Hemp seed protein hydrolysates and its bioactive peptides mitigate muscle atrophy in in vitro and in vivo models

We recently demonstrated that hemp seed protein hydrolysates (HPH), produced through enzymatic hydrolysis, protect against muscle atrophy in both in vitro and in vivo models. This study aimed to optimize the HPH production method and elucidate its mechanism of action in preventing muscle atrophy, including the identification of bioactive peptides within HPH. To optimize HPH production, we compared the degree of protein hydrolysis using alcalase and flavourzyme, both individually and in combination. Ultimately, we produced HPH by treating it with 2% flavourzyme for 2 h. Our results showed that HPH increased cell viability and normalized reactive oxygen species levels in H₂O₂-treated C2C12 myoblasts. In a mouse model of muscle atrophy induced by dexamethasone (DEX), HPH improved grip strength and increased muscle mass, demonstrating effects stronger than those of whey protein. Immunoblotting analysis indicated that HPH activates protein synthesis pathways while inhibiting protein degradation, apoptosis, and the production of inflammatory cytokines in skeletal muscle. Additionally, we analyzed the peptide composition of HPH and investigated the anti-atrophic effects of specific peptides in C2C12 myotubes. Among fourteen peptide candidates, peptides AERGF, VL, and GLK showed the most significant effects on enhancing myotube diameter and reducing the expression of ubiquitin ligases and cleaved PARP in DEX-treated C2C12 myotubes. These peptides also increased the phosphorylation of AMPK and FoxO3a, which were reduced by DEX. Notably, their protective effects against myotube atrophy diminished when the cells were co-treated with an AMPK inhibitor. These findings demonstrate that HPH and its bioactive peptides effectively mitigate DEX-induced muscle atrophy by positively regulating muscle protein synthesis and degradation pathways.