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

Abstract

Background: The exact signalling mechanism of the mTOR complex remains a subject of constant debate, even with some evidence that amino acids participate in the same pathway as used for insulin signalling during protein synthesis. Therefore, this work conducted further study of the actions of amino acids, especially leucine, in vivo, in an experimental model of cachexia. We analysed the effects of a leucine-rich diet on the signalling pathway of protein synthesis in muscle during a tumour growth time-course.

Methods: Wistar rats were distributed into groups based on Walker-256 tumour implant and subjected to a leucine-rich diet and euthanised at three different time points following tumour development (the 7th, 14th and 21st day). We assessed the mTOR pathway key-proteins in gastrocnemius muscle, such as RAG-A-GTPase, ERK/MAP4K3, PKB/Akt, mTOR, p70S6K1, Jnk, IRS-1, STAT3, and STAT6 comparing among the experimental groups. Serum WF (proteolysis-induced factor like from Walker-256 tumour) and muscle protein synthesis and degradation were assessed.

Results: The tumour-bearing group had increased serum WF content, and the skeletal-muscle showed a reduction in IRS-1 and RAG activation, increased PKB/Akt and Erk/MAP4K3 on the 21st day, and maintenance of p70S6K1, associated with increases in muscle STAT-3 and STAT-6 levels in these tumour-bearing rats.

Conclusion: Meanwhile, the leucine-rich diet modulated key steps of the mTOR pathway by triggering the increased activation of RAG and mTOR and maintaining JNK, STAT-3 and STAT-6 levels in muscle, leading to an increased muscle protein synthesis, reducing the degradation during tumour evolution in a host, minimising the cancer-induced damages in the cachectic state.

Keywords: Cancer cachexia, Leucine, mTOR cell signalling pathway, Eukaryotic Initialisation factors; Walker-256 tumour.

Fig. 1

A profile of serum insulin (pM), a proteolysis-induced factor (Walker factor, WF, % compared to the control group). The animals were euthanised at different times during tumour development to assess the profile of serum insulin including the 7th, 14th, and 21st days of the experiment. The Walker Factor was assessed at 21st day of the experiment. Legend: C, control group; W, Walker tumour-bearing group; L, rats fed a leucine-rich diet; WL, tumour-bearing rats fed a leucine-rich diet. Graphics represent mean ± standard deviation, and statistical significance is presented in Table 1 after two-way Anova analysis, followed by the Fisher’s LSD multiple comparison test; P < 0.05

Fig. 2

Key-proteins related to the mTOR pathway: phosphorylated Akt-PKB, Erk/MAPK, and IRS-1 in the gastrocnemius muscle under the influence of Walker-tumour development and nutritional supplementation with leucine. The minimum number of animals per group was 6. The animals were euthanised at different times during tumour development, including the 7th, 14th, and 21st days of the experiment. Legend: C, control group; W, Walker tumour-bearing group; L, rats fed a leucine-rich diet; WL, tumour-bearing rats fed a leucine-rich diet. Graphics represent mean ± standard deviation. Statistical significance is presented in Table 1 after two-way Anova analysis, followed by the Fisher’s LSD multiple comparison test; P < 0.05

Fig. 3

mTOR, p70S6K-1, mTOR/p70S6K ratio and RAG-A (arbitrary units). The animals were euthanised at different times during tumour development, including the 7th, 14th, and 21st days of the experiment. Legend: C, control group; W, Walker tumour-bearing group; L, rats fed a leucine-rich diet; WL, tumour-bearing rats fed a leucine-rich diet. Graphics represent mean ± standard deviation. Statistical significance is presented in Table 1 after two-way Anova analysis, followed by the Fisher’s LSD multiple comparison test; P < 0.05

Fig. 4

A profile of phosphorylated JNK, STAT3 and STAT6 in the gastrocnemius muscle under the influence of Walker-tumour development and nutritional supplementation with leucine. The minimum number of animals per group was 6. The animals were euthanised at different times during tumour development, including 7th, 14th, and 21st day of the experiment. Legend: C, control group; W, Walker tumour-bearing group; L, rats fed a leucine-rich diet; WL, tumour-bearing rats fed a leucine-rich diet. Graphics represent mean ± standard deviation. Statistical significance is presented in Table 1 after two-way Anova analysis, followed by the Fisher’s LSD multiple comparison test; P < 0.05

Fig. 5

Effects of a leucine-rich diet in gastrocnemius skeletal muscle from tumour-bearing rats. a Phenylalanine incorporation, b Tyrosine release, c Total muscle protein net (represented by phenylalanine/tyrosine ratio), d Leucine incorporation, e KIC incorporation into muscle protein in different groups. For details, see Methods. Abbreviations: C, control; L, leucine-rich diet group; W, tumour-bearing rats; WL, tumour-bearing rats fed a leucine-rich diet. Graphics represent mean ± standard deviation. Statistical significance is presented in Table 1 after two-way Anova analysis, followed by the Fisher’s LSD multiple comparison test; P < 0.05

Fig. 6

A scheme showing variations of key phosphorylated protein concentrations in the gastrocnemius muscle under the influence of tumour development on different days of the experimental protocol (7th, 14th and 21st day), and under the influence of nutritional supplementation with leucine