A leucine-rich diet modulates the mTOR cell signalling pathway in the gastrocnemius muscle under different Walker-256 tumour growth conditions

Branched-chain amino acids, especially leucine, play a prominent role in the regulation of protein metabolism. Both in vivo and in vitro studies corroborate the fact that a diet supplemented with leucine stimulates protein synthesis, particularly in skeletal muscle [1‐4], and may even reverse protein catabolism [5‐7].

Cellular signalling in the setting of protein synthesis involves the regulation of protein turnover in skeletal muscle, a complex process that generally involves an interaction between gene transcription and protein translation and degradation. The stimulus and signal of this process each come from outside the cell; the primary stimulating agents are anabolic hormones, such as insulin, growth factors, glucose and nutrients, such as amino acids, especially leucine [8‐10].

The mammalian protein kinase target of rapamycin, mTOR, plays a central role in the development of skeletal muscle stimulation through growth by nutrition. mTOR acts as a sort of strategic centre from which various cellular processes are regulated [11, 12]. This protein kinase is composed of the following two independently adjustable complexes: mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2), where only mTORC1 is involved in cell signalling stimulated by nutrition, as focused here mainly in muscle cells, especially because further studies about this pathway in the cachectic host are necessary.

Leucine is the primary amino acid that activates protein synthesis in skeletal muscle [8, 9]. Some evidence suggests that amino acids use the same pathway as that used for insulin signalling, although the exact mechanism by which signalling in the mTOR complex occurs remains a subject of constant debate [13]. Considerable evidence regarding the mechanism by which amino acids act on mTOR pathway has been acquired from in vitro studies using cell culture techniques. This information indicates the need for further studies on the action of amino acids in vivo, the primary purpose of our research. Therefore, the primary purposes of our research include investigating the time-course of tumour growth under a leucine-rich diet and the effects of this diet on the activation of upstream and down-stream proteins involved in protein synthesis in the gastrocnemius muscle of Walker-tumour-bearing rats.

Our results showed the consequences of tumour evolution in muscle protein signalling during a time-course of Walker-256 tumour growth, and as present here, we showed that leucine participated in signalling pathways in parallel to insulin stimulation, modulating the Akt-PKB pathway, and also mTOR via RAG GTPases.

The mTOR complex integrates various signalling pathways that are modulated by different stimuli, including insulin, hypoxia, energy stress and nutrients [23]; the last group stands out, as amino acids may serve as stimulants, particularly leucine. Recent studies have shown that leucine participates in signalling pathways in parallel to those used by other stimuli [11, 24]. Therefore, although stimulation by insulin modulates the Akt-PKB pathway, leucine modulates mTOR via RAG GTPases [25, 26].

The present work contributed to deepening further the results obtained in previous studies [27]. Therefore, the present work analysed components of the mTOR cell signalling pathway under the influence of leucine, in the setting of cachexia; as mentioned above, this is the first time that the effects of tumour development on muscle protein synthesis were analysed during the course of an experiment. Furthermore, the analysis of these proteins at different stages of tumour development provided us with more detailed insights into the time course of activation or modulation as a result of nutritional supplementation with leucine, which may be important in the management of co-adjuvant therapy in patients with cancer.

Briefly, the Walker-256 tumour as an experimental model of cachexia shows that its development increases the muscle protein degradation by increasing the proteolysis-induction factor-like – Walker-Factor - (WF) levels, but also decreases the muscle protein synthesis by reducing the insulin level, which was associated with lower activation of IRS-1 in W group, especially after the 14th and 21st day after implant. In parallel, we verified increases in the levels of phosphorylated Akt-PKB and ERK/MAPK only at 21 days of tumour growth. Although, these high levels of Akt did not lead to activation of mTOR via in muscle of these tumour-bearing hosts, which clearly showed that the mTOR pathway was compromised likely starting by the 14th day of tumour development. On the other hand, one of the downstream components of mTORC1, phospho-STAT3 had some variable profile with an expressive rise in the muscle of these tumour-bearing groups on day 21. Indeed, STAT3 mediates the expression of a variety of genes in response to cellular stimuli and therefore plays a key role in many cellular processes, including cell apoptosis and inflammatory processes [27, 28]. STAT3 promotes oncogenesis by being constitutively active in various pathways characterised by the activity of cytokines and growth factors, including interleukins IL-6, as previously noted in our study [29]. Therefore, we hypothesised that the presence of tumour cells impaired the function of the mTOR pathway, which paralleled the effects attributed to increased phosphor-STAT3 levels in muscle cells. Additionally, the levels of phospho-STAT6 showed a rising level, which was evidenced on the 21st day of tumour development (W group). This protein plays a central role in IL-4-mediated biological responses during the inflammatory process [30]. It induces the expression of BCL2L1/BCL-X, which is responsible for the anti-apoptotic activity of IL-4 [31]. Then, we likely suggested that on 21st day, the tumour-bearing group (21 W) was subjected to more intense effects of the inflammatory process, higher interleukin IL-4 are associated with increased levels of IL-6, TNF and INF in the W group [29]. In addition, the stress-activated c-Jun amino-terminal kinase (JNK), which plays a pivotal role in several metabolic conditions, increased on 21st day in the skeletal muscle of the W group. This protein participates in the cell-signalling pathway associated with stress and inflammatory processes [32]. Tumour-bearing hosts, particularly those of the animals in the W group, are in a process characterised by inflammation and stress in the setting of cachexia [29, 33, 34]. All these points closely connected with the inflammatory process associated with the higher Walker-Factor levels in these tumour-bearing rats inducing a low activity of mTOR pathway associated with an increase on inflammatory pathways (Fig. 6).

In order to investigate some of the benefits of leucine-rich diet over the damage effects of tumour development on muscle protein, here we analysed the profile of these same key proteins of mTOR signalling pathway, including phosphorylated proteins, from up-stream and down-stream steps, which provided some interesting point how the leucine-rich diet could modulate the tumour-induced damages in this experimental model of cachexia.

In fact, the leucine-rich diet clearly demonstrated that could ensure maintenance of serum insulin levels throughout the course of tumour development, improving the muscle protein synthesis. Indeed, as presented here, Petruzzelli et al. [35] observed a reduction in serum insulin levels, particularly reduced insulin secretion, after glucose stimulation, in islet cells isolated from Walker 256 tumour-bearing rats. Insulin is an important hormone and plays a prominent role in the physiologic changes that occur in the setting of cachexia. For this reason, we hypothesised that the WL group would most likely benefit from nutritional supplementation, as muscle protein was less wasted in W group (phenylalanine incorporation and total protein net). It may be noted that the administration of leucine notably modulated insulin and also the proteolysis-induction factor-like (WF) levels, which led to an improvement effect on the mTOR cell signalling. The presence of leucine causing increases in serum insulin content [11, 36, 37] and in part modulating the WF levels, likely suggests the effect leading to increased IRS-1 activation. On the other hand, we confirmed that the leucine-rich diet did not appear to have affected signalling, by unchanged levels of phosphorylated Akt-PKB or ERK/MAPK, during the course of tumour development. A recent study demonstrated that leucine uses parallel stimulation via mTOR and does not directly participate in the modulation of Akt-PKB [13].

Indeed, the analysis of the concentration of phosphor-mTOR in the gastrocnemius muscle revealed an increase in this concentration in the tumour-bearing group that received a leucine-rich diet between the 14th and the 21st day of the experiment compared with the control group and also with the non-supplemented tumour-bearing group. Interestingly, leucine has been described by some researchers as an inhibitor of protein degradation and a stimulator of protein synthesis in cachectic hosts [5, 38]. In this present study, we observed results that are consistent with those noted by other researchers, as we found that leucine most likely stimulates protein synthesis through the mTOR signalling pathway (as noted in the WL group), particularly over longer time, such as 14 or 21 days of tumour development. Indeed, the higher phospho-mTOR concentrations are associated with higher serum insulin concentration, as verified by other studies with leucine-rich diet [29, 39, 40]. This fact was also confirmed by the amino-acid-signalling to mTORC1, which is mediated by Rag GTPases [13]; additionally, we observed that leucine led to a higher level with the difference more evident on the 21st day of the experiment in tumour-bearing group WL (Fig. 3 and Table 1). The leucine supplementation clearly demonstrated activation at this point of the pathway, which indicates that the stimulus that acted on the mTOR complex most likely resulted from the activation of RAG GTPases (Fig. 3). Amino acids signal the mechanistic and mammalian TOR complex 1 (mTORC1) via the RAS-related GTP-binding protein (RAG) family of small GTPases ([12, 13, 26]. The RAGs are assembled into heterodimers containing RAG-A (as verified in this study), a process stimulated by amino acids, particularly leucine [12]. In spite of the evidence that mTOR was not permanently increased in the L group, suggesting an adaptive process that reached a steady state following 3 weeks of leucine-rich dietary supplementation, the WL tumour-bearing group showed that RAG-A facilitated the stimulation of the mTOR pathway, confirming the muscle response in the WL group that was noted in previous studies [7, 10, 39, 40]. Meanwhile, in order to answer the question regarding the modulatory effect of leucine supplementation, we analysed the downstream component of mTOR complex 1 (mTORC1), the phospho p70S6K protein demonstrated only limited variation among the groups, which indicated that this portion of the mTOR pathway was only minimally affected by leucine supplementation. As mentioned above, we hypothesised that the adaptive process involving leucine supplementation might stimulate the mTOR pathway; thus, the analysis of mTOR/p70S6K-1 ratio made it possible to determine that a stronger stimulus occurred in the L group on the 7th day and continued as the experiment progressed (14th and 21st day). Analysing the tumour effect, the mTOR/p70S6K-1 stimulus that occurred in WL group during the experiment differed from that observed in the W group, especially after the 14th day, which was perfectly fitted to the raising of phospho-mTOR and RAG-A expression, suggesting the positive effect of leucine supplementation.

Considering the activation of downstream mTOR pathway, the maintenance of STAT3 phosphorylation occurred in WL group showed the leucine influence, since the group without tumour cells or bearing a tumour had constant levels of phospho-STAT3 over the three study days compared with the C group. Indeed, STAT3 mediates the expression of a variety of genes in response to cellular stimuli, in this case being modulated by leucine effects. We suggest that the STAT3 could be likely activated in various pathways characterised by the activity of cytokines and growth factors, including interleukins IL-6, as previously noted in your study [29], but under leucine supplementation, this downstream protein could be reduced benefiting the skeletal muscle in these tumour-bearing animals (Fig. 4). Constitutive STAT3 activation is associated with various human cancers and often suggestive of a poor prognosis, mostly related to inflammatory processes [27]. However, leucine supplementation interfered with this process, as muscle STAT3 activation was similar to that noted in the control groups (C and L; Fig. 4). In this same view, the raises of phospho-STAT6 and JNK in muscles of tumour-bearing rats leads us to the thought that the muscle catabolic process was directly related to the increase of pro-inflammatory cytokines from day 14th of tumour development, as previously shown by Cruz and colleagues [29]; although the leucine-rich diet also contributed to reducing this tumour-induced damage over skeletal muscle decreasing these proteins especially after day 14 of the experiment (Fig. 4). We hypothesised that on 21st day, the tumour-bearing group was subjected to more intense effects of the inflammatory process [29]. This change was smaller in the tumour-bearing rats subjected to leucine supplementation. The stress-activated c-Jun amino-terminal kinase (JNK) participates in the cell-signalling pathway associated with stress and inflammatory processes, which in particularly those of the animals in the W group are in inflammation and stress processes in the setting of cachexia, particularly when the Walker-tumour cell burden exceeds 10% of an animal’s body weight (in this study, the tumour/body weight ratio reached more than 12%, [29]), as noted previously by several researchers [34, 41, 42]. As noted in our previous studies, leucine stimulates protein synthesis [10, 39, 40]; therefore, based on the results obtained in this study, it is possible that a component acting in parallel to the mTOR signalling pathway acts as a result of leucine stimulation, leading to increase in muscle protein synthesis, minimising the effects of the tumour cells as a result of decreasing WF levels.

After analyse those time-course experiments we choose the 21st day as a point of more pronounced effect of a tumour and leucine-rich diet on animals’ body. Therefore, radioactive experiments were performed only on the 21st day of tumour development.

Leucine stimulates muscle protein synthesis and modules the activity of various proteins involved in the control of mRNA translation [43]. Leucine can stimulate protein synthesis directly and/or via its metabolite, as α-ketoisocaproic acid (KIC). These findings are corroborated by the data on protein synthesis and degradation contained in Fig. 5. It can be noted that the presence of leucine stimulates protein synthesis through increased incorporation of phenylalanine and KIC (a ketoacid converted from Leucine). In contrast, the group fed a control diet and tumour-bearing presents lower protein synthesis and higher degradation rates. Since protein synthesis is reduced and protein degradation is increased throughout the body during cancer cachexia, the incorporation of amino acids, especially branched-chain amino acids, by muscle cells could supply important nutrients and influence protein synthesis. The lower incorporation of leucine into gastrocnemius muscle proteins in tumour-bearing rats indicated a decrease in protein synthesis that paralleled an increase in protein wasting [40, 44, 45]. As shown here, the leucine-rich diet reduced the protein catabolism and increased the protein synthesis in WL rats compared to W rats.

These finds clearly show the minimisation of tumour-induced damage effects when these tumour-bearing rats were subjected to leucine-rich diet. Leucine supplementation appeared to reduce the effects of stress in the host body, as well as effects of the inflammatory processes generated by the presence of the tumour cells improving the host’s responses and maintaining lean body mass (Fig. 6).

This work demonstrates for the first time a time-course study with action of leucine anticipating and enhancing the parameters analysed in the setting of tumour growth; this involves the stimulation of protein synthesis, which results in reduced cachexia and occurs primarily in the experiment that achieves the best values on the 21st day following the start of the experiment. These results are consistent with those observed in previous studies performed by our research group. It was also noted that leucine stimulates various points of the mTOR pathway, including different anabolic hormones via, such as insulin. Further study is necessary in order to completely understand the effects of leucine on protein synthesis and protein degradation at the molecular level.