Human duodenal proteome modulations by glutamine and antioxidants.

PURPOSE: Glutamine (Gln) has protective, anti-inflammatory effects in animal models and humans. Antioxidant nutrients may exert synergistic effects on intestinal functions. Therefore, these combined nutrients may have a therapeutic potential during intestinal inflammation. This study was designed to investigate in humans the effects of a supplement composed of Gln and high-dosed antioxidant micronutrients compared to isomolar Gln only, on duodenal proteome. EXPERIMENTAL DESIGN: Enteral perfusion of Gln (0.8 mmol x kg(-1) x h(-1)) or supplement was performed in two groups of six healthy volunteers during 5 h before taking endoscopic duodenal biopsies. Protein expression was analyzed by 2-DE and the relevant proteins identified by MS/MS. RESULTS: About 1500 protein spots were revealed in both supplement and Gln conditions. Comparative proteomics analysis indicated that 11 proteins were differentially and significantly (p≤0.05) expressed in response to the supplement. These proteins were essentially implicated in metabolism pathways, e.g. fatty acid binding protein-1 and 40S ribosomal protein SA expressions were downregulated while manganese superoxide dismutase and retinal dehydrogenase-1 expressions were upregulated. CONCLUSIONS AND CLINICAL RELEVANCE: 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.

Glutamine regulates the human epithelial intestinal HCT-8 cell proteome under apoptotic conditions.

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-alpha-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

Proteomic analysis of glutamine-treated human intestinal epithelial HCT-8 cells under basal and inflammatory conditions.

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.

Orderly pattern of development of the autoantibody response in (New Zealand White x BXSB)F1 lupus mice: characterization of target antigens and antigen spreading by two-dimensional gel electrophoresis and mass spectrometry.

Immunoblots of a two-dimensional PAGE-separated HL-60 cell proteomic map and mass spectrometry were combined to characterize proteins targeted by autoantibodies produced by male (New Zealand White x BXSB)F(1) (WB) mice that develop lupus and anti-phospholipid syndrome. Analysis of sera sequentially obtained from seven individual mice at different ages showed that six proteins, vimentin, heat shock protein 60, UV excision-repair protein RAD23, alpha-enolase, heterogeneous nuclear ribonucleoprotein L, and nucleophosmin, were the targets of the B cell autoimmune response, and that autoantibodies to them were synthesized sequentially in an orderly pattern that recurred in all the male WB mice analyzed: anti-vimentin first and anti-nucleophosmin last, with anti-RAD23 and anti-heat shock protein 60, then anti-alpha-enolase and anti-heterogeneous nuclear ribonucleoprotein L Abs occuring concomitantly. Anti-vimentin reactivity always appeared before anti-cardiolipin and anti-DNA Abs, suggesting that vimentin is the immunogen initiating the autoimmune process. The pattern of HL-60 proteins recognized by female WB sera differed from that of male sera, indicating that the Y chromosome-linked autoimmune acceleration gene is not an accelerator but a strong modifier of the autoimmune response. Thus, 1) combining two-dimensional PAGE and mass spectrometry constitutes a powerful tool to identify the set of Ags bound by autoantibodies present in a single serum and the whole autoantibody pattern of an autoimmune disease; 2) the diversification of the autoimmune response in male WB mice occurs in a predetermined pattern consistent with Ag spreading, and thus provides a useful model to further our understanding of the development of the autoantibody response in lupus.