Does enteral protein administration stimulate duodenal mucosa protein synthesis through an mTORC1-independent signaling pathway?

See corresponding article on page 286.

More than 40 y ago, Morgan et al (1, 2) reported that in the isolated perfused rat heart, elevating amino acid concentrations in the perfusate to 5 times the concentrations observed in the plasma of a fasted animal stimulated protein synthesis rates by enhancing the initiation phase of mRNA translation. Since that time, a plethora of studies have focused on delineating the mechanism or mechanisms through which amino acids act to upregulate translation initiation. The results of those studies have identified 2 steps in initiation as targets of amino acid signaling: the binding of initiator methionyl-tRNA (met-tRNA_i)⁴ to the 40S ribosomal subunit to form the 43S preinitiation complex and the binding of mRNA to the 43S preinitiation complex. The binding of met-tRNA_i to the 40S ribosomal subunit is mediated by eukaryotic initiation factor (eIF) 2 and regulated by the guanine nucleotide exchange factor (GEF) eIF2B. Both proteins are phosphorylated in cells in culture on specific subunits (eIF2 on its α-subunit and eIF2B on its ϵ-subunit) in response to changes in amino acid concentrations. Specifically, the deprivation of essential amino acids leads to activation of the protein kinase general control non-derepressing 2, which phosphorylates eIF2α, converting it from a substrate into a competitive inhibitor of eIF2B (3). Amino acid deprivation also leads to increased phosphorylation of eIF2B on its catalytic ϵ-subunit, resulting in inhibition of its GEF activity toward eIF2 (4). Together, phosphorylation of eIF2α and eIF2Bϵ leads to repressed rates of translation initiation by perturbing the assembly of the eIF2·GTP·met-tRNA_i complex that mediates transfer of met-tRNA_i to the 40S ribosomal subunit.

The deprivation of essential amino acids also results in development of an impairment in the binding of mRNA to the 43S preinitiation complex, an event mediated by the eIF4F complex composed of eIF4A, eIF4E, and eIF4G (5). For most mRNAs, binding to the 43S preinitiation complex occurs through the interaction of eIF4E with the m⁷GTP cap at the 5′-end of the message and the subsequent binding of eIF4G to eIF3 that is part of the 43S preinitiation complex. The deprivation of essential amino acids leads to the disassembly of the eIF4F complex by promoting the interaction of eIF4E with eIF4E binding proteins (eg, 4E-BP1) and the interaction of eIF4A with programmed cell death 4 (PDCD4), thereby preventing their interaction with eIF4G. The association of eIF4E with 4E-BP1 and eIF4A with PDCD4 is regulated by phosphorylation of 4E-BP1 and PDCD4 in response to the activation of a protein kinase complex known as mechanistic target of rapamycin complex 1 (mTORC1). Amino acids, and in particular the branched-chain amino acid leucine, activate mTORC1, which leads to phosphorylation of 4E-BP1 and PDCD4, releasing their companion proteins and allowing them to bind to eIF4G to form the active eIF4F complex. Thus, in response to amino acid deprivation, mTORC1 is inhibited and eIF4E and eIF4A are sequestered away from eIF4G, resulting in impaired binding of mRNA to the 43S preinitiation complex.

Studies that investigated the effects of amino acids on protein synthesis in vivo have focused primarily on skeletal muscle. In skeletal muscle, oral administration of either leucine alone or a protein-containing meal leads to a stimulation of the rate of protein synthesis concomitant with activation of mTORC1 and increased assembly of the eIF4F complex (eg, references 6 and 7). Treatment with the mTORC1 inhibitor rapamycin blocks both the amino acid–induced stimulation of protein synthesis and activation of mTORC1 signaling in animals and humans (6–8). Notably, neither phosphorylation of eIF2α nor changes in eIF2B GEF activity are typically observed in skeletal muscle in response to feeding or oral administration of leucine (9, 10), suggesting that activation of mTORC1 is the primary target responsible for the stimulation of protein synthesis by amino acids in vivo.

In a study reported in this issue of the Journal, Coëffier et al (11) present data showing that enteral delivery of a mixture of protein and maltodextrin stimulates protein synthesis in the duodenal mucosa compared with provision of maltodextrin alone. Surprisingly, they do not observe an elevation in phosphorylation of 2 direct targets of mTORC1, 4E-BP1 and p70S6K1, suggesting that, in contrast to skeletal muscle, mTORC1 was not activated by the protein mixture even though elevated plasma concentrations of the branched-chain amino acids persisted throughout the 5-h infusion period. The authors conclude that enteral delivery of the protein mixture stimulated duodenal mucosa protein synthesis in humans through an mTORC1-independent pathway. If this conclusion is correct, it would represent an important new mechanism in cell biology and one that would stimulate elucidation of potentially novel means by which amino acids modulate protein synthesis. It is therefore imperative that future studies not only confirm the results of the present one but also test possible alternative pathways that might be involved in the effect. For example, in the Coëffier et al study (11), measurements were taken at a single time point, 5 h after the start of enteral delivery of the protein mixture and intravenous infusion of [¹³C]leucine and [²H]phenylalanine. To exclude the possibility that the protein-induced increase in incorporation of [¹³C]leucine and [²H]phenylalanine into protein may have occurred transiently at a time when mTORC1 signaling was activated, and that both may have returned to basal values by the time biopsies were taken, a time course analysis would be necessary. Moreover, a study that uses rapamycin to inhibit mTOR signaling could be performed, as has been used by others to assess the role of mTORC1 in the amino acid– and exercise-induced stimulation of protein synthesis in humans (8, 12). It is important that Western blot analysis, rather than dot-blot analysis, be used to assess potential changes in p70S6K1 and mTOR phosphorylation, and changes in protein phosphorylation should be expressed relative to the respective target protein, rather than GAPDH, to account for potential changes in expression. Finally, possible mTORC1-independent pathways, such as those involving eIF2 and eIF2B, which were not assessed in the Coëffier et al study (11), should be evaluated in future studies. It is possible that such measurements were performed and not reported in the present study or are planned for a future report. If not, future studies should address such possibilities to increase confidence that an mTORC1-independent pathway is indeed involved in the observed changes in duodenal mucosa protein synthesis.

Overall, the data reported by Coëffier et al (11) are interpreted to support a model in which amino acids act to stimulate protein synthesis in the duodenal mucosa through a mechanism distinct from the established one involving activation of mTORC1. Future studies should aim to confirm the initial conclusion and to identify the mTORC1-independent pathway involved in regulating protein synthesis in the duodenal mucosa.