Abstract
In this study, we explored the coordinate regulation of mTORC1 by insulin and amino acids. Rat livers were perfused with medium containing various concentrations of insulin and/or amino acids. At fasting (1×) or 2× (2×AA) concentrations of amino acids, insulin maximally stimulated Akt phosphorylation but had no effect on global rates of protein synthesis. In the absence of insulin, 4×AA produced a moderate stimulation of protein synthesis and activation of mTORC1. The combination of 4×AA and insulin produced a maximal stimulation of protein synthesis and activation of mTORC1. These effects were accompanied by decreases in raptor and PRAS40 and an increase in RagC associated with mTOR (mammalian target of rapamycin). The studies were extended to a cell culture model in which mTORC1 activity was repressed by deprivation of leucine and serum, and resupplementation with the amino acid and insulin acted in an additive manner to restore mTORC1 activation. In deprived cells, mTORC1 was activated by expressing either constitutively active (ca) Rheb or a caRagB·caRagC complex, and coexpression of the constructs had an additive effect. Notably, resupplementation with leucine in cells expressing caRheb or with insulin in cells expressing the caRagB·caRagC complex was as effective as resupplementation with both leucine and insulin in non-transfected cells. Moreover, changes in mTORC1 activity correlated directly with altered association of mTOR with RagB/RagC, Rheb, raptor, and PRAS40. Overall, the results suggest that amino acids signal through the Rag complex and insulin through Rheb to achieve coordinate activation of mTORC1.
Highlights
Activation of Akt by insulin leads to the phosphorylation of at least two proteins involved in the regulation of mTORC1, PRAS40 and TSC2
The hypothesis being tested was that insulin and amino acids act in a coordinate manner to activate mTORC1 and to stimulate protein synthesis
The findings suggest that insulin and amino acids activate mTORC1 by altering its interaction with regulatory proteins such as raptor, PRAS40, Rheb, and the Rag GTPases
Summary
The stimulation of global rates of hepatic protein synthesis in response to refeeding is likely a consequence of increases in plasma concentrations of both insulin and amino acids acting in a coordinate manner to activate mTORC1. Phosphorylation of TSC2 by Akt results in inhibition of its GTPase activator activity, leading to increased GTP loading on Rheb and increased mTORC1 activity. Recent studies have implicated the heterodimeric Rag GTPases in the amino acid-induced activation of mTORC1 [7, 8] Based on those studies, a model has been proposed [9] in which the Rag GTPases bind to mTORC1 in an amino acid-dependent manner and, via interaction with a complex termed Ragulator, promote its translocation to lysosomal membranes, where. The findings suggest that insulin and amino acids activate mTORC1 by altering its interaction with regulatory proteins such as raptor, PRAS40, Rheb, and the Rag GTPases
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