The present study sought to evaluate the impact of HMβ supplementation on the expression of proteins involved in skeletal muscle hypertrophy in healthy and sedentary rats and proteins involved in insulin signalling. Our results show that HMβ supplementation increased mTOR expression and phosphorylation of p70S6K in the EDL muscle while increasing fasting insulin levels and testosterone/corticosterone ratios and decreasing fasting glucose and corticosterone levels in the serum.
As previously described, HMβ is a widely studied metabolite of leucine. Several reports have shown that branched-chain amino acids (BCAA), isolated leucine and HMβ can stimulate skeletal muscle protein synthesis and activate the mTOR pathway in skeletal muscle [9, 21] as well as in primary hepatocytes .
In the present study, we observed that, relative to the control group, the supplemented group demonstrated an increase in mTOR protein levels and activation of p70S6K, which are linked to increased skeletal muscle mass in the EDL muscle. Our findings are supported by a recent study by Lang's group  showing that gastrocnemius mass and protein synthesis were robustly decreased in mTOR heterozygous mice compared to wild type mice. Based on this information, we conclude that not only the activity but also the level of mTOR is an important regulator of skeletal muscle mass.
Contrary to what was observed in the present study, Ostaszewski et al.  and Holecek et al.  did not observe increased protein synthesis in the EDL and soleus muscles after HMβ supplementation but measured consistently decreased protein degradation, as estimated by the net release of tyrosine from incubated muscles.
In the present study, we analysed the expression of AMPK, which is known to be an important regulator of muscle protein synthesis [25, 26], but we found no differences between the groups.
In the present study, we found no alterations in the Akt/PKB pathway in the EDL muscle. Thus, we suggest that increased skeletal muscle protein mass by HMβ supplementation was induced directly via increased mTOR expression and activation of p70S6K and not via phosphorylation of Akt/PKB. However, as previously shown in several studies, constitutive activation of Akt/PKB is capable of inducing skeletal muscle hypertrophy [27–29], although we did not observe this effect in our study. Moreover, as the molecular analyses were performed 15-18 hours after HMβ oral gavage, we suggest that the activation of the mTOR/p70S6K pathway persists for many hours after supplementation. However, Laymans group has shown that peak mTOR and insulin signaling responses occur shortly after consumption of a meal (i.e. 1-3 hrs) ; considering that our measures were taken 15-18 hrs after the HMβ gavage, and after an overnight fast, it is possible that we missed certain signals. Therefore, the acute (1-3 hrs post gavage) effects of HMB on translation initiation and elongation factors, as well as insulin signaling warrant further investigation.
In the current study, we investigated the intracellular signalling pathways involved in increased protein synthesis induced by HMB; however, it is important to remember that potent hormones are secreted in response to nutrients and might exert robust increases in protein synthesis and metabolism in several tissues [31–33]. In the present study, we observed that increased insulin levels may have resulted in increased mTOR levels and phosphorylation of p70S6K; however, we have no data that directly address this idea, and previous studies [30, 34] do not support the idea that typical fasting insulin concentrations (above basal levels) can stimulate the mTOR/p70S6K pathway.
As discussed above, several studies [16, 22, 26] have been performed to determine the main mechanism of action of HMβ. Based on the results of this study, we have outlined the main mechanisms of HMβ action as being related to increases in mTOR/p70S6K pathway signalling and most likely leading to improved protein synthesis and muscle hypertrophy. However, it is important to note that protein synthesis pathways are extremely redundant, and other important proteins not evaluated in our study, such as the eukaryotic translation initiation factor 4E (eIF4E/2B), might play a role in the observed final response (i.e., muscle hypertrophy).
In accordance with this idea, hormones control anabolic/catabolic pathways that can favour (insulin, testosterone) or antagonise (glucocorticoid hormones) [35, 36] anabolism in skeletal muscle. In fact, we observed low serum corticosterone levels and high testosterone/corticosterone ratios after HMβ supplementation, which could contribute to skeletal muscle hypertrophy. Likewise, Olza et al.  observed that in elderly patients, a protein-enriched diet was able to increase protein synthesis and reduce protein degradation. Moreover, the reduction in corticosterone and increase in insulin levels may have favoured the reduction in fasting blood glucose after HMβ supplementation. Recently, Guo et al.  also found improvements in glucose homeostasis after leucine supplementation.
Hepatic lipid levels were not altered, as shown in Table 2. These results are consistent with the lack of change in serum lipid levels. Several studies [3, 17] have shown that HMβ is metabolised to HMG-CoA and used for de novo synthesis of cholesterol in certain tissues, including the liver. However, this conclusion is debatable; Holecek et al.  showed increases in serum cholesterol levels after HMβ administration, while Nissen and Abumrad  showed decreases in LDL-c.
In the present study, we also demonstrated an increase in skeletal muscle weight (EDL and soleus) in the absence of changes in total body mass, fat mass or liver weight. However, is suggesting that other body measures of body fat, such as the size of different fat deposits, e.g., epididymal fat, may have changed.
As stated previously, the effects of HMβ supplementation in peripheral tissues have not been intensely investigated. In accordance with this, Pedrosa et al.  showed that leucine supplementation increased liver protein content. However, in this study, we thoroughly investigated HMβ's effects on proteins involved in insulin signalling, starting with the insulin receptor (IR), and showed that HMβ supplementation stimulated IR expression in the liver, likely due to mTOR activation. However, the effects of long-term HMβ supplementation on this outcome are as yet unknown.
In summary, HMβ treatment leads to skeletal muscle hypertrophy via increases in mTOR expression and decreases in serum corticosterone concentrations. In addition, HMβ supplementation increases IR expression in the liver compared to control group. Thus, our results suggest that HMβ supplementation can be used to increase muscle mass without adverse health effects.