INTRODUCTION
Skeletal muscle is not only the primary organ responsible for body movement but also plays a central role in systemic metabolic homeostasis [1]. Clinically, immobilization, defined as the intentional or unintentional restriction of movement of a body part caused by fracture, injury or disease, is known to induce muscle atrophy [2]. Even a short period of immobilization (e.g., with a cast, splint, or brace) can lead to a rapid loss of muscle mass, and subsequent decline in strength and functional capacity [3,4]. Moreover, because skeletal muscle accounts for a substantial proportion of resting energy expenditure and serves as a primary site for carbohydrate and fat oxidation [5-7], immobilization-induced muscle atrophy (IMA) may profoundly alter whole-body energy expenditure and substrate utilization patterns [8-10].
Previous studies have demonstrated that IMA disrupts skeletal muscle protein homeostasis by reducing protein synthesis and increasing protein degradation, thereby driving atrophy [11-13]. In addition to these structural consequences, IMA is associated with metabolic disturbances, such as impaired mitochondrial biogenesis and function, shifts in fiber-type composition, changes in fuel utilization, and altered activity of key regulators (e.g., AMPK and PGC-1α), among other processes [14-17]. Collectively, these changes indicate broader metabolic reprogramming that highlights vulnerability in systemic energy regulation, given the central role of skeletal muscle in whole-body metabolism [18]. Consequently, muscle disturbances induced by immobilization have the potential to impair whole-body metabolic homeostasis, which in turn may predispose individuals to metabolic inefficiency or impaired recovery [19]. However, the systemic metabolic effects of immobilization on energy expenditure and substrate oxidation remain poorly characterized. Therefore, elucidating the metabolic alterations that occur during the atrophy stage of immobilization is essential to inform targeted intervention strategies.
Physical activity is a well-established intervention for maintaining or recovery skeletal muscle and function, and is therefore considered a central strategy during [20,21]. Previous studies have predominantly focused on the structural and functional benefits of exercise, demonstrating improvements in muscle size, strength, and performance following periods of immobilization [4,22]. However, given that IMA induces metabolic reprogramming that may influence recovery, the assessment of whole-body metabolism during the rehabilitation phase is equally important. Exercise has been shown to exert profound metabolic effects, including enhanced mitochondrial activity and biogenesis, increased oxidative enzyme capacity, and improved vascular adaptations [23-25]. In addition to supporting local muscular recovery, these systemic adaptations indicate that exercise could also play a critical role in normalizing whole-body energy expenditure and substrate utilization following immobilization. Nevertheless, most existing studies have been limited to muscle-specific or indirect metabolic markers, and few have directly examined how rehabilitative exercise influences whole-body expenditure and substrate utilization during recovery from IMA.
Therefore, in the present study we employed a mouse hindlimb immobilization model to longitudinally examine the effects of short-term immobilization and subsequent rehabilitative exercise on whole-body energy metabolism and muscle function. Specifically, we assessed whether immobilization alters energy expenditure and substrate utilization, and whether exercise during the recovery phase can modulate these changes whole facilitating the restoration of muscle mass and functional performance. By integrating systemic metabolic measurements with assessments of muscle mass and strength, this study provides a comprehensive evaluation of the metabolic and functional consequence IMA. Our findings aim to advance the understanding of how rehabilitative exercise influences whole-body energy metabolism following immobilization, thereby offering mechanistic insights that may inform the development of effective strategies for preventing and mitigating IMA.
METHODS
Animals
Eight-week-old male Institute of Cancer Research (ICR) mice (Orient Bio Inc., Seongnam, Republic of Korea) were acclimatized to the laboratory environment for at least 2 weeks under controlled conditions: a 12-hour light/dark cycle, an air-conditioned room maintained at 23-24 °C with 45-50% relative humidity. The mice were provided with a standard commercial diet (5L79, Orient Bio Inc., Seongnam, Republic of Korea) and water ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Konkuk University (Approval Number: KU230220).
Experimental design
This study was designed as a longitudinal investigation of the effects of short-term immobilization and subsequent rehabilitative exercise on energy metabolism and muscle function. This study was performed over a total period of four weeks (Figure 1). During the first two weeks (atrophy phase), all mice (n = 20) underwent right hindlimb immobilization by casting to induce IMA [26,27]. Following cast removal, the mice entered two weeks recovery phase and were randomly assigned to two groups using simple randomization: Non-exercise group (n = 10), which underwent recovery without exercise intervention, and Exercise group (n = 10), which performed rehabilitative exercise during the recovery phase. Randomization was performed by an investigator who was not involved in outcome assessment. All measurements of body composition, whole-body energy metabolism, grip strength, and gait were conducted by investigators blinded to group allocation during data collection. Measurements were performed at three time points: pre-IMA (before immobilization), post-IMA (immediately after immobilization), and recovery (after recovery phase).
Immobilization-induced muscle atrophy (IMA)
IMA was induced by immobilizing the right hindlimb of each mouse using a modified casting method based on the previous studies (Figure 2) [28,29]. The hindlimb was fixed with a plastic stick and secured with surgical tape. The health and well-being of the mice were monitored daily to ensure that any signs of distress or injury were promptly addressed.
Treadmill exercise protocol
Treadmill exercise was performed using a small-animal treadmill. The exercise regimen was designed to deliver low-intensity training (18 m/min, 15° incline, 30 minutes per session), 5 days per week, based on the methods our previous study [30]. Exercise was initiated 24 hours after cast removal to minimize musculoskeletal stress during the early recovery phase. The initial treadmill speed was set low and progressively increased over the first several sessions (10-12 m/min for 10-20 minutes) before reaching 18 m/min for 30 minutes, all performed at a 15° incline, to further reduce strain during recovery [31].
Dual-energy X-ray Absorptiometry (DXA)
DXA was used to evaluate the body composition and right calf lean mass. A specific region of interest (ROI) encompassing the whole-body and right calf muscle was defined, as shown in Figure 3. The ROI was carefully set to include the whole-body and the skeletal muscle of the hindlimb while excluding adjacent bone structures to ensure accurate measurement of lean mass.
Whole-body metabolic analysis at rest
The measurement of whole-body energy metabolism at rest was conducted using a metabolic analyzer for 24 h. The metabolic chambers were constructed using the open-circuit method, with an average flow rate of 1L/min. Each chamber was connected to an acrylic tube to control the air volume. Respiratory gases (oxygen uptake and carbon dioxide production) were analyzed using a mass spectrometer (ARCO-2000-GS-8, ARCO System, Chiba, Japan) equipped with a switching system from each chamber every 15 s. Based on the measured oxygen uptake and carbon dioxide production, respiratory exchange rate, carbohydrate oxidation, fat oxidation, and energy expenditure were calculated [32].
Grip strength
Grip strength was measured using strength meter (JDA-22, JEUNG DO BIO&PLANT, Seoul, Korea). The mice were positioned to grasp the grid of the apparatus securely with their fore and hindlimb. The tail was gently pulled backward until the grip was released. Each mouse underwent three measurements, and the average value was recorded as grip strength (gram-force, gf)
Gait analysis
As previously described, the gait test was performed to evaluate balance and motor coordination [33]. A runway, 15 cm wide and 42 cm length, was lined with white paper. Prior to the experiment, all mice were trained to walk smoothly across the runway without stopping. The hind paws of the mice were painted with non-toxic, washable paint during the experimental assessments. Each mouse was allowed to walk across runway, leaving a series of footprints. Measurements were repeated in two trials, and the collected footprints were analyzed to measure the intrastep distance.
Statistical analysis
All data were analyzed using IBM SPSS Statistics 25 and visualized using GraphPad Prism (version 10.2). The Shapiro-Wilk test was applied to assess the normality of data distribution. For the atrophy phase, paired t-tests were used to evaluate immobilization intervention (pre- vs post-IMA). Body weight across time was analyzed using repeated-measures analysis of variance (ANOVA). For the recovery phase, comparisons between two groups (Non-exercise vs Exercise) over time (post-IMA vs recovery) were analyzed two-way mixed ANOVA, followed by Bonferroni-adjusted post hoc tests for pairwise comparisons. When assumption of normality or homogeneity was violated, non-parametric alternatives (e.g., Wilcoxon signed-rank test or Kruskal-Wallis test, as appropriate) were applied. Daily food intake was recorded at the cage level, and therefore individual-level analysis was not possible; these data are presented descriptively without statistical testing. All data are expressed as mean ± standard deviation (SD) and statistical significance was set at p < 0.05.
RESULTS
Atrophy phase: Body weight and food intake
During the atrophy phase, body weight showed a progressive reduction across the immobilization period (Figure 4A). These findings indicated that short-term immobilization leads to a gradual but significant loss of body mass. In contrast, daily food intake did not exhibit differences across the immobilization period according to descriptive inspection (Figure 4B). These results indicated that reductions in body weight were likely driven by immobilization-induced tissue loss rather than changes in nutritional intake.
Atrophy phase: body composition of lean and fat mass
DXA analysis revealed significant changes in body composition following immobilization. Total fat mass and fat mass percentage did not show significant changes between the pre- and post-IMA (Figure 5A, B). In contrast, total lean mass was significantly reduced after immobilization compared with pre-IMA (p < 0.05) (Figure 5C). Calf lean mass, representing the immobilized limb, showed a marked reduction after immobilization (p < 0.05) (Figure 5D), further confirming localized muscle atrophy. These results demonstrated that the decline in body weight observed during immobilization was primarily attributable to losses in lean mass rather than changes in adiposity.
Atrophy phase: Muscle function of strength and locomotion
Grip strength was significantly decreased after immobilization compared with the pre-IMA (p < 0.05) (Figure 6A), indicating a decline in overall muscle force-generating capacity. Walking length also exhibited a significant reduction on both the left and right sides following immobilization (p < 0.05) (Figure 6B, C). These results demonstrate that short-term immobilization not only reduced muscle but also impaired functional performance, as reflected by diminished grip strength and locomotor ability.
Atrophy phase: Energy metabolism
Immobilization induced marked alterations in whole-body energy metabolism. Both oxygen uptake and carbon dioxide production were significantly increased after immobilization compared with pre-IMA (p < 0.05) (Figure 7A, B). The respiratory exchange ratio was significantly reduced (p < 0.05), suggesting a relative shift toward enhanced lipid utilization despite increasing oxygen uptake and carbon dioxide production (Figure 7C). Consistent with this interpretation, fat oxidation was significantly elevated following immobilization (p < 0.05) (Figure 7D), whereas carbohydrate oxidation remained unchanged (Figure 7E). Total energy expenditure also increased significantly after immobilization (p < 0.05) (Figure 7F), indicating that immobilization was accompanied by a paradoxical elevation in metabolic demand despite reduction in muscle mass and function.
Recovery phase: Body weight and food intake
During the recovery phase, body weight increased progressively in both groups over the 2-week recovery phase (time effect, p < 0.001) (Figure 8A). However, no significant group effect or interaction was detected, indicating that exercise did not produce an additional effect on overall body weight compared with the non-exercise group. Post hoc comparisons showed significant increase from post-IMA to days 14 in both (p < 0.05), confirming gradual recovery on body weight. Daily food intake increased descriptively over the course of recovery, with slightly higher values observed in the exercise group compared with eh non-exercise group (Figure 8B). However, these findings are based on descriptive observations, as no formal statistical analysis was performed.
Recovery phase: body composition of lean and fat mass
DXA analysis demonstrated significant changes in body composition during the recovery phase. Fat mass showed a significant interaction effect (p = 0.047) (Figure 9A), and fat mass percentage exhibited a significant time effect (p = 0.006) (Figure 9B). Post-hoc analysis indicated that significant decreases in both fat mass and fat percentage occurred only in the exercise group. Similarly, lean mass showed a significant time effect (p = 0.029), with recovery-associated increases observed in the exercise group whereas the non-exercise group showed no corresponding improvement (Figure 9C). Calf lean mass demonstrated both a significant time effect (p = < 0.001) and group effect (p = 0.031), with significant improvements detected in the exercise group and higher values compared with the non-exercise group (Figure 9D). Collectively, these findings indicate that although recovery contributed to some normalization of body composition, exercise was essential for restoring lean mass following immobilization.
Recovery phase: Muscle function of strength and locomotion
Grip strength showed a significant time effect (p < 0.001) and interaction effect (p = 0.028) (Figure 10A). Post-hoc analysis indicated that significant improvements from post-IMA to recovery occurred only in the exercise group. The exercise group also exhibited a greater numerical increase compared with the non-exercise group, which approached but did not reach statistical significance. Walking performance demonstrated partial recovery during the recovery period. For the left leg, a significant interaction effect (p = 0.011) was detected, and post-hoc analysis revealed a significant restoration in the exercise group, whereas the non-exercise group showed no significant change (Figure 10B). Right leg walking length followed a similar trend but did not show statistically significant effects (Figure 10C). Together, these results suggest that functional recovery following immobilization was primarily driven by exercise.
Recovery phase: Energy metabolism
Oxygen uptake showed significant effects of time (p < 0.001), group (p = 0.021), and interaction (p = 0.019) (Figure 11A). Post-hoc analysis revealed that oxygen uptake significantly decreased from post-IMA to recovery in both groups, with exercise group maintaining significantly higher values during recovery phase. Carbon dioxide production demonstrated significant time (p < 0.001), group (p = 0.043), and interaction (p = 0.023) effects (Figure 11B). Post-hoc comparison indicated that carbon dioxide production significantly decreased in non-exercise group, resulting in lower values compared with exercise group at recovery. Respiratory exchange ratio showed a significant time effect (p = 0.030), with the exercise group exhibiting significant increase from post-IMA to recovery, whereas no significant change was observed in the non-exercise group (Figure 11C). Fat oxidation revealed a significant effect of time (p < 0.001), decreasing in both groups during recovery, with no additional group or interaction effects (Figure 11D). Carbohydrate oxidation displayed a significant interaction effect (p = 0.043). Post-hoc analysis revealed a modest reduction in the non-exercise group during recovery (p = 0.064), whereas values were relatively maintained in the exercise group, leading to between-group differences at recovery (Figure 11E). Energy expenditure displayed significant time (p < 0.001), group (p = 0.024), and interaction (p = 0.019) effects (Figure 11F). Post-hoc analysis revealed that energy expenditure decreased significantly in both groups, although the decline was greater in the non-exercise group, resulting in significantly higher values in the exercise group at recovery. Collectively, these results indicate that rehabilitative exercise contributed to the modulation of whole-body energy metabolism during the recovery period.
DISCUSSION
This study demonstrated that short-term hindlimb immobilization induced pronounced reduction in body weight, lean mass, and muscle function, confirming the rapid onset of immobilization. In addition to these structural and functional changes, immobilization elicited marked alterations in systemic energy metabolism. Oxygen uptake, carbon dioxide production, and total energy expenditure increased, whereas the respiratory exchange ratio decreased and fat oxidation increased, indicating a shift toward greater lipid utilization. These unexpected metabolic alterations suggest a maladaptive response, potentially stemming from compromised movement economy and compensatory gait adjustments required due to the unilateral immobilization. During the recovery phase, rehabilitative exercise played a critical role in facilitating muscle mass and metabolic restoration. Exercise, but not passive recovery, promoted reductions in fat mass, increases in lean mass, and improvements in grip strength and walking performance. Moreover, exercise helped modulate the metabolic changes observed during passive recovery and was associated with the maintenance of higher carbohydrate utilization and energy expenditure.
In the present study, short-term immobilization induced decline whole body and right calf lean mass and impairment strength and locomotor, thereby confirming the rapid onset of IMA. These findings are consistent with previous studies indicating that immobilization induces muscle atrophy and functional impairments [3,4,14]. Interestingly, the results of increased energy expenditure following immobilization are noteworthy, as it contrasts with several previous studies reporting decreased metabolic demand during disuse [34,35]. This paradoxical rise in energy expenditure appears to result from locomotor inefficiency and compensatory gait alterations resulting from unilateral immobilization [36]. Consistent with this interpretation, previous studies have demonstrated that knee immobilization markedly increases the energy expenditure of movement [37,38]. Indeed, gait analysis revealed reduced stride length in both hindlimbs, indicating that biomechanical imbalance imposed additional energetic costs despite overall reductions in muscle mass. Thus, even localized immobilization may not simply reduce global activity but can increase systemic metabolic demand beyond the affected limb.
Moreover, the observed alterations in substrate utilization, accompanied by elevated fat oxidation, suggest an impairment in metabolic flexibility. Immobilization-induced insulin resistance compromises this crucial adaptability, as muscle atrophy has been closely linked to reduced insulin sensitivity in skeletal muscle [39-41]. Mechanistically, immobilization has been shown to depress skeletal muscle insulin signaling. Specifically, studies report reduced Akt phosphorylation and impaired GLUT4 translocation, which together limit glucose uptake even when circulating insulin levels are maintained [42,43]. Consistently, previous studies have reported diminished glucose disposal and enhanced lipid utilization during periods of immobilization [8,44].
In addition, hormonal and inflammatory perturbations may contribute to this shift in substrate preference and to the paradoxical increase in energy expenditure. The acute physical stress of immobilization, including inflammation and elevated glucocorticoids, can increase resting energy expenditure and whole-body protein turnover [45,46]. Elevated circulating glucocorticoids under physical stress can promote lipolysis, exert catabolic effects on muscle, and subsequently increase the availability and oxidation of fatty acids [47,48]. Immobilization acts as a potent physiological stressor that disrupts endocrine regulation and further reinforces the shift toward lipid metabolism. Collectively, these findings suggest that immobilization contributes to maladaptive metabolic reprogramming characterized by locomotor inefficiency and altered substrate utilization.
Exercise is well-established intervention for restoring skeletal muscle mass and reversing function impairments caused by immobilization [4,22]. Consistently, the results of the present study demonstrated that rehabilitative exercise exerted clear benefits for restoration of both muscle mass and function during the recovery phase. Beyond its effects on muscular restoration, exercise also improved the maladaptive metabolic responses observed during passive recovery, as reflected by the maintenance of higher value of oxygen uptake and carbon dioxide production, along with greater carbohydrate oxidation and overall energy expenditure. These results suggest that exercise restored metabolic flexibility by supporting carbohydrate utilization and balancing substrate oxidation.
In addition, preservation of carbohydrate oxidation observed in the exercise group is likely to attributable enhanced regenerative processes at the cellular level [49]. Improved insulin signaling following rehabilitative exercise may further contribute to this pattern by facilitating glucose uptake into recovering muscle, thereby sustaining glycolytic flux during periods of active regeneration [50]. The activation of satellite cells and myogenic progenitors has been shown to depend on glycolytic flux, with intermediates such as pyruvate and lactate contributing both energy supply and signaling pathways the regulate myogenic differentiation [51-53]. Furthermore, carbohydrate metabolism supplies rapid ATP generation to sustain the wide array of biochemical processes required for cellular regeneration [54,55]. Therefore, by maintaining carbohydrate utilization during the recovery phase, rehabilitative exercise may have fulfilled the energetic and biosynthetic demands of muscle regeneration, thereby promoting structural recovery in parallel with systemic metabolic restoration.
Despite these meaningful findings, several limitations should be acknowledged. First, this study employed a mouse model, which may not fully capture the complex physiological responses observed in humans. Second, while systemic metabolic parameters were assessed comprehensively, molecular mechanisms underlying altered substrate utilization (e.g., insulin sensitivity, mitochondrial biogenesis, or fiber-type specific adaptations) were not directly investigated. Future studies incorporating molecular analyses and extending these findings to human cohorts will be essential to elucidate the mechanisms driving immobilization-induced metabolic reprogramming and to refine rehabilitative interventions. Third, this experiment did not perform statistical comparisons between the Recovery endpoint and the Pre-IMA baseline, because animals were randomized into exercise and non-exercise groups only after the immobilization phase. This design limited our ability to formally evaluate complete restoration to baseline levels, and future studies should employ long-term follow-up designs that allow direct assessment of recovery kinetics and completeness.
Nevertheless, the present findings demonstrate that short-term immobilization induces both structural atrophy and maladaptive systemic metabolic alterations, whereas rehabilitative exercise effectively promotes muscle recovery and restores metabolic flexibility. Collectively, these results underscore the pivotal role of exercise in mitigating the consequences of disuse and provide mechanistic insights that may inform therapeutic strategies for populations at risk of immobilization-induced impairments. These findings have critical clinical implications for human populations, particularly given the rapid onset of both muscular and metabolic disturbances. This has particular relevance for metabolically vulnerable groups such as older adults with sarcopenia, patients with type 2 diabetes, and adolescents undergoing growth and development. In these populations, the observed maladaptive shift toward enhanced fat oxidation and compromised movement efficiency could significantly worsen existing insulin resistance and accelerate muscle loss. Notably, even brief periods of immobilization were sufficient to elicit systemic metabolic disturbances, highlighting the importance of early intervention. Timely initiation of low-intensity rehabilitative exercise may therefore represent a critical strategy to preserve metabolic flexibility, maintain muscular integrity, and prevent the compounding effects of disuse-induced metabolic stress during recovery.
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