This study established a mouse model of repeated intermittent administration of clenbuterol (CB), a β2-adrenergic receptor agonist known to increase skeletal muscle mass. The authors compared the long-term effects of a single CB exposure versus repeated intermittent CB exposure on the plantaris muscle. Proteomic profiling was performed using label-free quantitative proteomics to capture broad protein-level changes associated with the different exposure histories.
Under the same long-term experimental timeline, repeated intermittent CB exposure produced a more evident increase in muscle weight than did a single CB exposure. The authors report that repeated exposure produced a clearer muscle-weight response, indicating that exposure history modifies the phenotypic outcome of β2-adrenergic stimulation in skeletal muscle.
The plantaris muscle was subjected to label-free quantitative proteomic analysis to identify proteins whose abundance changed after CB exposure. The proteomic workflow allowed the authors to detect broad changes across structural, remodeling-related, trafficking-related, and metabolic proteins, enabling a systems-level view of how intermittent CB administration alters the muscle proteome.
Proteomic profiling revealed that the response to repeated CB exposure was not a simple reproduction or amplification of the single-exposure response. Instead, repeated exposure associated with distinct changes in multiple functional groups of proteins, including those linked to sarcomere structure, tissue remodeling, membrane trafficking, and metabolic processes. These groupings point to coordinated adjustments in contractile architecture, extracellular or interstitial remodeling, intracellular membrane dynamics, and cellular metabolism after repeated β2-adrenergic stimulation.
To characterize the diversity of protein-level responses, the authors performed an integrated response-class analysis. This analysis identified multiple regulatory patterns: a set of proteins that responded to any CB exposure (shared CB-responsive proteins) and subsets of proteins that were preferentially altered in the repeated CB condition. The classification highlighted that some molecular changes are common to acute and repeated exposure, whereas others emerge or become more pronounced only with repeated intermittent dosing.
Select proteomic findings were validated by immunoblotting. The authors report validation of representative proteins from different response classes: Klhl40 and Napa were validated as shared CB-responsive proteins; Galectin-3 was validated as a strongly CB-responsive protein associated with remodeling; and Rab18 was identified as a protein change more clearly detected under repeated CB conditions. These immunoblot results support the proteomic classification into shared and repeated-specific response patterns.
Collectively, the data indicate that repeated intermittent clenbuterol exposure induces exposure history–dependent proteomic remodeling in skeletal muscle. The repeated-exposure proteomic signature is distinct from that produced by a single exposure, implicating exposure history as an important determinant of molecular adaptation to β2-adrenergic stimulation. The authors present their dataset as a resource to improve understanding of the molecular consequences of repeated CB exposure and to support future mechanistic studies of β2-adrenergic–induced skeletal muscle adaptation.
This report is a preprint and has not been certified by peer review. Specific experimental details (such as dosing regimen, timelines, sample sizes, and statistical thresholds) are part of the full manuscript and supplementary files; if not detailed in this summary, those specifics were not reported in the abstract text used as the source. The authors declared no competing interests. Funding sources listed include Japan Society for the Promotion of Science (KAKENHI grants), NCNP intramural grants, and the Japan Sports Agency. The authors indicate the dataset and validations provide a resource for future mechanistic work on β2-adrenergic stimulation and skeletal muscle adaptation.