We previously demonstrated that the expression of the argininosuccinate synthetase (ASS) gene, a key step in nitric oxide production, is stimulated either by interleukin‐1β[Brasse‐Lagnel et al. (2005) Biochimie 87, 403–9] or by glutamine in Caco‐2 cells [Brasse‐Lagnel et al. (2003) J. Biol. Chem. 278, 52504–10], through the activation of transcription factors nuclear factor‐κB and Sp1, respectively. In these cells, the fact that glutamine stimulated the expression of a gene induced by pro‐inflammatory factors appeared paradoxical as the amino acid is known to exert anti‐inflammatory properties in intestinal cells. We therefore investigated the effect of simultaneous addition of both glutamine and interleukin‐1β on ASS gene expression in Caco‐2 cells. In the presence of both compounds for 4 h, the increases in ASS activity, protein amount and mRNA level were almost totally inhibited, implying a reciprocal inhibition between the amino acid and the cytokine. The inhibition was exerted at the level of the transcription factors Sp1 and nuclear‐κB: (a) interleukin‐1β inhibited the glutamine‐stimulated DNA‐binding of Sp1, which might be related to a decrease of its glutamine‐induced O‐glycosylation, and (b) glutamine induced per se a decrease in the amount of nuclear p65 protein without affecting the stimulating effect of interleukin‐1β on nuclear factor‐κB, which might be related to the metabolism of glutamine into glutamate. The present results constitute the first demonstration of a reciprocal inhibition between the effects of an amino acid and a cytokine on gene expression, and provide a molecular basis for the protective role of glutamine against inflammation in the intestine.
Glutamine plays a key role in the metabolism of rapidly dividing cells, including enterocytes and lymphocytes, which may contribute to its beneficial clinical effects. Gut mucosal homeostasis is achieved through a balance between cell proliferation and apoptosis. In T cells, glutamine up-regulates antiapoptotic proteins and down-regulates proapoptotic proteins. In gut mucosa, glutamine prevents apoptosis in rat epithelial cell lines, whereas glutamine starvation induces apoptosis through caspase activation. Finally glutamine specifically prevents tumor necrosis factor-␣-related apoptosis in the human intestinal cell line HT-29. Comparative functional proteomics enables the characterization of each differentially expressed protein in intestinal cells in response to modifications of nutritional environment. The influence of glutamine on intestinal proteome expression in apoptotic conditions has not been studied and evaluated. This comparative proteomics study was performed in the human epithelial intestinal cell line HCT-8 under experimental apoptotic conditions to investigate the influence of glutamine on protein expression during apoptosis. The pharmaconutritional effects of glutamine were determined under 2 mM (physiological concentration) and 10 mM (pharmaconutritional concentration) conditions. About 1,800 protein spots were revealed in both conditions. Comparative assessments indicated that 28 proteins were differentially expressed significantly (i.e. at least 2-fold modulated and Student's t test with p < 0.05) in response to an increase of glutamine concentration in the culture medium. Twenty-four proteins were identified by mass spectrometry and associated databases. From these proteins, 34% are involved in cell cycle and apoptosis mechanisms, 17% are involved in signal transduction, and 13% are involved in cytoskeleton organization. These data were integrated in a proposed schema of the interactome under apoptotic conditions. In conclusion, this study provides the first holistic picture of proteome mod-
Glutamine (Gln) promotes intestinal growth and maintains gut structure and function, especially in situations of injury and during inflammation. Several mechanisms could contribute to Gln protective effects on gut. Proteomics enable us to characterize differentially expressed proteins in tissues in response to modifications of the biological or nutritional environment. Gln effects on the human intestinal epithelial HCT-8 cell line proteome were assessed under basal and proinflammatory conditions. The 2-DE gels were obtained and compared. Proteins were identified by MS and using databases. About 1200 spots were detected in both 2- and 10-mM Gln concentrations. Under basal conditions, 24 proteins were differentially expressed in response to Gln. Half of these proteins were implicated in protein biosynthesis or proteolysis and 20% in membrane trafficking. Under proinflammatory conditions, 27 proteins were up- or down-regulated by Gln 10 mM. From these proteins, 40% were involved in protein biosynthesis or proteolysis, 16% in membrane trafficking, 8% in cell cycle and apoptosis mechanisms and 8% in nucleic acid metabolism. This study provides the first holistic picture of proteome modulation by Gln in a human enterocytic cell line under basal and proinflammatory conditions, and supports further evaluation of nutritional modulation of intestinal proteome in humans.
This study provides new information on human duodenal proteome and its nutritional modulation, and supports further clinical investigations designed to evaluate the effects of Gln plus antioxidants during intestinal inflammation and cancer.
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