The widespread adoption of high-fat diets (HFDs) in modern societies has contributed to a marked rise in obesity and related metabolic disorders (Abbasi and Khodadadi, 2025). Although the detrimental consequences of HFDs—such as adipose tissue expansion, chronic low-grade inflammation, and insulin resistance—are well documented, accumulating evidence indicates that skeletal muscle is also a direct target of diet-induced metabolic stress (He et al., 2020; Kawai et al., 2021; Kitessa and Abeywardena, 2016). Prolonged HFD consumption has been associated with decreased muscle mass, disorganization of myofiber structure, and impaired contractile function, changes that collectively compromise physical performance and metabolic health (Lee et al., 2015; Abrigo et al., 2016).

Resistance exercise is widely regarded as the most effective non-pharmacological strategy for preserving skeletal muscle mass. Its robust anabolic effects—classically mediated by activation of mechanistic target of rapamycin (mTOR) signaling, enhanced muscle protein synthesis, and increased expression of myogenic regulatory factors—have been consistently demonstrated in both clinical and experimental studies (Bodine et al., 2001; Fragala et al., 2019). In parallel, resistance training suppresses catabolic programs, including the ubiquitin–proteasome system and the autophagy–lysosome pathway, thereby shifting muscle toward an anabolic state (Kwon et al., 2018; Zanchi et al., 2009). These well-established adaptations underpin current recommendations that emphasize resistance training for the prevention and management of muscle-wasting conditions.

By contrast, aerobic (endurance) exercise has historically been viewed as less effective for preserving muscle mass, given that its canonical adaptations primarily involve improvements in cardiorespiratory fitness, mitochondrial biogenesis, and metabolic flexibility (An et al., 2024). Consequently, aerobic exercise has received comparatively limited attention in the context of muscle wasting. Nonetheless, emerging evidence indicates that aerobic exercise can attenuate muscle loss during metabolic stress, including that induced by HFD feeding (Xu et al., 2025; Zhang et al., 2021). However, the molecular mechanisms through which aerobic exercise mitigates HFD-induced muscle wasting remain insufficiently characterized. This gap is clinically relevant because aerobic exercise—particularly moderate-intensity continuous training (MICT)—is often more accessible and feasible than resistance training for individuals with joint limitations, severe obesity, or low baseline muscle strength. Defining the mechanisms underlying these benefits would therefore strengthen the biological rationale for considering aerobic exercise, especially MICT, as a practical strategy to manage obesity-associated muscle wasting.

To address this gap, we examined the effects of MICT on the gastrocnemius muscle in a mouse model of HFD-induced muscle wasting. RNA sequencing was performed to identify MICT-responsive transcriptional programs and signaling pathways associated with muscle preservation. By integrating physiological phenotyping with transcriptomic profiling, this study delineates the role of MICT in preserving skeletal muscle during HFD feeding and provides a mechanistic framework for its protective effects.

In the present study, 21 weeks of HFD feeding caused a significant reduction in normalized grip strength, a lower gastrocnemius muscle index, decreased muscle fiber cross-sectional area, and excessive lipid accumulation, collectively indicating muscle wasting.

At the molecular level, these impairments were accompanied by pronounced transcriptional remodeling, characterized by strong induction of genes involved in fatty acid uptake and trafficking (Cd36, Fabp3, Dbi) and lipid droplet scaffolding (Plin2, Plin5), suggesting an increased capacity for fatty acid import, binding, and storage (Sztalryd and Brasaemle, 2017; Ramos-Jiménez et al., 2022; Li et al., 2022; Knudsen et al., 1993). Consistently, enrichment analyses highlighted GO terms related to fatty acid metabolism, lipid catabolic processes, and fatty acid β-oxidation, as well as KEGG pathways including fatty acid degradation, PPAR signaling, and fat digestion and absorption, pointing to a transcriptional state dominated by lipid handling.

HFD also elicited coordinated upregulation of mitochondrial and peroxisomal β-oxidation programs. Key transporters and enzymatic components, such as Slc25a20 and Cpt2, together with multiple β-oxidation enzymes (Acadl, Hadha, Hadhb, Acaa2, Ech1, Eci2, Decr1, Etfdh), were significantly elevated, indicating a transcriptional shift toward enhanced fatty acid oxidation (Wanders et al., 2010; Wang et al., 2019; Tonazzi et al., 2021; Dissanayake et al., 2025). Consistent with this pattern, Pex11a and several fatty acid–modifying enzymes (Acot1, Acot2, Retsat, Ephx2, Hsdl2, Dhrs4) were also induced, paralleling enrichment of peroxisome-related and fatty acid elongation pathways. These features reflect an increased reliance on lipid utilization, a common adaptation to chronic lipid oversupply. While such remodeling may initially buffer nutrient overload, sustained activation of lipid oxidative pathways has been associated with the accumulation of reactive lipid intermediates, mitochondrial stress, and impaired insulin signaling, providing a plausible mechanistic link to insulin resistance (Aguer et al., 2015; Fazakerley et al., 2018; Muoio and Neufer, 2012).

Notably, enrichment of AMPK and insulin signaling pathways, together with altered expression of genes implicated in carbohydrate metabolism (Fbp2, Slc5a3, Impa2), suggests that carbohydrate handling and energy-sensing networks were engaged alongside the lipid-dominant shift (Bakshi et al., 2018; Brosch et al., 2022; Chatree et al., 2020). This pattern may indicate reduced metabolic flexibility, whereby skeletal muscle increasingly depends on fatty acid catabolism at the expense of efficient glucose utilization. Moreover, KEGG enrichment in valine, leucine and isoleucine degradation and butanoate metabolism suggests recruitment of branched-chain amino acid and short-chain fatty acid pathways, pointing to broader reprogramming of substrate oxidation under HFD feeding.

Beyond metabolic changes, transcriptomic signatures also suggested structural and microenvironmental remodeling. Enrichment of ECM–receptor interaction, together with upregulation of Mmp12, Itgad, and Angptl4, suggests activation of extracellular matrix turnover and low-grade inflammatory signaling, which may undermine contractile integrity (Alameddine and Morgan, 2016; Blythe et al., 2021; Zuo et al., 2023). Downregulation of Slc4a4, a bicarbonate transporter, may indicate reduced buffering capacity, potentially contributing to impaired contractile performance during repeated activity (Kurtz and Zhu, 2013).

Taken together, the transcriptomic profile of HFD-fed muscle reveals a lipid-centric metabolic state marked by coordinated upregulation of fatty acid uptake, storage, and oxidation pathways, accompanied by changes in carbohydrate handling, amino acid catabolism, and extracellular matrix remodeling. While these responses may be initially compensatory, they also establish a cellular milieu susceptible to lipotoxic stress, diminished metabolic flexibility, and compromised structural support, thereby providing a mechanistic framework for how chronic HFD predisposes skeletal muscle to dysfunction and insulin resistance.

Against the HFD-induced maladaptive changes described above, MICT markedly alleviated muscle wasting, as indicated by improvements in normalized grip strength, the gastrocnemius muscle index, fiber cross-sectional area, and lipid accumulation. At the molecular level, MICT reshaped the transcriptional landscape by suppressing genes associated with catabolic stress, atrophy, and metabolic inflexibility, while inducing genes linked to angiogenesis, insulin responsiveness, and structural remodeling, collectively reflecting exercise-mediated re-establishment of muscle homeostasis.

An important transcriptional signature of MICT was the repression of the FoxO-driven atrophy program. MICT decreased the expression of the transcription factor Foxo1 and concomitantly downregulated its downstream E3 ubiquitin ligases Trim63 and Fbxo32, key mediators of ubiquitin–proteasome–dependent proteolysis in muscle wasting (Peris-Moreno et al., 2020). In line with these gene-level changes, KEGG analysis showed significant enrichment of the FoxO signaling pathway, indicating that attenuation of FoxO-associated catabolic signaling is a major mechanism underlying MICT–mediated muscle preservation under HFD stress. In contrast, myogenic genes (Pax7, Myod1, Myog) exhibited only marginal alterations, indicating that MICT primarily reverses a pro-catabolic, wasting-associated transcriptional program rather than eliciting a robust pro-growth signature. In parallel, suppression of Pdk4—a key regulator that shifts substrate preference away from glucose oxidation toward fatty acid utilization—supports improved metabolic flexibility and potentially enhanced glucose handling (Pettersen et al., 2019). Reduced expression of Txnip, which is linked to oxidative stress and impaired insulin signaling, further suggests attenuation of redox stress and insulin-resistance pressure (Choi and Park, 2023). Likewise, downregulation of Angptl4, typically induced under lipotoxic and inflammatory conditions, is consistent with decreased intramuscular lipid burden.

In addition, MICT upregulated genes involved in cell survival, vascularization, and structural remodeling. For example, Cited4, a coactivator implicated in mitochondrial biogenesis and growth, was increased, potentially reflecting enhanced oxidative capacity (Lerchenmüller et al., 2020). Mrtfa, a regulator of actin dynamics and mechanosensitive transcription, together with Apold1, which mediates endothelial responses and angiogenic remodeling, points to vascular remodeling and support (Hyväri et al., 2020; Fan et al., 2023). In addition, the upregulation of Acvr1 and Ret suggests broader engagement of growth factor and survival signaling cascades (Pacifici and Shore, 2016; Carlomagno et al., 2025). These findings align with GO enrichment in sprouting angiogenesis, response to insulin, and regulation of cytosolic calcium ion concentration, as well as KEGG enrichment in PI3K–Akt, MAPK, Rap1, and calcium signaling pathways, indicating transcriptional adaptations that may enhance insulin responsiveness, excitation–contraction coupling, and vascular supply, thereby supporting endurance capacity and functional resilience.

Nevertheless, several limitations should be acknowledged. First, the mechanistic inferences in this study are based on transcriptomic profiling of the gastrocnemius muscle. While RNA-seq provides a comprehensive view of gene regulation, it does not necessarily reflect protein abundance, post-translational modifications, enzymatic activity, or metabolic flux. Future studies integrating proteomics and metabolomics, together with targeted assays of pathway activity and substrate utilization, will be important to strengthen causal links between the observed transcriptional programs and functional outcomes. Second, we implemented an 8-week MICT intervention with progressive increases in treadmill speed; however, the present design does not allow determination of the optimal training dose. Systematic comparisons across training durations and intensities will be required to define a dose–response relationship for MICT in alleviating obesity-associated muscle wasting. Third, experiments were conducted exclusively in male mice; given well-documented sex differences in hormonal milieu, substrate metabolism, and exercise adaptation, inclusion of female cohorts will be necessary to improve generalizability.

These results suggest that MICT mitigates HFD-induced muscle wasting primarily by reprogramming the transcriptome from a lipotoxic, atrophic state toward a more insulin-sensitive, pro-angiogenic profile, with limited myogenic activation.