GAA administration (p > 0.05). Organometric analysis of critical organ indices (heart, liver, and kidney) further demonstrated that chronic GAA treatment did not induce significant weight fluctuations in these vital organs, suggesting a physiological tolerance to the intervention (Table 1 and Figure 1).

TABLE 1.

Absolute organ weights of experimental animals.

Group/Organs	Heart (%)	Liver (%)	Kidney (%)
BC	0.44 ± 0.02	4.02 ± 0.14	1.30 ± 0.07
MC	0.43 ± 0.02	4.06 ± 0.31	1.27 ± 0.07
L‐GAA	0.43 ± 0.02	4.00 ± 0.18	1.28 ± 0.07
M‐GAA	0.46 ± 0.03	3.99 ± 0.18	1.29 ± 0.09
H‐GAA	0.46 ± 0.03	4.06 ± 0.21	1.30 ± 0.08

FIGURE 1.

Effects of GAA on body weight and organ index. (A) The experiment scheme, (B) body weight, (C) heart index, (D) liver index, (E) kidney index. Data are expressed as mean ± SD, N = 12.

3.2. Effects of GAA on Exhaustive Running Time of Mice

The exhaustive running time was not measured in the BC group because these mice were not subjected to the treadmill test. Compared with the MC group, the exhaustive running time of each dose of GAA treatment group was significantly longer, with statistically significant differences observed (p < 0.05, p < 0.01) (Figure 2). These results suggest that GAA has significant anti‐fatigue activity and can increase exercise tolerance in a dose‐dependent manner. In addition, Figures [Link], [Link] show the line graphs of the daily exercise time changes of each mouse in every experimental group throughout the entire experimental period, presenting the continuous characteristics of the exercise performance of the mice during the experiment in a visual manner.

FIGURE 2.

Effects of GAA on exhaustive running time of mice. Note: The BC group was not subjected to the treadmill test and therefore is not included. Data are expressed as mean ± SD, N = 12.*p < 0.05, **p < 0.01 compared with the MC group.

3.3. The Effects GAA on Serum Biochemical Parameters in Fatigue Mice

Standard serum biomarkers of exercise‐induced fatigue—including blood urea nitrogen (BUN), creatine kinase (CK), lactate dehydrogenase (LDH), and lactate acid (LD)—were analyzed to quantify physiological stress responses (Chen et al. 2022). As shown in Figure 3B, exhaustive exercise provoked a marked 2.1‐fold elevation in CK levels in the MC group compared to BC group (p < 0.01). GAA intervention elicited significant dose‐dependent attenuation of CK activity, with high‐dose administration (H‐GAA) achieving 63% normalization compared to MC values (p < 0.01). This pattern was consistently replicated across other biomarkers: exhaustive exercise induced significant elevations in BUN (187% increase), LDH (2.3‐fold increase), and LD (209%) relative to BC (all p < 0.01). However, all GAA‐treated cohorts demonstrated progressive metabolic recovery, with medium‐ and high‐dose groups (M‐GAA/H‐GAA) exhibiting reductions in BUN (34%–58%, p < 0.05), LDH normalization (41%–67%, p < 0.01), and decrements in LD (39%–72%, p < 0.01) compared to MC counterparts (Figure 3A,C,D). Notably, the high‐dose regimen restored all parameters to near‐baseline levels, suggesting a potent dose‐dependent normalization of exercise‐perturbed metabolic homeostasis. It is worth noting that the relatively high serum lactate in the BC group may be attributed to handling stress during blood collection, but this did not affect intergroup comparisons as all animals underwent similar procedures. In conclusion, all mice eventually reached exhaustion. GAA treatment prolonged the time to exhaustion and improved metabolic parameters, including reduced lactate accumulation.

FIGURE 3.

The effects of GAA on serum biochemical parameters. (A) Blood urea nitrogen (BUN), (B) Creatine kinase (CK), (C) Lactic dehydrogenase (LDH), (D) Lactic acid (LD). Data are expressed as mean ± SD. *p < 0.05 and **p < 0.01 compared with the BC group; #p < 0.05 and ##p < 0.01 compared with the MC group.

3.4. Regulation of GAA on Energy Metabolism in Fatigue Mice

Perturbations in energy homeostasis, particularly the depletion of diminished glycogen reserves in striated muscle and hepatic tissue, are recognized as significant contributors to exercise‐associated fatigue. In the present study, post‐exertional analysis revealed that glycogen stores in the model control cohort were significantly suppressed compared to baseline controls (p < 0.01), highlighting the occurrence of energy depletion during exercise. In contrast, chronic administration of GAA led to the restoration of these energy reserves, with a dose‐dependent increase observed in muscle glycogen content (p < 0.05, p < 0.01). However, liver glycogen levels did not exhibit a clear dose‐dependent trend across the GAA‐treated groups. Similar trends were observed in ATP metabolism. Untreated models exhibited severely compromised Ca²⁺‐Mg²⁺‐ATPase activity (p < 0.01), and while GAA intervention effectively normalized this activity (p < 0.05, p < 0.01), the response did not show a distinct dose‐dependent relationship.

The results of succinate dehydrogenase (SDH) activity further support this notion. SDH, a pivotal enzyme in the TCA cycle, was suppressed post‐exercise in the MC group (p < 0.01). However, under GAA treatment, SDH activity demonstrated a dose‐responsive reactivation (p < 0.05, p < 0.01) (Figure 4).

FIGURE 4.

Regulation by GAA of energy metabolism. (A) Muscle glycogen (MG), (B) Liver glycogen (LG), (C) Succinate dehydrogenase activity (SDH), (D) Ca²⁺/Mg²⁺‐ATPase. Data are expressed as mean ± SD. *p < 0.05 and **p < 0.01 compared with the BC group; #p < 0.05 and ##p < 0.01 compared with the MC group.

3.5. Correlation Analysis of Biochemical Parameters

The study revealed significant associations between key biochemical parameters and exhaustive running time, with distinct positive and negative correlations observed (Figure 5). Energy metabolism markers, including LG, MG, SDH, and ATPase, demonstrated positive relationships with exercise duration (r > 0.5, p < 0.01 or p < 0.001), indicating that enhanced energy substrate storage and metabolic enzyme activity are closely linked to improved exercise endurance. In contrast, indicators of metabolite accumulation, namely blood BUN, LD, LDH, and CK, showed significant inverse correlations with exhaustive running time (r < −0.4, p < 0.01 or p < 0.001), reflecting that reduced accumulation of metabolic waste products contributes to alleviated fatigue. Notably, metabolite clearance efficiency was found to interact with energy metabolism regulation, as evidenced by the inverse relationship between BUN and both MG (r = −0.42, p < 0.01) and ATPase levels (r = −0.38, p < 0.05). These findings suggest that GAA may exert anti‐fatigue effects through modulation of energy metabolism pathways, potentially enhancing exercise capacity by optimizing the metabolic microenvironment in fatigued organisms.

FIGURE 5.

Biochemical parameters correlation analysis. BUN, blood urea nitrogen; CK, creatine kinase; LDH, lactate dehydrogenase; LD, lactate; MG, muscle glycogen; LG, liver glycogen; SDH, succinate dehydrogenase; ATPase, Ca²⁺‐Mg²⁺‐ATPase. Correlation coefficients were calculated using Pearson's correlation analysis. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

3.6. Histopathological Analysis

Exhaustive exercise‐induced skeletal structural alterations in skeletal muscle, consistent with the pathophysiology of exercise‐related fatigue (Duan et al. 2017). Histological analysis revealed a dose‐dependent mitigation of muscle tissue damage across GAA intervention groups compared to untreated models. While control specimens maintained normal myofiber organization and cellular architecture, exercise‐challenged subjects exhibited characteristic structural disruptions (Figure 6). Notably, high‐dose GAA administration demonstrated optimal preservation of histological integrity, approaching baseline tissue organization. Lower GAA concentrations partially attenuated damage progression, though residual myofibril disorganization persisted. These findings indicate GAA's dose‐responsive protective effects against exercise‐induced musculoskeletal deterioration, potentially contributing to its anti‐fatigue efficacy through structural preservation mechanisms.

FIGURE 6.

Histopathological analysis of muscle tissue. (A) BC, (B) MC, (C) L‐GAA, (D) M‐GAA, (E) H‐GAA. Magnification ×100.

3.7. Molecular Mechanism of GAA Regulating Glycometabolism in Gastrocnemius Muscle Tissue of Fatigue Mice

AMPK serves as a master regulator of energy metabolism within the body. Its downstream factor PGC‐1α, which is primarily localized in high‐energy metabolizing tissues, including the heart, skeletal muscle, and kidneys (Shen et al. 2019). To elucidate the molecular mechanism by which GAA influences glycometabolism, Western blotting was employed to examine the effects of GAA on the expression levels of AMPK, p‐AMPK, PGC‐1α and Glut4 proteins. The results indicated that the expression of AMPK protein remained relatively unchanged across all experimental groups. Compared with the BC group，the exhaustive treadmill test significantly reduced the protein levels of p‐AMPK, PGC‐1α and Glut4 (p < 0.05 and p < 0.01). Following GAA treatment, the protein levels of p‐AMPK in the muscle of fatigue mice significantly increased across all groups (p < 0.01), while the protein expression of PGC‐1α and Glut4 were significantly elevated in the medium and high dose groups (p < 0.05 and p < 0.01) (Figure 7).

FIGURE 7.

Molecular mechanisms of GAA regulation of energy metabolism in fatigued mouse muscle tissue. (A) AMPK, p‐AMPK, PGC‐1α, Glut4 and GAPDH protein electrophoresis images, (B) relative expression of muscle AMPK protein, (C) relative expression of muscle p‐AMPK protein, (D) relative expression of muscle PGC1‐α protein, (E) relative expression of muscle GLUT4 protein. Data are expressed as mean ± SD. *p < 0.05 and **p < 0.01 compared with the BC group; #p < 0.05 and ##p < 0.01 compared with the MC group.

4. Discussion

Exercise‐induced fatigue represents a transient physiological impairment of bodily functions resulting from prolonged or intensive physical activity, which may precipitate various pathophysiological complications. Failure to achieve timely recovery from this condition can lead to diminished athletic performance, increased injury risk, and adverse health outcomes (Tan et al. 2020; Paillard ²⁰¹²). Our experimental findings demonstrate that GAA exerts significant anti‐fatigue effects through multimodal mechanisms involving metabolic regulation, tissue protection, and modulation of energy homeostasis.

The exhaustive treadmill test, a validated preclinical model for evaluating exercise endurance (Yan et al. 2022), revealed that GAA administration dose‐dependently extended the duration of exhaustive running in murine subjects. This enhancement in exercise capacity occurred independently of variations in body weight, confirming that the observed effects derive specifically from GAA's pharmacological activity rather than nonspecific physiological changes. The dose–response relationship provides crucial guidance for potential clinical translation, suggesting that optimal dosing parameters require careful stratification.

Comprehensive serum biomarker analysis elucidated GAA's capacity to mitigate exercise‐induced metabolic perturbations. The compound significantly reduced levels of lactate dehydrogenase and blood urea nitrogen, indicating a reduction in the dominance of anaerobic glycolysis and protein catabolism during strenuous activity (Coco et al. 2020; Westerblad et al. ²⁰¹⁰; Zhu et al. ²⁰²⁰). These biochemical improvements correlated with histopathological findings, which showed preserved muscle architecture in GAA‐treated subjects. The observed reductions in serum CK and LDH leakage further confirm GAA's cytoprotective effects against exercise‐induced myocyte damage (Robergs et al. 2004; Li et al. ²⁰¹⁸; Zhao et al. ²⁰¹⁵; Xu et al. ²⁰¹⁸), substantiating its role in maintaining membrane integrity during metabolic stress.

The analysis of glycogen metabolism underscores the critical role of GAA in preserving energy reserves. Exhaustive exercise resulted in the depletion of both hepatic and muscular glycogen stores; however, GAA intervention effectively maintained these essential carbohydrate reservoirs (Kjaer 1998; Kent et al. ²⁰¹⁶; Vigh‐Larsen et al. ²⁰²¹). This glycogen‐sparing effect likely delays the transition to anaerobic metabolism during prolonged activity, thereby enhancing endurance capacity. The extended time to exhaustion and improved metabolic parameters observed in GAA‐treated mice suggest that GAA may mitigate metabolic dysfunction by enhancing mitochondrial bioenergetics and TCA cycle function, thereby delaying exhaustion onset. Moreover, GAA has been shown to enhance key enzymatic regulators of cellular energetics, particularly succinate dehydrogenase and Ca²⁺‐Mg²⁺‐ATPase, which are pivotal in regulating tricarboxylic acid cycle flux and calcium homeostasis, respectively (Glancy and Balaban 2012; Cao et al. ²⁰²³; Berti et al. ²⁰¹⁷). The restoration of ATP‐generating capacity, coupled with improved calcium handling, collectively contributes to sustained muscle contractility during physical exertion. It is worth noting that the increase in SDH activity suggests enhanced mitochondrial function, while upregulated SDH and PGC‐1α indicate enhanced mitochondrial biogenesis signaling; direct evidence such as mitochondrial DNA copy number or electron microscopy is needed to confirm increased mitochondrial quantity. Future research could further explore this aspect to comprehensively elucidate the mechanisms underlying GAA's regulatory effects on energy metabolism in fatigue mice. Notably, the present study found a dose‐dependent increase in muscle glycogen in model animals after GAA intervention, but liver glycogen did not show a clear trend, and the medium‐dose group instead showed the lowest level. The differential glycogen response between muscle and liver, particularly the non‐dose‐dependent trend in hepatic glycogen, may reflect tissue‐specific metabolic roles. The gastrocnemius muscle, as the primary site of energy consumption during exercise, likely prioritized glycogen replenishment via the activated AMPK/GLUT4 pathway. In contrast, hepatic glycogen metabolism is subject to more complex systemic regulation, and the observed fluctuation could be related to dynamic energy partitioning between tissues under GAA intervention at different doses.

Mitochondrial bioenergetics has emerged as a central mechanism underlying the anti‐fatigue properties of GAA. The activation of the AMPK/PGC‐1α signaling pathway is consistent with findings that demonstrate its role in mitochondrial biogenesis (Herzig and Shaw 2018). Our investigation extends these observations by establishing GAA's capacity to potentiate this pathway, particularly through increasing AMPK phosphorylation, which is associated with pathway activation. This upstream regulation increases PGC‐1α expression, subsequently enhancing mitochondrial biosynthesis and functional capacity (Yap et al. 2020; Martinez‐Redondo et al. ²⁰¹⁵). Furthermore, our results show increased total GLUT4 protein expression. However, this does not confirm functional translocation. Future studies should examine membrane GLUT4 localization and glucose uptake. The compound's dual action on glucose transport, via GLUT4 membrane trafficking, and on mitochondrial efficiency creates synergistic metabolic advantages, optimizing substrate availability while improving ATP yield per glucose molecule (Katz and Westerblad 2014; Feng et al. ²⁰²²).

The limitations of this study should be considered. This study provides supportive but indirect evidence for the activation of the AMPK/PGC‐1α/GLUT4 pathway. Specifically, the current Western blot analysis does not include more direct functional readouts, such as the phosphorylation status of PGC‐1α (p‐PGC‐1α), direct evidence of GLUT4 translocation, or the identification of the specific AMPK phosphorylation site(s) involved. At the same time, only one mouse model (the outbred KM strain) was used in this study, which introduces genetic variability, and future studies should validate these findings in standardized inbred models with human trials to assess their translational relevance. Although rodent models provide essential mechanistic insights, interspecies differences in exercise physiology and metabolic regulation necessitate confirmation in human subjects. Furthermore, additional investigations should explore potential sex‐specific responses, long‐term safety profiles, and optimal dosing regimens across diverse populations.

These collective findings position GAA as a multifaceted therapeutic candidate for managing exercise‐induced fatigue. By concurrently addressing energy substrate availability, mitochondrial efficiency, and myocyte preservation, GAA demonstrates unique potential for athletes and occupational workers engaged in high‐intensity physical activities. The elucidation of its AMPK/PGC‐1α‐mediated mechanisms provides a pharmacological foundation for the development of natural product‐derived ergogenic aids.

This study confirmed that GAA could increase the protein expression of p‐AMPK, PGC‐1α and Glut4 in gastrocnemius muscle of tired mice, suggesting that the improvement in glucose metabolism is associated with AMPK pathway activation. However, due to the limitation of experimental conditions, the phosphorylation site of AMPK was not identified, and the expression of P‐PGC‐1α and plasma membrane Glut4 or glucose transport was not detected. In the future, specific antibodies will be used to determine the activated form of AMPK, and relevant indicators will be further explored to fully reveal the mechanism of GAA regulating glucose metabolism. While the current study demonstrates GAA‐mediated improvements in glycometabolic markers (e.g., p‐AMPK, PGC‐1α, Glut4) and energy metabolism, the absence of direct GAA concentration data in muscle tissue represents a limitation. Future studies could employ mass spectrometry to quantify GAA levels in gastrocnemius muscle and perform correlative analyses with metabolic parameters (e.g., glycogen content, SDH activity, ATPase function). Such investigations would help establish whether GAA exerts its effects in a dose‐dependent manner within the muscle microenvironment, thereby providing a more comprehensive understanding of its therapeutic mechanism.

5. Conclusions

This study investigated the effects of GAA, an active ingredient in Ganoderma lucidum, on exercise‐induced fatigue. GAA effectively enhances exercise performance by improving endurance duration while simultaneously reducing metabolic byproducts and muscle fiber damage. Furthermore, GAA increases glycogen storage and enhances energy metabolism. Additionally, GAA appears to stimulate AMPK and PGC1‐α, which induces GLUT4 expression, thereby improving glucose uptake and mitochondrial function. These findings suggest that GAA may have the potential to serve as an effective anti‐fatigue dietary supplement in the future. However, it is important to emphasize that these results are derived from a preclinical murine model. The significant interspecies physiological differences between mice and humans necessitate further validation through clinical trials before any definitive translational claims can be made. Future research will continue to explore additional molecular mechanisms underlying the anti‐fatigue effects of GAA, as well as determine the biologically effective and optimal dosage of GAA for producing anti‐fatigue effects in humans. Furthermore, we aim to investigate the mechanisms by which GAA influences abnormal glucose metabolism, thereby supporting its clinical application in metabolic diseases such as diabetes and insulin resistance.

Author Contributions

Jialin Zhu: data curation (equal), methodology (equal), software (equal), visualization (equal), writing – original draft (equal). Fenglin Peng: conceptualization (equal), data curation (equal). Lin Zhang: investigation (equal), methodology (equal). Yanju Guo: project administration (equal), resources (equal). Weiguo Liu: software (equal). Jingrong Li: validation (equal). Shuju Shang: visualization (equal). Bohan Zhang: writing – review and editing (equal). Taotao Qiu: conceptualization (equal), funding acquisition (equal), writing – review and editing (equal).

Funding

This work was supported by the Innovation Project of Guangxi Graduate Education, YCSW2024212. The Exercise Intervention Key Technology and Product Development Project, SYS2024002.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Daily running time of each rat in the EC group.

Figure S2: Daily running time of each rat in the L‐GAA group.

Figure S3: Daily running time of each rat in the L‐MAA group.

Figure S4: Daily running time of each rat in the H‐MAA group.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.