Effects of essential amino acids or glutamine deprivation on intestinal permeability and protein synthesis in HCT-8 cells: involvement of GCN2 and mTOR pathways

Abstract

GCN2 and mTOR pathways are involved in the regulation of protein metabolism in response to amino acid availability in different tissues. However, regulation at intestinal level is poorly documented. The aim of the study was to evaluate the effects of a deprivation of essential amino acids (EAA) or glutamine (Gln) on these pathways in intestinal epithelial cells. Intestinal epithelial cell, HCT-8, were incubated during 6 h with 1/DMEM culture medium containing EAA, non EAA and Gln, 2/with saline as positive control of nutritional deprivation, 3/DMEM without EAA, 4/DMEM without Gln or 5/DMEM without Gln and supplemented with a glutamine synthase inhibitor (MSO, 4 mM). Intestinal permeability was evaluated by the measure of transepithelial electric resistance (TEER). Using [L-(2)H(3)]-leucine incorporation, fractional synthesis rate (FSR) was calculated from the assessed enrichment in proteins and free amino acid pool by GCMS. Expression of eiF2α (phosphorylated or not), used as marker of GCN2 pathway, and of 4E-BP1 (phosphorylated or not), used as a marker of mTOR pathway, was evaluated by immunoblot. Results were compared by ANOVA. Six-hours EAA deprivation did not significantly affect TEER and FSR but decreased p-4E-BP1 and increased p-eiF2α. In contrast, Gln deprivation decreased FSR and p-4E-BP1. MSO induced a marked decrease of TEER and FSR and an increase of p-eiF2α, whereas mTOR pathway remained activated. These results suggest that both mTOR and GCN2 pathways can mediate the limiting effects of Gln deprivation on protein synthesis according to its severity.

Enteral delivery of proteins stimulates protein synthesis in human duodenal mucosa in the fed state through a mammalian target of rapamycin–independent pathway

“…To better understand the underlying mechanisms involved in the effects of protein delivery, we studied intracellular phosphokinases as previously described (19,34). We did not observe the activation of the mTOR pathway that usually mediates the effects of amino acids on protein translation in muscle (35) and intestinal cells (12,16,20,36). Neither mTOR nor downstream components such as the p70S6kinase or factor 4E binding protein 1 were affected by the protein supply.…”

“…Glutamine, which is the preferential substrate of rapidly dividing cells (ie, enterocytes and immune cells), is associated with less-infectious complications in critically ill patients (10) that could be partly due to the maintenance of gut barrier function (11). In vitro data showed that glutamine increased protein synthesis in intestinal epithelial cells (12)(13)(14)(15)(16). In animals, glutamine enhanced intestinal protein synthesis in hypercatabolic dogs (17) but not in malnourished rats (18).…”

“…One major pathway involved in the regulation of the initiation and elongation of protein synthesis by amino acids is the mammalian target of rapamycin (mTOR) 4 pathway. In intestinal epithelial cells, recent in vitro studies showed that glutamine enhanced protein synthesis through an activation of the mTOR pathway (12,16), but data in the human gut are lacking. We reported that enteral leucine infusion failed to activate the mTOR pathway in human duodenal mucosa (19), whereas leucine activated mTOR pathway in intestinal epithelial cells in vitro (20)(21)(22) or in animals (23,24).…”

Glutamine metabolism in advanced age

“…Cytosolic glutamine synthetase, which is a longevityrelated gene, is responsible for this synthesis Small intestine and kidneys are the major sites of glutamine utilization under normal or acidotic conditions Intracellular glutamine concentration generally decreases in catabolic states. In humans, this change can be prevented or attenuated by glutamine supplementation (enteral or parenteral) The liver can synthesize or degrade glutamine, depending on the nutritional conditions (2)Regulatory roles of glutamine in cell-specific processes Boukhettala et al (2012), 25 Brasse-Lagnel et al (2009), 26 Yi et al (2015) 27 , BrasseLagnel (2010), 28 Curi et al (2005) 29 (2007), 30 Deniel et al (2007), 31 Larson 34 Matés et al (2006), 37 Nakajo et al (2005), 38 Naomoto et al (2005), 39 Nishikawa et al (2007), 40 Papaconstantinou (2000), 41 Rhoads & Wu (2009), 42 Roth et al (2002), 45 Roth (2007), 43 (2008), 44 Rutten et al Abbreviations: ATP, adenosine triphosphate; EAAs, essential amino acids; NEAAs, nonessential amino acids; NF-jB, nuclear factor jB.…”

Mechanism of enhanced oral absorption of hydrophilic drug incorporated in hydrophobic nanoparticles

“…12,13 The dialysis bag allowed the transfer of the released drug molecules into the release media while intercepting the nanoparticles. The dialysis bag (molecular weight range: 8,000 to 14,000; Sigma-Aldrich) was boiled for more than 30 minutes and soaked in the release media overnight prior to the experiment; the release media was double-distilled water.…”