Hitachi, K.; Yamaguchi, H.; Kiyofuji, Y.; Tsuchida, K.

Clenbuterol (CB), a {beta}2-adrenergic receptor agonist, is known to increase skeletal muscle mass. However, because its effects are influenced by treatment duration, dose, and {beta}-adrenergic receptor responsiveness, it remains unclear how extended intermittent CB exposure alters the skeletal muscle proteome. In this study, we established a repeated intermittent CB administration model in mice and compared the effects of single and repeated CB exposure on plantaris muscle using label-free quantitative proteomics. Repeated CB exposure produced a more evident muscle-weight response than single exposure under the same long-term experimental timeline. Proteomic profiling revealed that repeated CB exposure was not a simple reproduction or amplification of the single CB response, but was associated with distinct changes in sarcomere-associated, tissue-remodeling-related, membrane-trafficking-related, and metabolic proteins. Integrated response-class analysis further identified multiple patterns of protein regulation, including shared CB-responsive proteins and proteins preferentially identified as changed under repeated CB conditions. Immunoblotting validated representative proteins from these response classes: Klhl40 and Napa as shared CB-responsive proteins, Galectin-3 as a strongly CB-responsive remodeling-related protein, and Rab18 as a protein change more clearly detected under repeated CB conditions. Together, these findings indicate that repeated intermittent CB exposure induces exposure history-dependent proteomic remodeling in skeletal muscle. This study provides a resource for understanding the molecular consequences of repeated CB exposure and for future mechanistic studies of {beta}2-adrenergic stimulation-induced skeletal muscle adaptation.