The effectiveness of protein, leucine and [beta]-hydroxy-[beta]-methylbutyrate on cell-signaling pathways controlling protein turnover in red and white gastrocnemius muscles of rats

Whey protein supplementation, containing large amount of leucine, has been a traditional intervention to maintain net protein balance in the past decades. It has been recognized that leucine alone is able to stimulate protein synthesis by activating mTOR and its related downstream pathway without affecting protein degradation, whereas its metabolite β-hydroxy-β-methylbutyrate (HMB) is known to attenuate protein degradation when provided chronically. However, the mechanism of HMB’s benefit remains unclear. To address how HMB regulates protein synthesis and degradation signaling pathways, we compared one dose of whey protein (187.5mg/kg), HMB (400mg/kg) or leucine (1.4g/kg) by oral gavage. Blood was collected at 0, 45 and 90 min for blood glucose and plasma insulin analysis. Red and white gastrocnemius muscle was taken separately 90 min after gavage. Blood glucose was reduced by leucine at 45 and 90 min post gavage. Plasma insulin was enhanced by leucine at 45 min and then decreased at 90 min post gavage, whereas HMB decreased plasma insulin through 90 min post gavege. Western blot analysis showed that HMB phosphorylated Akt in red gastronemius, and enhanced phosphorylation of mTOR in both types of muscles. Leucine phosphorylated mTOR, p70s6k and 4E-BP1 in both red and white gastronemius. Regarding protein degradation signals, phosphorylation of FOXO3A was enhanced by HMB, but not in the other treatment groups. Whey protein had no effect on those cellular signaling. Our results indicate that both HMB and leucine may stimulate protein synthesis through the mTOR pathway in red and white gastrocnemius muscles by different degrees with leucine more effective than HMB. HMB may have a greater effect than leucine on limiting protein degradation by phosphorylating Akt and FOXO3A in red and white gastrocnemius muscles. A combination of HMB and leucine, as a new interventional strategy, is predicted to maximize protein accretion by increasing protein synthesis as well as inhibiting protein degradation.