Next Article in Journal
The Effects of SGLT2 Inhibitors on Lipid Profile and Kidney Function in Patients with Chronic Kidney Disease Regardless of Diabetes and Hypertension Status
Previous Article in Journal
Metabolomics Insights into Gut Microbiota and Functional Constipation
Previous Article in Special Issue
Exerkines and Sarcopenia: Unveiling the Mechanism Behind Exercise-Induced Mitochondrial Homeostasis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Metabolites
Volume 15
Issue 4
10.3390/metabo15040270
metabolites-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Combined Effects of Ketogenic Diet and Aerobic Exercise on Skeletal Muscle Fiber Remodeling and Metabolic Adaptation in Simulated Microgravity Mice

by
Jun Chen
1,2,†,
Wenjiong Li
3,†,
Liang Yu
4,
Bowei Zhang
4,
Zhili Li
3,
Peng Zou
3,
Bai Ding
3,
Xiaoqian Dai
3,* and
Qirong Wang
2,*
1
School of Exercise and Health, Shanghai University of Sport, Shanghai 200438, China
2
Sports Nutrition Center, National Institute of Sports Medicine, Beijing 100029, China
3
National Key Laboratory of Space Medicine, Beijing 100094, China
4
Department of Exercise Physiology, Beijing Sport University, Beijing 100084, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Metabolites 2025, 15(4), 270; https://doi.org/10.3390/metabo15040270
Submission received: 13 March 2025 / Revised: 11 April 2025 / Accepted: 11 April 2025 / Published: 13 April 2025
(This article belongs to the Special Issue Muscle Metabolic Response and Adaptation to Exercise, Diet, and Environment)
Browse Figures
Versions Notes

Abstract

:
Objective: Prolonged microgravity environments impair skeletal muscle homeostasis by triggering fiber-type transitions and metabolic dysregulation. Although exercise and nutritional interventions may alleviate disuse atrophy, their synergistic effects under microgravity conditions remain poorly characterized. This study investigated the effects of an 8-week ketogenic diet combined with aerobic exercise in hindlimb-unloaded mice on muscle fiber remodeling and metabolic adaptation. Methods: Seven-week-old male C57BL/6J mice were randomly divided into six groups: normal diet control (NC), normal diet with hindlimb unloading (NH), normal diet with hindlimb unloading and exercise (NHE), ketogenic diet control (KC), ketogenic diet with hindlimb unloading (KH), and ketogenic diet with hindlimb unloading and exercise (KHE). During the last two weeks of intervention, hindlimb unloading was applied to simulate microgravity. Aerobic exercise groups performed moderate-intensity treadmill running (12 m/min, 60 min/day, and 6 days/week) for 8 weeks. Body weight, blood ketone, and glucose levels were measured weekly. Post-intervention assessments included the respiratory exchange ratio (RER), exhaustive exercise performance tests, and biochemical analyses of blood metabolic parameters. The skeletal muscle fiber-type composition was evaluated via immunofluorescence staining, lipid deposition was assessed using Oil Red O staining, glycogen content was analyzed by Periodic Acid–Schiff (PAS) staining, and gene expression was quantified using quantitative real-time PCR (RT-qPCR). Results: Hindlimb unloading significantly decreased body weight, induced muscle atrophy, and reduced exercise endurance in mice. However, the combination of KD and aerobic exercise significantly attenuated these adverse effects, as evidenced by increased proportions of oxidative muscle fibers (MyHC-I) and decreased proportions of glycolytic fibers (MyHC-IIb). Additionally, this combined intervention upregulated the expression of lipid metabolism-associated genes, including CPT-1b, HADH, PGC-1α, and FGF21, enhancing lipid metabolism and ketone utilization. These metabolic adaptations corresponded with improved exercise performance, demonstrated by the increased time to exhaustion in the KHE group compared to other hindlimb unloading groups. Conclusions: The combination of a ketogenic diet and aerobic exercise effectively ameliorates simulated microgravity-induced skeletal muscle atrophy and endurance impairment, primarily by promoting a fiber-type transition from MyHC-IIb to MyHC-I and enhancing lipid metabolism gene expression (CPT-1b, HADH, and PGC-1α). These findings underscore the potential therapeutic value of combined dietary and exercise interventions for mitigating muscle atrophy under simulated microgravity conditions.

1. Introduction

In recent years, advancements in space exploration and long-term microgravity research have increasingly drawn the attention of sports scientists toward the effects of weightlessness on muscle health. Numerous studies indicate that microgravity conditions can cause significant structural and functional alterations in skeletal muscles, particularly in antigravity muscles, including muscle atrophy, changes in muscle fiber types, and reduced motor function [1], subsequently impairing overall health. Consequently, developing effective interventions to mitigate or reverse the adverse effects of microgravity on muscles has become a critical research area in sports science.
The ketogenic diet (KD), characterized by high fat, moderate protein, and very low carbohydrate intake [2,3], stimulates ketone body production—primarily, β-hydroxybutyrate (β-HB) and acetoacetate—as alternative energy substrates [4]. These metabolites enhance muscular endurance and strength by providing efficient energy sources for skeletal muscles, thereby potentially improving athletic performance [5,6]. Emerging evidence suggests that KD may further support muscular adaptive changes by promoting muscle tissue remodeling under metabolic stress, including shifts in the muscle fiber-type composition, which is critical for optimizing endurance and muscular function [7]. For instance, studies on aged mice have demonstrated that KD interventions increase skeletal muscle mass and enhance functional capacity. Specifically, prolonged KD reduces the cross-sectional area of Myosin Heavy Chain (MyHC)-IIb fibers while increasing the area of MyHC-IIa fibers in senescent muscles [8], highlighting its potential in mitigating age-related muscle disorders, such as sarcopenia. These findings align with prior observations that KD preserves muscle mass in sedentary animals and facilitates MyHC isoform transitions from type IIb to IIx [9]. Conversely, reduced skeletal muscle activity triggers metabolic remodeling, characterized by decreased fat oxidation capacity, enhanced glycolytic metabolism, and a slow-to-fast twitch myosin isoform transition [10,11]. Notably, aerobic exercise, a conventional intervention against sarcopenia, has also been shown to stimulate ketogenesis [12], suggesting potential synergy when combined with KD. Animal studies further report enhanced endurance performance following KD regimens lasting 5 [13], 8 [14], and 12 [15] weeks. Previous research has also indicated that combining aerobic exercise with KD preferentially enhances fatty acid utilization and lipid oxidation without negatively affecting muscle glucose utilization or glycogen content [16], holding promise for optimizing lipid metabolism to improve endurance performance. Although prior studies highlight that either exercise or nutritional interventions alone can counteract muscle atrophy across various pathophysiological conditions [17,18,19], the combined effects of KD and aerobic exercise on microgravity-induced muscular atrophy remain underexplored.
Based on existing evidence, we hypothesize that combining KD with aerobic exercise under simulated microgravity conditions will synergistically enhance skeletal muscle mass and exercise capacity in mice. We propose that this combined intervention modulates the muscle fiber-type composition and metabolic adaptations, thus delaying or preventing muscle atrophy progression. This study aims to elucidate the integrated effects of KD and aerobic exercise on skeletal muscle adaptability, providing novel insights for aerospace medicine, sports science, and nutritional interventions. Using 7-week-old C57BL/6J mice, this study investigates how an 8-week KD combined with aerobic exercise influences skeletal muscle fiber characteristics and exercise performance in simulated microgravity, aiming to establish foundational insights for targeted interventions against microgravity-induced muscle atrophy.

2. Materials and Methods

2.1. Animals and Diets

Seven-week-old male C57BL/6J mice (20–25 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed under standard laboratory conditions. Mice were housed in cages containing 4–5 animals each and maintained in a controlled environment with a temperature of 20–24 °C, relative humidity of 45–55%, and a 12 h light/dark cycle. All experimental procedures were approved by the Ethics Committee of the National Key Laboratory of Space Medicine (approval no.: ACC-IACUC-2021-010). Following a one-week acclimation period, mice were stratified by body weight and randomly assigned to six experimental groups using SPSS 22.0 (IBM, Armonk, NY, USA) to ensure a balanced allocation. The experimental design comprised two dietary conditions (normal diet (ND) and ketogenic diet (KD)) and three intervention types: control (C), hindlimb unloading (HU), and hindlimb unloading combined with aerobic exercise (HU + EX). Sample sizes were determined as follows: the control groups (NC and KC) contained 8 mice each, while the HU and HU + EX groups (NH, NHE, KH, and KHE) were expanded to 12–14 mice to account for potential attrition during prolonged interventions. The detailed group allocation is presented in Table 1.
Mice in the normal diet groups received a standard diet (AIN93G, Shanghai Puloteng Biotechnology Co., Ltd., Shanghai, China) containing 7% fat, 17.8% protein, and 64.3% carbohydrates (3.601 kcal/g). Mice in ketogenic diet groups were fed a ketogenic formula (TP-201450, Beijing BioPeak Biotechnology Co., Ltd., Beijing, China) containing 76.1% fat, 8.9% protein, and 3.5% carbohydrates (4.056 kcal/g). All animals had ad libitum access to food and water. Following a one-week acclimation period, mice were randomly allocated into experimental groups as described above. Body weight was measured weekly every Sunday between 9:00 and 10:00 AM, and blood samples were collected from tail veins for the determination of glucose and ketone concentrations using FreeStyle Optium Neo Blood Glucose/β-Ketone Test Strips (Abbott Diabetes Care Ltd., Manchester, UK). A schematic overview of the experimental design is presented in Figure 1.

2.2. Hindlimb Unloading Model

Simulated weightlessness was achieved through hindlimb unloading (HU) using the tail suspension method. Briefly, medical adhesive tape was applied 2–3 cm from the tip of the tail to attach it to an overhead chain, suspending the animal’s hindlimbs to avoid contact with the cage floor. The forelimbs maintained contact, allowing for free movement within the cage. Environmental conditions were maintained as described above. The hindlimb unloading intervention lasted for 2 weeks.

2.3. Aerobic Exercise Protocol

Aerobic exercise was conducted using a motor-driven treadmill (JD-PT, Jide Technology Co., Ltd., Beijing, China), based on the experimental protocol described by Ma et al. [14]. Mice in the exercise groups (NHE and KHE) underwent a 3-day treadmill acclimation period (5 m/min, 10 min/day, and 0° incline). Subsequently, formal training consisted of daily treadmill exercise at 12 m/min for 40 min/day, 5 days per week, over an 8-week intervention period. During the last 2 weeks, exercise intervention was carried out under simulated microgravity conditions, as described by Wang et al. [20] (Figure 1).

2.4. Respiratory Exchange Ratio (RER) Test

Respiratory metabolism was assessed before and after hindlimb unloading. Mice were placed individually in sealed respiratory chambers (Shenzhen Kerano Electronics Technology Co., Ltd., Shenzhen, China), and oxygen (O2) consumption and carbon dioxide (CO2) production were continuously recorded until the CO2 concentration reached approximately 5000 ppm. RER was calculated as VCO2/VO2.

2.5. Assessment of Endurance Exercise Performance

Exercise endurance capacity was evaluated using an incremental treadmill running protocol. The exercise capacity test was adapted from a study by Dougherty et al. [21], with minor modifications. Following a 1-week adaptation phase (5 m/min for 10 min/day), mice underwent incremental treadmill exercise starting at 14 m/min for 2 min, followed by stages of 16 m/min (3 min), 18 m/min (25 min), 20 m/min (15 min), 22 m/min (15 min), 24 m/min (15 min), and 26 m/min until exhaustion. Exhaustion was defined as the inability of mice to maintain pace despite repeated gentle encouragement [22].

2.6. Tissue Sampling

Upon completion of the final endurance exercise test, mice were anesthetized using sodium pentobarbital. Blood samples were collected via retro-orbital sinus puncture and centrifuged to obtain serum, which was stored at −20 °C. Left soleus muscles were immediately excised, weighed, snap-frozen in liquid nitrogen, and stored at −80 °C for subsequent analysis. The right soleus muscle was embedded in an optimal cutting temperature compound (OCT, Sakura, Tokyo, Japan), rapidly frozen in liquid nitrogen, and stored for subsequent sectioning and staining.

2.7. Plasma Biochemical Assessment

Serum levels of triglycerides (TG, A110-1-1); total cholesterol (TC, A111-1-1); high-density lipoprotein cholesterol (HDL-C, A112-1-1); low-density lipoprotein cholesterol (LDL-C, A113-1-1); insulin; creatine kinase (CK, A032-1-1); lactate dehydrogenase (LDH, A020-2-2); blood urea nitrogen (UREA, C013-2-1); insulin (A019-2-1); and blood lactate (LA, H203-1-1) were measured using an automated biochemical analyzer (Huake Bio, Beijing, China). All procedures were conducted in strict accordance with the manufacturer’s instructions provided by the reagent kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.8. Oil Red O Staining

The OCT-embedded tissue samples were retrieved from liquid nitrogen and sectioned to a thickness of 8 µm using a cryostat (Leica, Wetzlar, Germany). The sections were mounted onto slides, with six sections per tissue sample, and placed in a slide box. The slides were stored at −20 °C until further use. Lipid staining was performed following the instructions provided by the Oil Red O Staining Kit (G1260, Solarbio, Beijing, China). Tissue sections were initially washed in 60% isopropanol for 2 min, followed by staining with the Oil Red O working solution for 2–5 min. After staining, the sections were rapidly differentiated in 60% isopropanol and washed with distilled water. Subsequently, the sections were counterstained with hematoxylin for 30 s, differentiated with acid alcohol for 1–5 s, and rinsed under running water until a blue color was achieved. Finally, the sections were mounted with glycerol gelatin and observed under a light microscope, with images captured.

2.9. Periodic Acid–Schiff (PAS) Staining

The prepared frozen sections were retrieved and sequentially stained with periodic acid, Schiff’s reagent, and hematoxylin, in accordance with the instructions provided in the Periodic Acid–Schiff (PAS) Staining Kit (G1281, Solarbio, Beijing, China). Following washing and dehydration, the sections were mounted with neutral gum. The tissue sections were observed under an optical microscope, and images were captured and saved.

2.10. Quantitative Real-Time Polymerase Chain Reaction

A total of 10 mg of soleus muscle tissue was weighed, and 1 mL of the TRIzol reagent was added (Invitrogen, Carlsbad, CA, USA) to homogenize the sample. After separating the supernatant, 200 µL of chloroform was added and mixed thoroughly. The mixture was left to stand at room temperature for 5 min. Then, it was centrifuged at 12,000 rpm at 4 °C for 10 min to collect the aqueous phase. A total of 500 µL of isopropanol was added and mixed gently. Then, the mixture was left to stand at room temperature for 10 min. It was centrifuged again at 12,000 rpm at 4 °C for 10 min, the supernatant was discarded, and the RNA pellet was washed with 1 mL of pre-cooled 75% ethanol. The mixture was centrifuged at 7500 rpm at 4 °C for 5 min, the supernatant was removed, and the pellet was left to air-dry for 15 min to remove residual ethanol. An appropriate volume of DEPC-treated water was added to fully dissolve the RNA pellet and measure the RNA concentration. Genomic DNA was removed using the Master Mix Kit RR047A, Takara Bio USA, Mountain View, CA, USA), and RNA was reverse transcribed using the same kit. Quantitative RT-PCR was performed using TB Green Premix Ex Taq (RR420A, Takara Bio USA, Mountain View, CA, USA). The 18S rRNA gene was used as the internal reference, and relative mRNA expression was calculated using the 2−ΔΔCt method. The RT-PCR primers used in this study are listed in Table 2.

2.11. Histological Staining

For staining, the cryosections were blocked with 3% bovine serum albumin (BSA) in DPBS for 1 h at room temperature, followed by incubation overnight at 4 °C with a cocktail of primary antibodies, including MyHC I (BA-D5, DSHB, Iowa City, IA, USA); MyHC IIa (SC-71, DSHB); MyHC IIb (BF-F3, DSHB); and Laminin (ab11575, Abcam, Cambridge, UK). After washing, the cryosections were incubated with a cocktail of secondary antibodies, including Alexa Fluor 647-conjugated goat anti-mouse IgG2b (A21242, Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA); Alexa Fluor 568-conjugated goat anti-mouse IgG1 (A21124, Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA); Alexa Fluor 488-conjugated goat anti-mouse IgM (A21042, Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA); and Alexa Fluor 405-conjugated goat anti-rabbit IgG (A31556, Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), for 1 h at room temperature. Images of the cryosections were acquired using a Leica Thunder Imager 3D Assay (Leica Application Suite X (LAS X) 3.6.0, Leica) and analyzed with the Image J software (version 1.53t; NIH, Bethesda, MD, USA). To minimize observer bias, the researchers performing image acquisition and analysis were blinded to group assignments. Tissue sections were labeled with anonymous codes (e.g., A1–F6) prior to staining, and group identities were revealed only after statistical comparisons were completed.

2.12. Controlled Variables and Measurement Validation

To ensure reproducibility and transparency, all experimental variables, measurement instruments, and validation references are summarized in Table 3. Key biochemical assays followed standardized protocols provided by reagent manufacturers, while histological and molecular analyses were validated against established methodologies.

2.13. Statistical Analysis

Statistical analysis was performed via GraphPad Prism 10.0 (GraphPad Software, San Diego, CA, USA) and the SPSS 22.0 software (IBM, Armonk, NY, USA). The experimental results are presented as means ± standard deviations (means ± SD). After confirming a normal distribution, one-way analysis of variance (ANOVA) was employed for statistical analysis, with pairwise comparisons conducted via the least significant difference (LSD) method, and post hoc tests were performed via Duncan’s multiple range test. A value of p < 0.05 was considered statistically significant.

3. Results

3.1. Establishment of the Ketogenic Diet Model

As shown in Figure 2A, the weight difference among the mice from all groups gradually increased with age. During the first 4 weeks, the weight gain in the KD group was significantly lower than that in the normal control (NC) group (p < 0.05). However, after week 5, no significant changes in the weight difference were observed between the dietary groups. Figure 2B demonstrates that compared to the NC group, the ketone levels in the KD group were significantly higher from week 1 to week 8 (0.73 ± 0.06 mmol/L, p < 0.01), while blood glucose levels remained unchanged (Figure 2C). Research indicates that when ketone levels reach 0.5–3.0 mmol/L, the body is considered to be in a state of nutritional ketosis [26]. The RER, which represents the ratio of carbon dioxide produced to oxygen consumed during metabolism, reflects the balance between fat and carbohydrate metabolism. As seen in Figure 2D, the RER in the KD group was significantly lower than that in the NC group (p < 0.05), maintaining a value of around 0.7. Studies suggest that an RER value between 0.7 and 1.0 indicates a mixed utilization of fat and carbohydrates for energy [27]. These results suggest that a short-term ketogenic diet intervention in mice significantly reduces weight gain, increases ketone production, enhances the process of ketone body generation from fat breakdown, and lowers the RER, indicating successful establishment of the ketogenic diet in a mouse model.

3.2. Effects on Body Weight and Skeletal Muscle Mass in Simulated Weightlessness Mice

As shown in Figure 3A, both the KC and NC groups exhibited significant body weight reduction (p < 0.05) after two weeks of tail suspension. Compared to the NC group, the soleus muscle wet weight-to-body weight ratio decreased significantly in the NH and NHE groups (p < 0.05; Figure 3B). Notably, the NH group showed a marked elevation in skeletal muscle Atrogin1 mRNA expression (p < 0.05; Figure 3D). Previous studies have established that reduced muscle wet weight-to-body weight ratios and increased expression of skeletal muscle atrophy markers (MuRF-1 or Atrogin1) are hallmarks of muscle atrophy [28]. These results confirm the successful establishment of a weightlessness-induced skeletal muscle atrophy model. In contrast, the KH and KHE groups displayed significant reductions in the soleus muscle wet weight-to-body weight ratio compared to the KC group (p < 0.05), though no significant changes were observed in MuRF-1 or Atrogin1 mRNA expression (Figure 3C,D). Intergroup comparisons further revealed significant decreases in soleus muscle MuRF-1 and Atrogin1 mRNA expression between the NC and KC, NH and KH, and NKE and KHE groups (p < 0.05). Collectively, these findings indicate that weightlessness conditions induce muscle atrophy in both standard and ketogenic diet groups, whereas a ketogenic diet alone or combined with exercise intervention effectively counteracts this atrophy.

3.3. Effects on Glucose and Lipid Metabolism in Mice

To examine changes in glycogen and lipid droplet content in skeletal muscle after a single bout of exhaustive exercise, PAS and Oil Red O staining were performed on skeletal muscle sections from the mice. PAS staining results (Figure 4A,C) show no significant changes in glycogen content across all groups. Additionally, Oil Red O staining results (Figure 4B,D) indicate that there were no significant differences in lipid droplet content within each dietary group. However, intergroup comparisons revealed that the lipid droplet content in skeletal muscle was significantly higher in the NC group compared to the KC group, the NH group compared to the KH group, and the NKE group compared to the KHE group (p < 0.05). To assess changes in blood ketone and glucose levels before and after exhaustive exercise, test strips were used. These results suggest that neither a regular nor a ketogenic diet, in combination with a weightless environment or aerobic exercise alone, significantly alters lipid droplet content in skeletal muscle. However, the ketogenic diet significantly increased lipid droplet content in skeletal muscle. Moreover, the increase in circulating ketone levels at rest indicates that the ketogenic diet enhances fat metabolism, elevating blood ketone concentrations to support prolonged exercise by promoting the utilization of ketones as an energy source.
To further examine changes in the metabolic levels of mice, biochemical analyses were conducted to measure blood metabolic parameters. As shown in Table 4, compared to the NC group, serum levels of TC, TG, LDL-C, and HDL-C in the NH and NHE groups showed no significant changes, but insulin levels were significantly increased (p < 0.05). Compared to the KC group, serum TC and HDL levels in the KH and KHE groups were significantly reduced (p < 0.05), and serum LDL levels in the KHE group were also significantly decreased (p < 0.05). Interestingly, the serum TC level in the KH group was significantly increased (p < 0.05). Notably, there were no significant changes in urea and insulin levels between the groups. Furthermore, intergroup comparisons revealed that compared to the NC group, both the KC and NH groups had significantly lower levels of serum TC, TG, LDL-C, HDL-C, urea, and insulin (p < 0.05). Additionally, compared to the NHE group, the KHE group had significantly lower insulin levels (p < 0.05), along with significantly higher levels of TC and HDL-C (p < 0.05), while the remaining parameters showed no significant differences. Therefore, simulated weightlessness did not affect serum lipid levels in mice on a regular diet. However, a ketogenic diet alone led to increased serum lipid levels, whereas a ketogenic diet combined with exercise partially mitigated the lipid increase induced by the ketogenic diet. These results suggest that simulated weightlessness impairs the blood glucose regulation ability in mice after exhaustive exercise. Furthermore, a ketogenic diet intervention alone did not significantly influence the reduction in blood glucose after exhaustive exercise in weightless mice, and the combination of exercise with a ketogenic diet did not show any improvement in this regard.

3.4. Effects on Skeletal Muscle Fiber-Type Composition

To investigate changes in muscle fiber types, immunofluorescent staining was performed on skeletal muscle to analyze the percentage of each muscle fiber type relative to the total fiber count. As shown in Figure 5, compared to the NC group, the proportion of MyHC-I muscle fibers in the NH group was significantly reduced (p < 0.05), while the proportion of MyHC-IIb muscle fibers was significantly increased (p < 0.05). In the NHE group, the proportion of MyHC-I fibers was significantly decreased (p < 0.05), while no significant change was observed in the proportion of MyHC-IIb fibers. Compared to the KC group, the KH group showed a significant decrease in the proportion of MHC-I fibers (p < 0.05) and a significant increase in the proportion of MyHC-IIb fibers (p < 0.05). However, no significant changes were observed in the fiber proportions in the KHE group. Additionally, intergroup comparisons revealed that compared to the NC group, the KC group had a significantly higher proportion of MyHC-I fibers (p < 0.05) and a significantly lower proportion of MyHC-IIb fibers (p < 0.05). The proportion of MyHC-I fibers in the KHE group was significantly higher than that in the KH group (p < 0.05), while the decrease in MyHC-IIb fibers showed a trend but did not reach statistical significance. Notably, no significant changes in the proportion of MyHC-IIa fibers were observed across all groups. In conclusion, both regular and ketogenic diet interventions in mice under simulated weightlessness lead to a shift from slow-twitch to fast-twitch fibers in the soleus muscle. While aerobic exercise can partially suppress the increase in MyHC-IIb fibers in the soleus muscle of mice on a regular diet, it does not effectively prevent the reduction in slow-twitch fibers. However, the combination of a ketogenic diet and exercise has a significantly greater effect than exercise alone, substantially improving the muscle fiber-type transition induced by simulated weightlessness. Furthermore, under normal conditions, the ketogenic diet promotes the conversion of fast-twitch fibers to slow-twitch fibers in the soleus muscle.

3.5. Effects on Skeletal Muscle-Related Gene Expression

Previous studies have shown that FGF21, SIRT1, and PGC-1α are key regulators of aerobic metabolism in skeletal muscle and play pivotal roles in facilitating ketone body breakdown and fatty acid oxidation [29,30]. As shown in Figure 6A–C, compared with the NC group, the NH group exhibited significantly higher FGF21 mRNA expression in skeletal muscle (p < 0.05), whereas both PGC-1α and FGF21 mRNA levels were significantly upregulated in the NHE group (p < 0.05), with no notable changes in SIRT1 mRNA. Compared with the KC group, skeletal muscle PGC-1α and FGF21 mRNA levels were significantly decreased in the KH group (p < 0.05), whereas SIRT1 mRNA expression was markedly increased in the KHE group (p < 0.05). Furthermore, intergroup comparisons revealed that PGC-1α, SIRT1, and FGF21 mRNA expression in the KC and KHE groups were significantly higher than those in the NC and NHE groups, respectively (p < 0.05), whereas FGF21 mRNA in the KH group was significantly lower than that in the NH group (p < 0.05).
In addition, OXCT is an essential ketolytic enzyme in skeletal muscle and reflects the ketone body breakdown process [31]. CPT-1b serves as a crucial fatty acid transporter involved in β-oxidation [32,33], whereas HADH is the rate-limiting enzyme in the β-oxidation of fatty acids in skeletal muscle [34]. Changes in the expression of these enzymes can thus indicate the extent of lipid metabolism in murine skeletal muscle. As shown in Figure 6D–F, there were no significant changes in CPT-1b, HADH, or OXCT mRNA expression in the NH and NHE groups compared with the NC group. However, relative to the KC group, OXCT mRNA in the KH group was significantly reduced (p < 0.05), and CPT-1b mRNA in the KHE group was also markedly decreased (p < 0.05). Moreover, intergroup comparisons indicated that CPT-1b, HADH, and OXCT mRNA levels in the KC, KH, and KHE groups were significantly higher than those in the NC, NH, and NHE groups, respectively (p < 0.05).

3.6. Effects on Exercise Capacity in Mice

To investigate the fatigue recovery capacity of mice following a single bout of exhaustive exercise, a biochemical analyzer was used to measure blood markers associated with fatigue. As shown in Table 5, compared to the NC group, the serum LA levels in the NH and NHE groups were significantly elevated (p < 0.05), with no significant difference observed between the NH and NHE groups. Additionally, the serum LD levels in the KHE group were significantly lower than those in the KC group (p < 0.05), while no significant differences were observed for other markers. Intergroup comparisons revealed that the serum LA levels in the KC group were significantly higher than those in the NC group, suggesting that the ketogenic diet leads to increased lactate accumulation post-exercise, which may be associated with the metabolic shifts induced by the ketogenic diet.
The single exhaustive exercise test serves as a measure of exercise endurance. As shown in Table 3, compared to the NC group, the exercise exhaustion time was significantly reduced in the NH and NHE groups (p < 0.05). When compared to the KC group, the exercise exhaustion time was also significantly reduced in the KH and KHE groups (p < 0.05), although the KHE group exhibited a significantly longer exhaustion time than the KH group (p < 0.05). Furthermore, intergroup comparisons revealed no significant differences in exercise exhaustion times between the NC and KC groups or between the NH and KH groups. However, the exercise exhaustion time in the KHE group was significantly longer than that in the NHE group (p < 0.05). In conclusion, both standard and ketogenic diets led to a reduction in exercise endurance under simulated weightlessness. Neither aerobic exercise nor ketogenic diet interventions alone were able to counteract the decline in exercise endurance induced by simulated weightlessness, whereas the combination of a ketogenic diet and aerobic exercise significantly mitigated the decrease in exercise endurance in mice.

4. Discussion

4.1. Effects of Ketogenic Diet Combined with Aerobic Exercise on Body Weight and Skeletal Muscle in Simulated Weightlessness Mice

The ketogenic diet (KD), characterized by high-fat, low-carbohydrate, and moderate-protein intake, has gained considerable attention for its potential health benefits [35,36]. Previous research has demonstrated that a 4-week KD intervention effectively prevented age-related weight gain in rodents [9], consistent with other published reports [37,38,39,40,41,42,43,44]. Similarly, the present study showed that after 4 weeks of dietary intervention, C57BL/6 mice in the KD group exhibited significantly lower body weight gains compared to controls, beginning from the second week, corroborating earlier findings [45,46,47]. However, this weight difference progressively diminished with a longer intervention duration, suggesting the development of keto-adaptation, potentially explaining the phased weight changes observed. Notably, these results contrast with findings from Shimizu et al. [16], Wallace et al. [8], and Zhou et al. [48], possibly due to differences in intervention duration, dietary protein content, or animal strain.
Currently, limited research has explored the effects of KD under simulated microgravity conditions. Typically, body weight, muscle wet weight, and the muscle-to-body-weight ratio are standard parameters for evaluating muscle atrophy in rodent models, with MuRF-1 and Atrogin1 recognized as biomarkers for skeletal muscle wasting [49]. In the present study, 2 weeks of hindlimb unloading significantly decreased body weight and the soleus muscle wet weight-to-body weight ratio and elevated Atrogin1 expression in mice on a normal diet, confirming muscle atrophy induction. Notably, subsequent dietary and exercise interventions revealed that KD alone or combined with exercise significantly downregulated MuRF-1 and Atrogin1 expression in soleus muscles, mitigating muscle atrophy progression. Intriguingly, KD alone also demonstrated protective effects on soleus muscle integrity and mass under normal conditions. However, further extensive studies are required to validate these findings comprehensively.

4.2. Effects of Ketogenic Diet Combined with Aerobic Exercise on Skeletal Muscle Fiber Remodeling in Simulated Weightlessness Mice

Sandonà et al. [50] demonstrated that prolonged microgravity exposure results in approximately a 35% reduction in the cross-sectional area of soleus muscle fibers (anti-gravity, slow-twitch muscles) in mice, accompanied by homogeneous atrophy across all fiber types (MyHC-I, MyHC-IIa, and MyHC-IIx). Concurrently, a significant slow-to-fast fiber phenotype transition was observed, characterized by decreased proportions of MyHC-I fibers and increased proportions of fast isoforms (MyHC-IIa and MyHC-IIb). This shift may impair oxidative metabolic capacity and consequently reduce muscular endurance [51]. Consistent with these observations, our results reveal that a 2-week HU intervention significantly reduced the proportion of MyHC-I fibers while increasing MyHC-IIb fibers in mice, correlating with decreased aerobic endurance, as evidenced by exhaustive exercise tests. Previous research has indicated that KD could induce beneficial skeletal muscle remodeling by promoting the conversion of MyHC-IIb to MyHC-I fibers, potentially counteracting muscle fiber loss associated with sarcopenia [8,9,52,53,54]. Furthermore, exercise-induced reductions in MyHC-IIb isoforms have been associated with improved muscular aerobic capacity [55,56,57]. Our data support these findings, demonstrating that the combination of KD and aerobic exercise intervention produced superior outcomes compared to exercise alone, effectively ameliorating fiber-type transitions induced by simulated microgravity.
Notably, the FGF21-SIRT1-PGC-1α signaling pathway plays a critical role in promoting myocyte differentiation and facilitating anaerobic-to-oxidative fiber-type conversions [30]. Consistent with this mechanism, our study found significantly increased mRNA expression levels of PGC-1α, SIRT1, and FGF21 in the soleus muscles of mice receiving the ketogenic diet compared to regular diet controls. Immunofluorescence staining further confirmed a higher proportion of MyHC-I fibers and reduced MyHC-IIb fibers in the KD groups relative to regular diet groups. However, KD alone did not completely counteract the fiber-type shift induced by hindlimb unloading, as evidenced by the persistent slow-to-fast fiber conversion in the KH group. These pathophysiological alterations significantly impaired aerobic endurance, as demonstrated by reduced performance in exhaustive exercise tests. In contrast, the combined KD and aerobic exercise intervention yielded superior outcomes, effectively mitigating fiber-type transitions induced by simulated microgravity and significantly improving exercise performance.

4.3. Effects of Ketogenic Diet Combined with Aerobic Exercise on Metabolic Adaptations in Skeletal Muscle in Simulated Weightlessness Mice

Our study demonstrated that KD significantly increased lipid droplet content in skeletal muscle and enhanced systemic lipid metabolism, consistent with the findings of Ogura et al. [9] and Huang et al. [58]. KD promotes greater utilization of lipids as a fuel source, thereby reducing reliance on glucose metabolism [14,59,60]. Our experimental data show that KD effectively maintained blood glucose levels and preserved intramuscular glycogen content, suggesting improved metabolic flexibility. The observed increase in intramuscular lipid droplets provides a mechanistic explanation for the enhanced aerobic capacity observed with KD and its combination with aerobic exercise interventions. Chronic KD combined with aerobic exercise specifically promoted the remodeling of the muscle fiber composition toward aerobic fiber predominance in hindlimb-unloaded mice, optimizing metabolic efficiency by increasing lipid droplets and maintaining glycogen stores. These coordinated adaptations facilitated a metabolic shift toward enhanced lipid and ketone body utilization, significantly improving endurance performance and reducing fatigue biomarkers following exhaustive exercise.
As the mitochondrial transporter for fatty acid β-oxidation [61], CPT-1b cooperates with HADH, a key rate-limiting enzyme in this process [34]. Ketogenic diet upregulated CPT-1b and HADH expression in response to increased circulating free fatty acids and intramuscular lipid accumulation [62]. Enhanced β-oxidation elevated NAD+ levels, which activated SIRT1 expression. Subsequently, SIRT1 deacetylated and activated PGC-1α [63], forming a positive regulatory loop that further amplified lipid-metabolizing enzyme expression [64,65]. Concurrently, the FGF21-SIRT1-PGC-1α axis promoted a fast-to-slow fiber transition [30], counteracting an HU-induced fiber-type shift and establishing a slow-twitch dominant phenotype. This fiber-type remodeling synergized with persistent ketogenic intervention to augment intramuscular triglyceride storage—a recognized biomarker of aerobic capacity [58,66] that enhances endurance performance through sustained energy provision [67]. This adaptive cascade ultimately elevated CPT-1b and HADH expression in soleus muscles, collectively enhancing lipid metabolism efficiency. The ketogenic diet thus establishes a self-reinforcing cycle where improved aerobic capacity drives ketogenesis [68,69], with hepatic ketone bodies entering skeletal muscles via circulation. Notably, PGC-1α-mediated OXCT upregulation (the rate-limiting enzyme in ketolysis) facilitated ketone conversion into TCA cycle intermediates [70], completing the metabolic adaptation that significantly improved aerobic endurance. Remarkably, despite profound lipid/ketone metabolic alterations, the combined intervention maintained stable glycogen reserves and glycemic control (albeit with slight reductions), enabling sustained exercise capacity. This metabolic flexibility—characterized by ketone-dominated energy production, enhanced lipid utilization, and preserved glucose homeostasis—collectively optimized murine aerobic endurance.

5. Conclusions

This study demonstrated that a ketogenic diet combined with aerobic exercise effectively mitigates simulated microgravity-induced skeletal muscle atrophy and endurance impairments. The beneficial effects were mediated through the following: (1) increasing the proportion of MyHC-I fibers, (2) upregulating lipid metabolism-related genes (CPT-1b and HADH), and (3) suppressing muscle atrophy markers (MuRF-1). Future studies should incorporate larger sample sizes, utilize multi-omics approaches (e.g., metabolomics and proteomics), and perform detailed mechanistic validations. Moreover, exploring gender-specific responses and long-term efficacy will further advance translational applications in aerospace medicine, sports science, and nutritional interventions.

Author Contributions

J.C., X.D. and L.Y. conceived the study and designed the experiments. J.C. and W.L. carried out the experiments. J.C., B.Z., W.L., Z.L., P.Z., B.D. and X.D. contributed to the sample collection. J.C. and W.L. analyzed the data. J.C. wrote the manuscript. X.D., Q.W. and L.Y. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Special Fund for Basic Scientific Research for Central Universities (Grant No. 2022YB019); the National Natural Science Foundation of China (Grant No. 32071168); the Open Project of the State Key Laboratory of Basic and Application of Aerospace Medicine (Grant No. SMFA20K04); and the National Key Laboratory of Space Medicine, China Astronaut Research and Training Center (Grant No. SMFA18B04).

Institutional Review Board Statement

The animal study protocols were approved by the Ethics Committee of the National Key Laboratory of Space Medicine (approval no.: ACC-IACUC-2021-010).

Informed Consent Statement

Not applicable.

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article.

Acknowledgments

The authors gratefully acknowledge the excellent assistance of Yaxuan Liu, Xiangsheng Pang, and Shiming Li.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Trappe, T.A.; Minchev, K.; Perkins, R.K.; Lavin, K.M.; Jemiolo, B.; Ratchford, S.M.; Claiborne, A.; Lee, G.A.; Finch, W.H.; Ryder, J.W.; et al. NASA SPRINT Exercise Program Efficacy for Vastus Lateralis and Soleus Skeletal Muscle Health during 70 Days of Simulated Microgravity. J. Appl. Physiol. 2024, 136, 1015–1039. [Google Scholar] [CrossRef] [PubMed]
  2. Li, R.-J.; Liu, Y.; Liu, H.-Q.; Li, J. Ketogenic Diets and Protective Mechanisms in Epilepsy, Metabolic Disorders, Cancer, Neuronal Loss, and Muscle and Nerve Degeneration. J. Food Biochem. 2020, 44, e13140. [Google Scholar] [CrossRef]
  3. Zhou, Z.; Hagopian, K.; López-Domínguez, J.A.; Kim, K.; Jasoliya, M.; Roberts, M.N.; Cortopassi, G.A.; Showalter, M.R.; Roberts, B.S.; González-Reyes, J.A.; et al. A Ketogenic Diet Impacts Markers of Mitochondrial Mass in a Tissue Specific Manner in Aged Mice. Aging 2021, 13, 7914–7930. [Google Scholar] [CrossRef] [PubMed]
  4. Fukao, T.; Lopaschuk, G.D.; Mitchell, G.A. Pathways and Control of Ketone Body Metabolism: On the Fringe of Lipid Biochemistry. Prostaglandins Leukot. Essent. Fatty Acids 2004, 70, 243–251. [Google Scholar] [CrossRef] [PubMed]
  5. McSwiney, F.T.; Wardrop, B.; Hyde, P.N.; Lafountain, R.A.; Volek, J.S.; Doyle, L. Keto-Adaptation Enhances Exercise Performance and Body Composition Responses to Training in Endurance Athletes. Metabolism. 2018, 83, e1–e2. [Google Scholar] [CrossRef]
  6. Kephart, W.C.; Pledge, C.D.; Roberson, P.A.; Mumford, P.W.; Romero, M.A.; Mobley, C.B.; Martin, J.S.; Young, K.C.; Lowery, R.P.; Wilson, J.M.; et al. The Three-Month Effects of a Ketogenic Diet on Body Composition, Blood Parameters, and Performance Metrics in CrossFit Trainees: A Pilot Study. Sports 2018, 6, 1. [Google Scholar] [CrossRef]
  7. Bloemberg, D.; Quadrilatero, J. Rapid Determination of Myosin Heavy Chain Expression in Rat, Mouse, and Human Skeletal Muscle Using Multicolor Immunofluorescence Analysis. PLoS ONE 2012, 7, e35273. [Google Scholar] [CrossRef]
  8. Wallace, M.A.; Aguirre, N.W.; Marcotte, G.R.; Marshall, A.G.; Baehr, L.M.; Hughes, D.C.; Hamilton, K.L.; Roberts, M.N.; Lopez-Dominguez, J.A.; Miller, B.F.; et al. The Ketogenic Diet Preserves Skeletal Muscle with Aging in Mice. Aging Cell 2021, 20, e13322. [Google Scholar] [CrossRef]
  9. Ogura, Y.; Kakehashi, C.; Yoshihara, T.; Kurosaka, M.; Kakigi, R.; Higashida, K.; Fujiwara, S.-E.; Akema, T.; Funabashi, T. Ketogenic Diet Feeding Improves Aerobic Metabolism Property in Extensor Digitorum Longus Muscle of Sedentary Male Rats. PLoS ONE 2020, 15, e0241382. [Google Scholar] [CrossRef]
  10. Stein, T.P.; Wade, C.E. Metabolic Consequences of Muscle Disuse Atrophy. J. Nutr. 2005, 135, 1824S–1828S. [Google Scholar] [CrossRef]
  11. Ciciliot, S.; Rossi, A.C.; Dyar, K.A.; Blaauw, B.; Schiaffino, S. Muscle Type and Fiber Type Specificity in Muscle Wasting. Int. J. Biochem. Cell Biol. 2013, 45, 2191–2199. [Google Scholar] [CrossRef] [PubMed]
  12. Evans, M.; Cogan, K.E.; Egan, B. Metabolism of Ketone Bodies during Exercise and Training: Physiological Basis for Exogenous Supplementation. J. Physiol. 2017, 595, 2857–2871. [Google Scholar] [CrossRef]
  13. Miller, W.C.; Bryce, G.R.; Conlee, R.K. Adaptations to a High-Fat Diet That Increase Exercise Endurance in Male Rats. J. Appl. Physiol. 1984, 56, 78–83. [Google Scholar] [CrossRef] [PubMed]
  14. Ma, S.; Huang, Q.; Yada, K.; Liu, C.; Suzuki, K. An 8-Week Ketogenic Low Carbohydrate, High Fat Diet Enhanced Exhaustive Exercise Capacity in Mice. Nutrients 2018, 10, 673. [Google Scholar] [CrossRef]
  15. Simi, B.; Sempore, B.; Mayet, M.H.; Favier, R.J. Additive Effects of Training and High-Fat Diet on Energy Metabolism during Exercise. J. Appl. Physiol. 1991, 71, 197–203. [Google Scholar] [CrossRef] [PubMed]
  16. Shimizu, K.; Saito, H.; Sumi, K.; Sakamoto, Y.; Tachi, Y.; Iida, K. Short-Term and Long-Term Ketogenic Diet Therapy and the Addition of Exercise Have Differential Impacts on Metabolic Gene Expression in the Mouse Energy-Consuming Organs Heart and Skeletal Muscle. Nutr. Res. 2018, 60, 77–86. [Google Scholar] [CrossRef]
  17. Haba, Y.; Fujimura, T.; Oyama, K.; Kinoshita, J.; Miyashita, T.; Fushida, S.; Harada, S.; Ohta, T. Effect of Oral Branched-Chain Amino Acids and Glutamine Supplementation on Skeletal Muscle Atrophy After Total Gastrectomy in Rat Model. J. Surg. Res. 2019, 243, 281–288. [Google Scholar] [CrossRef]
  18. Moro, T.; Ebert, S.M.; Adams, C.M.; Rasmussen, B.B. Amino Acid Sensing in Skeletal Muscle. Trends Endocrinol. Metab. 2016, 27, 796–806. [Google Scholar] [CrossRef]
  19. Pedersen, B.K.; Saltin, B. Exercise as Medicine—Evidence for Prescribing Exercise as Therapy in 26 Different Chronic Diseases. Scand. J. Med. Sci. Sports 2015, 25 (Suppl. S3), 1–72. [Google Scholar] [CrossRef]
  20. Wang, D.; Li, Z.; Zhang, J.; Liu, S.; Chen, W.; Wang, H.; Wang, Y.; Wang, H.; Wang, L.; Li, Y. A Rat Weightlessness Simulation Treadmill and Exercise Method. Chinese Patent CN103999794B, 24 August 2016. [Google Scholar]
  21. Dougherty, J.P.; Springer, D.A.; Gershengorn, M.C. The Treadmill Fatigue Test: A Simple, High-Throughput Assay of Fatigue-like Behavior for the Mouse. J. Vis. Exp. JoVE 2016, 111, e54052. [Google Scholar]
  22. Burch, N.; Arnold, A.-S.; Item, F.; Summermatter, S.; Santos, G.B.S.; Christe, M.; Boutellier, U.; Toigo, M.; Handschin, C. Electric Pulse Stimulation of Cultured Murine Muscle Cells Reproduces Gene Expression Changes of Trained Mouse Muscle. PLoS ONE 2010, 5, e10970. [Google Scholar] [CrossRef] [PubMed]
  23. Liu, J.; Nie, C.; Xue, L.; Yan, Y.; Liu, S.; Sun, J.; Fan, M.; Qian, H.; Ying, H.; Wang, L.; et al. Growth Hormone Receptor Disrupts Glucose Homeostasis via Promoting and Stabilizing Retinol Binding Protein 4. Theranostics 2021, 11, 8283–8300. [Google Scholar] [CrossRef] [PubMed]
  24. Wang, M.-D.; Li, L.-F.; Yan, Y.-J.; Wang, X.-C.; Chen, L.; Dang, K.; Wang, Z.; Wang, H.-P. Metabolic Adaptation Mechanisms of Glycogen Reduction and Lipid Accumulation in Testicular Protection in Daurian Ground Squirrels during Hibernation. Biochimie 2025, 232, 133–141. [Google Scholar] [CrossRef]
  25. Liu, Q.; Chen, L.; Liang, X.; Cao, Y.; Zhu, X.; Wang, S.; Li, J.; Gao, J.; Xiao, J. Exercise Attenuates angiotensin II-Induced Muscle Atrophy by Targeting PPARγ/miR-29b. J. Sport Health Sci. 2022, 11, 696–707. [Google Scholar] [CrossRef] [PubMed]
  26. Venturini, C.; Mancinelli, L.; Matacchione, G.; Olivieri, F.; Antonicelli, R. The Cardioprotective Effects of Nutritional Ketosis: Mechanisms and Clinical Implications. Nutrients 2024, 16, 4204. [Google Scholar] [CrossRef]
  27. Jeukendrup, A.E.; Wallis, G.A. Measurement of Substrate Oxidation during Exercise by Means of Gas Exchange Measurements. Int. J. Sports Med. 2005, 26 (Suppl. S1), S28–S37. [Google Scholar] [CrossRef]
  28. Fernando, R.; Drescher, C.; Nowotny, K.; Grune, T.; Castro, J.P. Impaired Proteostasis during Skeletal Muscle Aging. Free Radic. Biol. Med. 2019, 132, 58–66. [Google Scholar] [CrossRef]
  29. Fisher, F.M.; Maratos-Flier, E. Understanding the Physiology of FGF21. Annu. Rev. Physiol. 2016, 78, 223–241. [Google Scholar] [CrossRef]
  30. Liu, X.; Wang, Y.; Hou, L.; Xiong, Y.; Zhao, S. Fibroblast Growth Factor 21 (FGF21) Promotes Formation of Aerobic Myofibers via the FGF21-SIRT1-AMPK-PGC1α Pathway. J. Cell. Physiol. 2017, 232, 1893–1906. [Google Scholar] [CrossRef]
  31. Abdurrachim, D.; Woo, C.C.; Teo, X.Q.; Chan, W.X.; Radda, G.K.; Lee, P.T.H. A New Hyperpolarized 13C Ketone Body Probe Reveals an Increase in Acetoacetate Utilization in the Diabetic Rat Heart. Sci. Rep. 2019, 9, 5532. [Google Scholar] [CrossRef]
  32. Angelini, A.; Saha, P.K.; Jain, A.; Jung, S.Y.; Mynatt, R.L.; Pi, X.; Xie, L. PHDs/CPT1B/VDAC1 Axis Regulates Long-Chain Fatty Acid Oxidation in Cardiomyocytes. Cell Rep. 2021, 37, 109767. [Google Scholar] [CrossRef] [PubMed]
  33. Dang, Y.; Xu, J.; Zhu, M.; Zhou, W.; Zhang, L.; Ji, G. Gan-Jiang-Ling-Zhu Decoction Alleviates Hepatic Steatosis in Rats by the miR-138-5p/CPT1B Axis. Biomed. Pharmacother. 2020, 127, 110127. [Google Scholar] [CrossRef] [PubMed]
  34. Maltais, F.; Simard, A.A.; Simard, C.; Jobin, J.; Desgagnés, P.; LeBlanc, P. Oxidative Capacity of the Skeletal Muscle and Lactic Acid Kinetics during Exercise in Normal Subjects and in Patients with COPD. Am. J. Respir. Crit. Care Med. 1996, 153, 288–293. [Google Scholar] [CrossRef]
  35. Xiang, Y.; Wang, Q.-Q.; Lan, X.-Q.; Zhang, H.-J.; Wei, D.-X. Function and Treatment Strategies of β-Hydroxybutyrate in Aging. Smart Mater. Med. 2023, 4, 160–172. [Google Scholar] [CrossRef]
  36. Wang, Q.; Lan, X.; Ke, H.; Xu, S.; Huang, C.; Wang, J.; Wang, X.; Huang, T.; Wu, X.; Chen, M.; et al. Histone β-Hydroxybutyrylation Is Critical in Reversal of Sarcopenia. Aging Cell 2024, 23, e14284. [Google Scholar] [CrossRef]
  37. Beckett, T.L.; Studzinski, C.M.; Keller, J.N.; Paul Murphy, M.; Niedowicz, D.M. A Ketogenic Diet Improves Motor Performance but Does Not Affect β-Amyloid Levels in a Mouse Model of Alzheimer’s Disease. Brain Res. 2013, 1505, 61–67. [Google Scholar] [CrossRef]
  38. Bielohuby, M.; Menhofer, D.; Kirchner, H.; Stoehr, B.J.M.; Müller, T.D.; Stock, P.; Hempel, M.; Stemmer, K.; Pfluger, P.T.; Kienzle, E.; et al. Induction of Ketosis in Rats Fed Low-Carbohydrate, High-Fat Diets Depends on the Relative Abundance of Dietary Fat and Protein. Am. J. Physiol. Endocrinol. Metab. 2011, 300, E65–E76. [Google Scholar] [CrossRef]
  39. Bielohuby, M.; Sawitzky, M.; Stoehr, B.J.M.; Stock, P.; Menhofer, D.; Ebensing, S.; Bjerre, M.; Frystyk, J.; Binder, G.; Strasburger, C.; et al. Lack of Dietary Carbohydrates Induces Hepatic Growth Hormone (GH) Resistance in Rats. Endocrinology 2011, 152, 1948–1960. [Google Scholar] [CrossRef] [PubMed]
  40. Ellenbroek, J.H.; van Dijck, L.; Töns, H.A.; Rabelink, T.J.; Carlotti, F.; Ballieux, B.E.P.B.; de Koning, E.J.P. Long-Term Ketogenic Diet Causes Glucose Intolerance and Reduced β- and α-Cell Mass but No Weight Loss in Mice. Am. J. Physiol. Endocrinol. Metab. 2014, 306, E552–E558. [Google Scholar] [CrossRef]
  41. Fukushima, A.; Ogura, Y.; Furuta, M.; Kakehashi, C.; Funabashi, T.; Akema, T. Ketogenic Diet Does Not Impair Spatial Ability Controlled by the Hippocampus in Male Rats. Brain Res. 2015, 1622, 36–42. [Google Scholar] [CrossRef]
  42. Parker, B.A.; Walton, C.M.; Carr, S.T.; Andrus, J.L.; Cheung, E.C.K.; Duplisea, M.J.; Wilson, E.K.; Draney, C.; Lathen, D.R.; Kenner, K.B.; et al. β-Hydroxybutyrate Elicits Favorable Mitochondrial Changes in Skeletal Muscle. Int. J. Mol. Sci. 2018, 19, 2247. [Google Scholar] [CrossRef] [PubMed]
  43. Parry, H.A.; Kephart, W.C.; Mumford, P.W.; Romero, M.A.; Mobley, C.B.; Zhang, Y.; Roberts, M.D.; Kavazis, A.N. Ketogenic Diet Increases Mitochondria Volume in the Liver and Skeletal Muscle without Altering Oxidative Stress Markers in Rats. Heliyon 2018, 4, e00975. [Google Scholar] [CrossRef]
  44. Roberts, M.N.; Wallace, M.A.; Tomilov, A.A.; Zhou, Z.; Marcotte, G.R.; Tran, D.; Perez, G.; Gutierrez-Casado, E.; Koike, S.; Knotts, T.A.; et al. A Ketogenic Diet Extends Longevity and Healthspan in Adult Mice. Cell Metab. 2018, 27, 1156. [Google Scholar] [CrossRef] [PubMed]
  45. Martín-Moraleda, E.; Delisle, C.; Collado Mateo, D.; Aznar-Lain, S. Weight loss and body composition changes through ketogenic diet and physical activity: A methodological and systematic review. Nutr. Hosp. 2019, 36, 1196–1204. [Google Scholar]
  46. Sahagun, E.; Ward, L.M.; Kinzig, K.P. Attenuation of Stress-Induced Weight Loss with a Ketogenic Diet. Physiol. Behav. 2019, 212, 112654. [Google Scholar] [CrossRef] [PubMed]
  47. Zhang, J.; Chen, B.; Zou, K. Effect of Ketogenic Diet on Exercise Tolerance and Transcriptome of Gastrocnemius in Mice. Open Life Sci. 2023, 18, 20220570. [Google Scholar] [CrossRef]
  48. Zhou, Z.; Vidales, J.; González-Reyes, J.A.; Shibata, B.; Baar, K.; Rutkowsky, J.M.; Ramsey, J.J. A 1-Month Ketogenic Diet Increased Mitochondrial Mass in Red Gastrocnemius Muscle, but Not in the Brain or Liver of Middle-Aged Mice. Nutrients 2021, 13, 2533. [Google Scholar] [CrossRef]
  49. Bilodeau, P.A.; Coyne, E.S.; Wing, S.S. The Ubiquitin Proteasome System in Atrophying Skeletal Muscle: Roles and Regulation. Am. J. Physiol. Cell Physiol. 2016, 311, C392–C403. [Google Scholar] [CrossRef]
  50. Sandonà, D.; Desaphy, J.-F.; Camerino, G.M.; Bianchini, E.; Ciciliot, S.; Danieli-Betto, D.; Dobrowolny, G.; Furlan, S.; Germinario, E.; Goto, K.; et al. Adaptation of Mouse Skeletal Muscle to Long-Term Microgravity in the MDS Mission. PLoS ONE 2012, 7, e33232. [Google Scholar] [CrossRef]
  51. Gundersen, K. Excitation-Transcription Coupling in Skeletal Muscle: The Molecular Pathways of Exercise. Biol. Rev. 2011, 86, 564–600. [Google Scholar] [CrossRef]
  52. Roberts, B.M.; Deemer, S.E.; Smith, D.L.; Mobley, J.A.; Musi, N.; Plaisance, E.P. Effects of an Exogenous Ketone Ester Using Multi-Omics in Skeletal Muscle of Aging C57BL/6J Male Mice. Front. Nutr. 2022, 9, 1041026. [Google Scholar] [CrossRef] [PubMed]
  53. Aare, S.; Spendiff, S.; Vuda, M.; Elkrief, D.; Perez, A.; Wu, Q.; Mayaki, D.; Hussain, S.N.A.; Hettwer, S.; Hepple, R.T. Failed Reinnervation in Aging Skeletal Muscle. Skelet. Muscle 2016, 6, 29. [Google Scholar] [CrossRef]
  54. Rowan, S.L.; Rygiel, K.; Purves-Smith, F.M.; Solbak, N.M.; Turnbull, D.M.; Hepple, R.T. Denervation Causes Fiber Atrophy and Myosin Heavy Chain Co-Expression in Senescent Skeletal Muscle. PLoS ONE 2012, 7, e29082. [Google Scholar] [CrossRef]
  55. Demirel, H.A.; Powers, S.K.; Naito, H.; Hughes, M.; Coombes, J.S. Exercise-Induced Alterations in Skeletal Muscle Myosin Heavy Chain Phenotype: Dose-Response Relationship. J. Appl. Physiol. 1999, 86, 1002–1008. [Google Scholar] [CrossRef] [PubMed]
  56. Ogura, Y.; Naito, H.; Kakigi, R.; Akema, T.; Sugiura, T.; Katamoto, S.; Aoki, J. Different Adaptations of Alpha-Actinin Isoforms to Exercise Training in Rat Skeletal Muscles. Acta Physiol. 2009, 196, 341–349. [Google Scholar] [CrossRef]
  57. Ogura, Y.; Naito, H.; Kakigi, R.; Ichinoseki-Sekine, N.; Kurosaka, M.; Katamoto, S. Alpha-Actinin-3 Levels Increase Concomitantly with Fast Fibers in Rat Soleus Muscle. Biochem. Biophys. Res. Commun. 2008, 372, 584–588. [Google Scholar] [CrossRef]
  58. Huang, T.-Y.; Linden, M.A.; Fuller, S.E.; Goldsmith, F.R.; Simon, J.; Batdorf, H.M.; Scott, M.C.; Essajee, N.M.; Brown, J.M.; Noland, R.C. Combined Effects of a Ketogenic Diet and Exercise Training Alter Mitochondrial and Peroxisomal Substrate Oxidative Capacity in Skeletal Muscle. Am. J. Physiol. Endocrinol. Metab. 2021, 320, E1053–E1067. [Google Scholar] [CrossRef]
  59. Nakao, R.; Abe, T.; Yamamoto, S.; Oishi, K. Ketogenic Diet Induces Skeletal Muscle Atrophy via Reducing Muscle Protein Synthesis and Possibly Activating Proteolysis in Mice. Sci. Rep. 2019, 9, 19652. [Google Scholar] [CrossRef] [PubMed]
  60. Burke, L.M.; Ross, M.L.; Garvican-Lewis, L.A.; Welvaert, M.; Heikura, I.A.; Forbes, S.G.; Mirtschin, J.G.; Cato, L.E.; Strobel, N.; Sharma, A.P.; et al. Low Carbohydrate, High Fat Diet Impairs Exercise Economy and Negates the Performance Benefit from Intensified Training in Elite Race Walkers. J. Physiol. 2017, 595, 2785–2807. [Google Scholar] [CrossRef]
  61. Ji, S.; You, Y.; Kerner, J.; Hoppel, C.L.; Schoeb, T.R.; Chick, W.S.H.; Hamm, D.A.; Sharer, J.D.; Wood, P.A. Homozygous Carnitine Palmitoyltransferase 1b (Muscle Isoform) Deficiency Is Lethal in the Mouse. Mol. Genet. Metab. 2008, 93, 314–322. [Google Scholar] [CrossRef]
  62. Ma, S.; Huang, Q.; Tominaga, T.; Liu, C.; Suzuki, K. An 8-Week Ketogenic Diet Alternated Interleukin-6, Ketolytic and Lipolytic Gene Expression, and Enhanced Exercise Capacity in Mice. Nutrients 2018, 10, 1696. [Google Scholar] [CrossRef] [PubMed]
  63. Fritzen, A.M.; Lundsgaard, A.-M.; Kiens, B. Tuning Fatty Acid Oxidation in Skeletal Muscle with Dietary Fat and Exercise. Nat. Rev. Endocrinol. 2020, 16, 683–696. [Google Scholar] [CrossRef]
  64. Espinoza, D.O.; Boros, L.G.; Crunkhorn, S.; Gami, H.; Patti, M.-E. Dual Modulation of Both Lipid Oxidation and Synthesis by Peroxisome Proliferator-Activated Receptor-Gamma Coactivator-1alpha and -1beta in Cultured Myotubes. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 2010, 24, 1003–1014. [Google Scholar]
  65. Wong, K.E.; Mikus, C.R.; Slentz, D.H.; Seiler, S.E.; DeBalsi, K.L.; Ilkayeva, O.R.; Crain, K.I.; Kinter, M.T.; Kien, C.L.; Stevens, R.D.; et al. Muscle-Specific Overexpression of PGC-1α Does Not Augment Metabolic Improvements in Response to Exercise and Caloric Restriction. Diabetes 2015, 64, 1532–1543. [Google Scholar] [CrossRef]
  66. Miller, V.J.; LaFountain, R.A.; Barnhart, E.; Sapper, T.S.; Short, J.; Arnold, W.D.; Hyde, P.N.; Crabtree, C.D.; Kackley, M.L.; Kraemer, W.J.; et al. A Ketogenic Diet Combined with Exercise Alters Mitochondrial Function in Human Skeletal Muscle While Improving Metabolic Health. Am. J. Physiol. Endocrinol. Metab. 2020, 319, E995–E1007. [Google Scholar] [CrossRef] [PubMed]
  67. Li, X.; Li, Z.; Zhao, M.; Nie, Y.; Liu, P.; Zhu, Y.; Zhang, X. Skeletal Muscle Lipid Droplets and the Athlete’s Paradox. Cells 2019, 8, 249. [Google Scholar] [CrossRef] [PubMed]
  68. Cox, P.J.; Clarke, K. Acute Nutritional Ketosis: Implications for Exercise Performance and Metabolism. Extreme Physiol. Med. 2014, 3, 17. [Google Scholar] [CrossRef]
  69. Sasai, H.; Aoyama, Y.; Otsuka, H.; Abdelkreem, E.; Naiki, Y.; Kubota, M.; Sekine, Y.; Itoh, M.; Nakama, M.; Ohnishi, H.; et al. Heterozygous Carriers of Succinyl-CoA:3-Oxoacid CoA Transferase Deficiency Can Develop Severe Ketoacidosis. J. Inherit. Metab. Dis. 2017, 40, 845–852. [Google Scholar] [CrossRef]
  70. Puchalska, P.; Crawford, P.A. Multi-Dimensional Roles of Ketone Bodies in Fuel Metabolism, Signaling, and Therapeutics. Cell Metab. 2017, 25, 262–284. [Google Scholar] [CrossRef]
Metabolites 15 00270 g001
Figure 1. Schematic overview of experimental design.
Figure 1. Schematic overview of experimental design.
Metabolites 15 00270 g001
Metabolites 15 00270 g002
Figure 2. Successful establishment of the ketogenic diet in a mouse model. (A) Weekly changes in mouse body weight. (B) Weekly variations in blood ketone levels. (C) Weekly fluctuations in blood glucose levels. (D) Changes in the respiratory exchange ratio. The data are presented as means ± SD. * p < 0.05; ** p < 0.01, compared with the normal diet control group. NC: normal diet control group; KC: ketogenic diet control group; RER: respiratory exchange ratio.
Figure 2. Successful establishment of the ketogenic diet in a mouse model. (A) Weekly changes in mouse body weight. (B) Weekly variations in blood ketone levels. (C) Weekly fluctuations in blood glucose levels. (D) Changes in the respiratory exchange ratio. The data are presented as means ± SD. * p < 0.05; ** p < 0.01, compared with the normal diet control group. NC: normal diet control group; KC: ketogenic diet control group; RER: respiratory exchange ratio.
Metabolites 15 00270 g002
Metabolites 15 00270 g003
Figure 3. Effects of simulated weightlessness on muscle atrophy markers. (A) Body weight fluctuations in the mice before and after hindlimb unloading. (B) Ratio of soleus muscle wet weight to body weight across groups. mRNA expression of MuRF-1 (C) and Atrogin1 (D) in the soleus muscle. The data are presented as means ± SD. aa p < 0.01, compared with before HU; * p < 0.05; ** p < 0.01 compared with the control group; # p < 0.05; ## p < 0.01, compared with the diet groups. BW: body weight; NC: normal diet control group; NH: normal diet + hindlimb unloading group; NHE: normal diet + hindlimb unloading + exercise group; KC: ketogenic diet control group; KH: ketogenic diet + hindlimb unloading group; KHE: ketogenic diet + hindlimb unloading + exercise group.
Figure 3. Effects of simulated weightlessness on muscle atrophy markers. (A) Body weight fluctuations in the mice before and after hindlimb unloading. (B) Ratio of soleus muscle wet weight to body weight across groups. mRNA expression of MuRF-1 (C) and Atrogin1 (D) in the soleus muscle. The data are presented as means ± SD. aa p < 0.01, compared with before HU; * p < 0.05; ** p < 0.01 compared with the control group; # p < 0.05; ## p < 0.01, compared with the diet groups. BW: body weight; NC: normal diet control group; NH: normal diet + hindlimb unloading group; NHE: normal diet + hindlimb unloading + exercise group; KC: ketogenic diet control group; KH: ketogenic diet + hindlimb unloading group; KHE: ketogenic diet + hindlimb unloading + exercise group.
Metabolites 15 00270 g003
Metabolites 15 00270 g004
Figure 4. Effects of KD and KD combined with exercise on skeletal muscle tissue in mice. PAS (A) and Oil Red O (B) staining of the soleus muscle. Percentage of PAS- (C) and Oil Red O-stained (D) area in the soleus muscle. The data are presented as means ± SD. ** p < 0.01, compared between diet groups.
Figure 4. Effects of KD and KD combined with exercise on skeletal muscle tissue in mice. PAS (A) and Oil Red O (B) staining of the soleus muscle. Percentage of PAS- (C) and Oil Red O-stained (D) area in the soleus muscle. The data are presented as means ± SD. ** p < 0.01, compared between diet groups.
Metabolites 15 00270 g004
Metabolites 15 00270 g005
Figure 5. Effects of KD or KD combined with exercise on muscle fiber composition in the soleus muscles of mice. Representative immunofluorescence images of MyHC-I (blue (A)), MyHC-IIa (red (B)), and MyHC-IIb (green (C)) muscle fibers in the soleus muscle. The proportion of MyHC-I (D), MyHC-IIa (E), and MyHC-IIb (F) muscle fibers in the soleus muscle. The data are presented as means ± SD. * p < 0.05; ** p < 0.01 indicate comparisons between diet groups; # p < 0.05; ## p < 0.01 indicate comparisons within each diet group.
Figure 5. Effects of KD or KD combined with exercise on muscle fiber composition in the soleus muscles of mice. Representative immunofluorescence images of MyHC-I (blue (A)), MyHC-IIa (red (B)), and MyHC-IIb (green (C)) muscle fibers in the soleus muscle. The proportion of MyHC-I (D), MyHC-IIa (E), and MyHC-IIb (F) muscle fibers in the soleus muscle. The data are presented as means ± SD. * p < 0.05; ** p < 0.01 indicate comparisons between diet groups; # p < 0.05; ## p < 0.01 indicate comparisons within each diet group.
Metabolites 15 00270 g005
Metabolites 15 00270 g006
Figure 6. Effects of the ketogenic diet or the ketogenic diet combined with exercise on skeletal muscle mRNA expression. mRNA expression of PGC-1α (A), SIRT1 (B), FGF21 (C), CPT-1b (D), OXCT (E), and HADH (F) in the soleus muscle. The data are presented as means ± SD. * p < 0.05; ** p < 0.01, denoting comparisons between diet groups; # p < 0.05; ## p < 0.01, reflecting comparisons within diet groups.
Figure 6. Effects of the ketogenic diet or the ketogenic diet combined with exercise on skeletal muscle mRNA expression. mRNA expression of PGC-1α (A), SIRT1 (B), FGF21 (C), CPT-1b (D), OXCT (E), and HADH (F) in the soleus muscle. The data are presented as means ± SD. * p < 0.05; ** p < 0.01, denoting comparisons between diet groups; # p < 0.05; ## p < 0.01, reflecting comparisons within diet groups.
Metabolites 15 00270 g006
Table 1. Experimental group assignment and sample sizes.
Table 1. Experimental group assignment and sample sizes.
DietInterventionGroupn
Normal diet (ND)ControlNC8
Hindlimb unloading (HU)NH12
HU + exerciseNHE14
Ketogenic diet (KD)ControlKC8
Hindlimb unloading (HU)KH12
HU + exerciseKHE14
Table 2. Description of primers used for quantitative real-time PCR.
Table 2. Description of primers used for quantitative real-time PCR.
Gene NameForward PrimerReverse Primer
Atrogin15′-TCAGCAGCCTGAACTACGAC-3′5′-GCGCTCCTTCGTACTTCCTT-3′
MURF-15′-GTGTGAGGTGCCTACTTGCT-3′5′-GACTTTTCCAGCTGCTCCCT-3′
PGC-1α5′-AGCCGTGACCACTGACAACGAG-3′5′-GCTGCATGGTTCTGAGTGCTAAG-3′
SIRT15′-TACCTTGGAGCAGGTTGCAG-3′5′-GCACCGAGGAACTACCTGAT-3′
FGF215′-GCATACCCCATCCCTGACTC-3′5′-GGATCAAAGTGAGGCGATCC-3′
CPT-1b5′-TTCAACACTACACGCATCCC-3′5′-GCCCTCATAGAGCCAGACC-3′
HADH5′-ACACCTTCATTCGCCATATTGC-3′5′-TCGGTGAATTTTCTGTAGACCAC-3′
OXCT5′-CCCATACCCACTGAAAGACGAA-3′5′-CTGGAGAAGAAAGAGGCTCCTG-3′
18s5′-GGGAGCCTGAGAAACGGC-3′5′-GGGTCGGGAGTGGGTAATTT-3′
Table 3. Controlled variables and measurement instruments.
Table 3. Controlled variables and measurement instruments.
VariableMeasurement InstrumentsValidation Reference
Body weightElectronic BalanceManufacturer’s protocol
Blood ketone/glucoseFreeStyle Optium Neo Blood β-Ketone/Glucose Test StripsAbbott technical manual [23]
RERIndirect CalorimetryManufacturer’s protocol
TGThe reagent kitsManufacturer’s protocol
TCThe reagent kitsManufacturer’s protocol
HDL-CThe reagent kitsManufacturer’s protocol
LDL-CThe reagent kitsManufacturer’s protocol
InsulinThe reagent kitsManufacturer’s protocol
Glycogen contentThe Periodic Acid–Schiff Staining KitSolarbio technical manual [24]
Lipid depositionThe Oil Red O Staining KitSolarbio technical manual [24]
LDThe reagent kitsManufacturer’s protocol
UREAThe reagent kitsManufacturer’s protocol
CKThe reagent kitsManufacturer’s protocol
LDHThe reagent kitsManufacturer’s protocol
Muscle fiber typesImmunofluorescence (MyHC Antibodies)Manufacturer’s protocol [25]
Table 4. Plasma metabolic parameters.
Table 4. Plasma metabolic parameters.
TC
(mmol/L)		TG
(mmol/L)		HDL-C
(mmol/L)		LDL-C
(mmol/L)		UREA
(mmol/L)		Insulin
(ng/mL)
NC1.35 ± 0.150.38 ± 0.071.18 ± 0.090.17 ± 0.039.57 ± 0.370.97 ± 0.21
NH1.29 ± 0.230.58 ± 0.181.12 ± 0.130.18 ± 0.069.28 ± 0.341.72 ± 0.58 ##
NHE1.24 ± 0.210.59 ± 0.291.09 ± 0.210.20 ± 0.068.33 ± 0.301.44 ± 0.56 ##
KC2.16 ± 0.20 **0.63 ± 0.20 **1.90 ± 0.12 **0.27 ± 0.04 **9.31 ± 0.80 **1.51 ± 0.59 *
KH1.96 ± 0.23 #**0.99 ± 0.16 ##**1.64 ± 0.27 ##**0.25 ± 0.03 **9.53 ± 0.85 **1.38 ± 0.51 **
KHE1.79± 0.36 ##**0.74 ± 0.29 #1.55 ± 0.38 ##**0.23 ± 0.03 ##9.25 ± 0.42 **1.65 ± 0.71 **
Note: The data are presented as means ± SD. * p < 0.05; ** p < 0.01, compared between diet groups; # p < 0.05; ## p < 0.01, compared within diet groups. TC: total cholesterol; TG: triglycerides; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol.
Table 5. The effects of exercise capacity in mice.
Table 5. The effects of exercise capacity in mice.
LA (mmol/L)UREA (mmol/L)CK
(U/L)		LDH
(U/L)		Time
(min)
NC1.81 ± 0.269.57 ± 0.37850 ± 310513 ± 166136 ± 15
NH2.41 ± 0.53 ##9.27 ± 0.34866 ± 260655 ± 9783 ± 24 ##
NHE2.32 ± 0.34 ##8.33 ± 0.30749 ± 275547 ± 11384 ± 30 ##
KC2.43 ± 0.39 **9.20 ± 0.71981 ± 233521 ± 180152 ± 18
KH2.23 ± 0.469.26 ± 0.61863 ± 315615 ± 27281 ± 36 ##
KHE2.03 ± 0.34 ##9.26 ± 0.49955 ± 288486 ± 286136 ± 26 ##*
Note: Values are expressed as means ± SD. * p < 0.05; ** p < 0.01, denoting comparisons between diet groups; ## p < 0.01, reflecting comparisons within diet groups. LA: lactate; UREA: urea nitrogen; CK: creatine kinase; LDH: lactate dehydrogenase.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Chen, J.; Li, W.; Yu, L.; Zhang, B.; Li, Z.; Zou, P.; Ding, B.; Dai, X.; Wang, Q. Combined Effects of Ketogenic Diet and Aerobic Exercise on Skeletal Muscle Fiber Remodeling and Metabolic Adaptation in Simulated Microgravity Mice. Metabolites 2025, 15, 270. https://doi.org/10.3390/metabo15040270

AMA Style

Chen J, Li W, Yu L, Zhang B, Li Z, Zou P, Ding B, Dai X, Wang Q. Combined Effects of Ketogenic Diet and Aerobic Exercise on Skeletal Muscle Fiber Remodeling and Metabolic Adaptation in Simulated Microgravity Mice. Metabolites. 2025; 15(4):270. https://doi.org/10.3390/metabo15040270

Chicago/Turabian Style

Chen, Jun, Wenjiong Li, Liang Yu, Bowei Zhang, Zhili Li, Peng Zou, Bai Ding, Xiaoqian Dai, and Qirong Wang. 2025. "Combined Effects of Ketogenic Diet and Aerobic Exercise on Skeletal Muscle Fiber Remodeling and Metabolic Adaptation in Simulated Microgravity Mice" Metabolites 15, no. 4: 270. https://doi.org/10.3390/metabo15040270

APA Style

Chen, J., Li, W., Yu, L., Zhang, B., Li, Z., Zou, P., Ding, B., Dai, X., & Wang, Q. (2025). Combined Effects of Ketogenic Diet and Aerobic Exercise on Skeletal Muscle Fiber Remodeling and Metabolic Adaptation in Simulated Microgravity Mice. Metabolites, 15(4), 270. https://doi.org/10.3390/metabo15040270

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop