Introduction
Exercise is known to cause physiological alterations in skeletal muscle, particularly in muscle glycogen reduction and regulation of muscle protein synthesis (MPS) [
1,
2]. Several studies have shown that the combination of carbohydrates and protein after exercise replenishes muscle glycogen more efficiently than consumption of carbohydrates alone [
3,
4]. Wang et al. [
4] reported that post-exercise dextrose and whey protein supplementation increased MPS after exercise compared to placebo, with whey protein probably initiating greater activation of the mammalian target of rapamycin (mTOR) signaling pathway. The branched-chain amino acids (BCAAs) comprised of leucine, isoleucine, and valine are commonly known as the nutrients with the strongest anabolic effect in mammals [
5]. Various studies have shown that amino acids, particularly BCAAs and specifically leucine, stimulate MPS in muscle [
6‐
9]. If the body does not supply free amino acids, the protein degradation that occurs after exercise may persist for a longer period of time, thus restricting MPS [
10]. During the first hour following exercise, MPS is increased and may continue for 24–48 h, and therefore supplementation with essential amino acids following exercise is vital for MPS [
11,
12]. Moreover, studies have shown that consumption of a protein-containing meal directly after exercise promotes MPS in the same manner as complex protein or full-mixed amino acids [
13,
14].
Although the anabolic structure of insulin is well described, the role of insulin in the molecular mechanism of MPS in exercised rats is still unclear. Many studies suggest that insulin can play an important role in enhancing net protein balance by reducing protein degradation [
10,
15]. In the presence of adequate whole protein and/or essential amino acids (EAAs), insulin stimulates MPS, while at lower blood EAA levels, insulin inhibits protein breakdown [
16,
17]. At the molecular level, the phosphoinositide-3-kinase (PI3K/Akt/mammalian mechanistic) target of the rapamycin (mTOR) pathway has a critical role in regulating skeletal muscle protein metabolism and mass [
18]. In particular, mTOR facilitates the effects of nutrients and insulin on protein synthesis. Activated mTOR promotes mRNA translation initiation and protein synthesis by phosphorylating eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). This supports the formation of eIF4E from the inhibitor eIF4E•4E-BP1 complex and the mRNA translation initiation formation, promoting the eIF4E•eIF4G complex [
19]. The translation is also stimulated by mTOR via the phosphorylation of the serine-threonine kinase ribosomal protein S6 kinase 1 (S6K1 or p70S6K1) [
20]. When amino acids are available, insulin regulates mTOR signaling through activation of upstream kinases such as Protein Kinase B, which phosphorylates serine 2448 of mTOR. Specifically, leucine is known to act with insulin to stimulate mTOR signaling proteins including ribosomal protein 6 (rpS6) and eIF4E [
21].
Chromium (Cr) has an important effect on improving insulin action and increasing the metabolism of nutrients including carbohydrates, lipids, proteins, and nucleic acids by activation of enzymes involved in linked pathways including glucose transporters (GLUTs), insulin receptor substrate-1 (IRS-1), and fatty acid synthase (FAS) [
22‐
25]. Because amylopectin may also lead to a rapid increase in blood glucose and insulin concentrations [
26], an amylopectin/chromium complex (ACr) has been studied for its ability to augment exercise-induced MPS. In a previous clinical study, it was shown that intake of ACr with WP directly before exercise doubles the rate of MPS compared to the same dose of WP alone [
17]. To date, however, no studies have reported on the detailed mechanisms of action (e.g. mTOR pathway) of BCAAs combined with ACr. Thus, the aim of the present study was to investigate the effects BCAAs combined with ACr on MPS, insulin concentration, and the mTOR signaling pathway.
Discussion
Previous studies have shown that supplementation with BCAAs or leucine alone results in significant rises in MPS rates with a concomitant increase in the phosphorylation of downstream targets of mTOR signaling in exercised rats [
30,
31]. Increases in MPS due to EAAs are associated with an increase in signaling activity in the mTOR / p70S6K pathway [
32]. In the present study, results showed that ACr combined with BCAAs significantly enhanced MPS, serum insulin levels, and amino acid levels compared to BCAAs alone and exercise alone. While this is the first study to examine ACr in combination with BCAAs, prior research has investigated the effects of ACr on MPS when combined with whey protein. A clinical model demonstrated that ingestion of ACr + 6 g of whey protein prior to a leg extension exercise protocol resulted in significantly greater MPS compared to 6 g of whey protein alone [
17]. Moreover, in a preclinical study of similar design to the present study, researchers found that ACr significantly increased MPS when combined with increasing doses of whey protein compared to whey protein alone. When comparing these findings to the present data, it is evident that the combination of BCAAs and ACr increased FSR by a similar amount as 20 g (HED) of whey protein alone [
33]. Although it has been suggested that the use of BCAAs alone for MPS is unwarranted [
34], the results from the present study support previous clinical research showing that BCAA supplementation promotes MPS [
35].
Results from the present study also demonstrated that ACr combined with BCAAs improved phosphorylation of mTOR, S6K1, and 4E-BP1 compared to BCAAs alone and exercise alone. These effects may be due to the ACr complex providing an extra advantageous biochemical situation through which proteins such as mTOR can be synthesized. Moreover, the phosphorylation states of mTOR, S6K1 and 4E-BP1 were greatly associated with insulin concentrations in this study. Based on these current results and earlier studies, it can be speculated that the elevated MPS stimulated via the ACr complex is mediated by the activation of the mTOR signaling pathway. The mTOR pathway, a significant controlling factor for MPS, stimulates the kinase activity of the complex and leads to the phosphorylation of 4E-BP1 and p70S6K, which are two enzymes that also modulate protein synthesis at the level of mRNA translation initiation [
36,
37]. The capacity of 4E-BP1 to release eIF4E from the 4E-BP1eIF4E complex to induce mRNA translation is mainly dependent on the 4E-BP1 hyperphosphorylation level. Several nutrients, such as amino acids and carbohydrates, stimulate protein synthesis via the activation of the mTOR/p70S6K and e4E-BP1 pathway [
38]. Previous studies showed that EAAs plus carbohydrates promoted better MPS post-exercise than control and that this MPS was linked to more robust phosphorylation of mTOR and p70S6k [
39,
40]. Additionally, leucine and insulin have been stated to stimulate phosphorylation of mTOR at Ser2448 [
41,
42]. Similar to our results, Wang et al. [
4] reported that MPS was significantly increased by whey protein plus carbohydrates compared with placebo, and approached significance compared to whey protein alone. They also reported that whey protein plus carbohydrates produced superior phosphorylation of mTOR and p70S6K compared with the sedentary and placebo groups. In addition, Yoshida et al. [
7] reported that treadmill exercise elevated the phosphorylation of p70S6 kinase in the muscle of low protein diet-fed chronic kidney disease (CKD) rats. They also reported that the BCAAs of the CKD rats restored the phosphorylation of p70S6 kinase to the same level detected in the sham group, however the rise in MPS and muscle mass was marginal. Morrison et al. [
39] reported that supplementing rats with a solution comprised of either carbohydrates, protein, or carbohydrates plus protein immediately following exercise rapidly improved the phosphorylation of mTOR, 4E-BP1 and p70S6K compared with exercise control rats. In a study investigating protein and carbohydrate (50% sucrose plus 50% maltodextrose) supplementation, both carbohydrate plus soy protein and carbohydrate plus whey protein improved formation of the mRNA cap-binding complex eIF4F and stimulated phosphorylation of the translational repressor, 4E-BP1, S6K1, and mTOR compared with carbohydrates alone [
31]. In another study, however, it was reported that neither hindlimb suspension nor chromium treatment changed the protein levels of myostatin, phospho-Forkhead box O-, or mTOR [
43].
The purported ability of chromium to favorably enhance insulin metabolism is possibly a mechanism by which ACr enhances the mTOR mediated MPS pathway during muscle recovery when combined with BCAAs [
22,
23,
44]. Previous studies have shown that chromium enhances GLUT-4 translocation by increasing insulin receptor activation, which results in improved insulin sensitivity and glucose uptake [
23]. For instance, studies done by our groups and others have reported that chromium picolinate (CrPic)/chromium histidinate (CrHis) may enhance carbohydrate and lipid metabolism by regulation of glucose transporters, PPAR-γ and p-IRS-1 expression, and other insulin metabolism aspects [
22‐
24,
45]. It has also been reported that supplemental CrHis/CrPic elevates liver GLUT-2 levels, as well as muscle Nrf2 and GLUT-4 levels, and reduces muscle NF-κB levels, with CrHis being superior to CrPic [
46]. Chromium’s mechanism of action is crucial for MPS because when insulin binds to muscle cells, it stimulates the transportation of EAAs into the muscle cells. It has been established that insulin has a stimulating effect on MPS when acceptable EAA precursors are present and works to reduce muscle protein degradation when EAA levels are decreased [
16,
17]. The transportation of EAAs into muscle cells is important for the activation of certain mTOR signaling proteins, such as S6K1 and 4E-BP1, that are responsible for regulating muscular growth [
31]. For instance, one study found that when orally administering leucine or a carbohydrate meal to rats after exercise, MPS only increased in the leucine group, demonstrating the importance of adequate levels of amino acids for MPS stimulation. These increases in MPS were also correlated with changes in the phosphorylation of S6K1 and 4E-BP1, demonstrating that leucine stimulates MPS through the mTOR signaling pathway [
30]. The connection between amino acid concentration and insulin-induced mTOR signaling is supported by clinical research showing that an increase in plasma amino acid levels by amino acid infusion increases insulin-stimulated mTORC1/S6K1 activity [
47]. Additionally, leucine and insulin have been stated to stimulate phosphorylation of mTOR at Ser2448 [
41]. Therefore, the mTOR signaling pathway is mediated by the presence and action of both amino acids and insulin, and lack of either may result in reduced MPS. By combining BCAAs with ACr, a factor that is known to positively impact insulin metabolism, the mTOR signaling pathway is provided with two of its critical components.
The anabolic benefits of post-exercise ACr and BCAA supplementation may be attributed to the fact that physical exercise normally causes an amino acid imbalance by promoting proteolysis relative to protein synthesis in skeletal muscle, leading to a decrease in plasma BCAA levels [
48]. Interestingly, results from the current experiment showed that ACr increased serum concentrations of BCAAs and other amino acids despite their known utilization in muscle during exercise. Because animals were fasted overnight, de novo synthesis of certain amino acids may have occurred to compensate for a shortage of non-essential amino acids in the gut, as suggested by Wolfe et al. [
34]. Furthermore, treatment with BCAAs, along with ACr, may have led to selective utilization of amino acids by muscle tissue, sparing glucogenic amino acids and S-containing amino acids.
While further clinical research is needed, results of the present experiment, along with data from previous preclinical and clinical studies, may be of interest to athletic and fitness communities who are interested in supplementing with a dietary supplement (ACr; Velositol®) in combination with a source of protein or amino acids to stimulate muscle anabolism and in turn, result in greater muscular outcomes from exercise. These results could also be of high relevance to aging populations who may have a harder time gaining and maintaining muscle, as aging can result in resistance to the anabolic effects of amino acids and negative alterations in glucose metabolism [
17,
49]. Chromium has been clinically shown to improve cholesterol and glucose levels in non-diabetic and diabetic subjects, as well as result in body fat loss and increased muscle gains in resistance trained men [
23,
50]. Consequently, ACr may enable older populations to gain muscle mass, reduce the risk of injury, and support overall health.
To further explore the effect of ACr on augmenting MPS, clinical outcomes studies should be carried out in the future to examine the effect of ACr and BCAAs on muscular growth and strength. Furthermore, because the present study was conducted as a single dose experiment, it would be of interest to examine the effect of consuming BCAAs and ACr after exercise over a longer period. Moreover, because the present study only examined the effect of the addition of ACr to a 6 g HED of BCAAs, it would be of interest to examine how ACr would affect MPS when added to various doses of BCAAs. Because MPS is strongly reliant on EAA levels, it is possible that adding ACr to increasing doses of BCAAs or EAAs will magnify the rates of MPS seen with intake of ACr plus a 6 g HED of BCAAs.
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