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 相似文献   

Inhibition of MCL‐1 by obatoclax sensitizes Sp2/0‐Ag14 hybridoma cells to glutamine deprivation‐induced apoptosis

For several cancer cell types, the lack of an adequate supply of the amino acidl ‐glutamine (Gln) triggers apoptosis, a phenomenon termed Gln addiction. In this report, we examined the role of the anti‐apoptotic proteins of the B‐cell lymphoma 2 (BCL‐2) protein family in the survival of Sp2/0‐Ag14 (Sp2/0) mouse hybridoma cells, a cell line that undergoes apoptosis within minutes of Gln deprivation. Western blot analysis revealed that myeloid cell leukaemia 1 (MCL‐1) was expressed at much higher levels than BCL‐2, B‐cell lymphoma extra‐large and BCL‐2‐like protein 2 making it the prominent pro‐survival BCL‐2 family member in this hybridoma. Gln deprivation triggered a progressive decrease in MCL‐1 protein levels, which coincided with the decrease in Sp2/0 cell survival. Moreover, Sp2/0 cells were much more sensitive to the broad Bcl‐2 homology domain‐3 (BH3) mimetic obatoclax (which targets MCL‐1) than to the more selective drug ABT‐737 (which does not target MCL‐1). Finally, we show that obatoclax sensitizes Sp2/0 cells to apoptosis following Gln starvation. All together, the data presented here reveal that modulation of the pro‐survival protein MCL‐1 is an important step in the sequence of events leading to the initiation of apoptosis in Gln‐starved Sp2/0 cells. Cancer cells require an adequate supply ofl ‐glutamine for their survival. Using a mouse hybridoma cell line that is exquisitely sensitive to glutamine starvation, we show that the levels of the pro‐survival BCL‐2 family protein MCL‐1 decrease upon glutamine starvation in a manner that correlates with the loss of cell viability. Moreover, inhibiting MCL‐1 with the drug obatoclax sensitizes hybridoma cells to glutamine starvation. Thus, in some cancer cells, glutamine starvation triggers the inactivation of pro‐survival proteins. Our data suggest that the combined inhibition of glutamine biosynthesis pathways and BCL‐2 proteins may prove effective against some cancers. Copyright © 2015 John Wiley & Sons, Ltd.     

Molecular mechanisms contributing to glutamine-mediated intestinal cell survival

Glutamine, the most abundant amino acid in the bloodstream, is the preferred fuel source for enterocytes and plays a vital role in the maintenance of mucosal growth. The molecular mechanisms regulating the effects of glutamine on intestinal cell growth and survival are poorly understood. Here, we show that addition of glutamine (1 mmol/l) enhanced rat intestinal epithelial (RIE)-1 cell growth; conversely, glutamine deprivation increased apoptosis as noted by increased DNA fragmentation and caspase-3 activity. To delineate signaling pathways involved in the effects of glutamine on intestinal cells, we assessed activation of extracellular signal-related kinase (ERK), protein kinase D (PKD), and phosphatidylinositol 3-kinase (PI3K)/Akt, which are important pathways in cell growth and survival. Addition of glutamine activated ERK and PKD in RIE-1 cells after a period of glutamine starvation; inhibition of ERK, but not PKD, increased cell apoptosis. Conversely, glutamine starvation alone increased phosphorylated Akt; inhibition of Akt enhanced RIE-1 cell DNA fragmentation. The role of ERK was further delineated using RIE-1 cells stably transfected with an inducible Ras. Apoptosis was significantly increased following ERK inhibition, despite Ras activation. Taken together, these results identify a critical role for the ERK signaling pathways in glutamine-mediated intestinal homeostasis. Furthermore, activation of PI3K/Akt during periods of glutamine deprivation likely occurs as a protective mechanism to limit apoptosis associated with cellular stress. Importantly, our findings provide novel mechanistic insights into the antiapoptotic effects of glutamine in the intestine.     

Kinetics of the sodium-dependent glutamine transporter in human intestinal cell confluent monolayers.

The intestinal epithelium metabolism of glutamine plays a critical role in inter-organ nitrogen flow. Although it is known that glutamine is the primary oxidative energy source and nucleotide precursor in intestinal cells, the luminal uptake of glutamine by the apical surface of enterocytes is poorly understood. In this study we have uncovered the sodium-dependent transporter system responsible for L-glutamine uptake by the apical membrane of a human intestinal epithelial cell line. The sodium-dependent Michaelis constant (Km) = 247 +/- 45 microM glutamine, and Jmax = 4.44 +/- 0.65 x 10(-9) mole min-1(mg protein)-1 (37 degrees C). Glutamine shares the transporter with alanine, as demonstrated by unlabeled glutamine inhibition of [3H]alanine uptake kinetics with a purely competitive-type inhibition pattern, and glutamine inhibition Ki = 205 +/- 18 microM by Dixon analysis. The inhibition pattern for a series of amino acid analogs indicated that this intestinal apical membrane sodium-dependent transporter for glutamine is distinct from any other transport system found in membranes of non-intestinal cells.     

Increased apoptosis and accelerated epithelial migration following inhibition of hedgehog signaling in adaptive small bowel postresection

The intestinal epithelium undergoes a marked adaptive response following loss of functional small bowel surface area characterized by increased crypt cell proliferation and increased enterocyte migration from crypt to villus tip, resulting in villus hyperplasia and enhanced nutrient absorption. Hedgehog (Hh) signaling plays a critical role in regulating epithelial-mesenchymal interactions during morphogenesis of the embryonic intestine. Our previous studies showed that blocking Hh signaling in neonatal mice results in increased small intestinal epithelial crypt cell proliferation and altered enterocyte fat absorption and morphology. Hh family members are also expressed in the adult intestine, but their role in the mature small bowel is unclear. With the use of a model of intestinal adaptation following partial small bowel resection, the role of Hh signaling in the adult gut was examined by determining the effects of blocking Hh signaling on the regenerative response following loss of functional surface area. Hh-inactivating monoclonal antibodies or control antibodies were administered to mice that sustained a 50% intestinal resection. mRNA analyses of the preoperative ileum by quantitative real-time PCR revealed that Indian hedgehog was the most abundant Hh family member. The Hh receptor Patched was more abundant than Patched 2. Analyses of downstream targets of Hh signaling demonstrated that Gli3 was twofold more abundant than Gli1 and Gli2 and that bone morphogenetic protein (BMP)2 was most highly expressed compared with BMP1, -4, and -7. Following intestinal resection, the expression of Hh, Patched, Gli, and most BMP genes was markedly downregulated in the remnant ileum, and, in anti-Hh antibody-treated mice, expression of Patched 2 and Gli 1 was further suppressed. In Hh antibody-treated mice following resection, the enterocyte migration rate from crypt to villus tip was increased, and by 2 wk postoperation, apoptosis was increased in the adaptive gut. However, crypt cell proliferation, villus height, and crypt depth were not augmented. These data indicate that Hh signaling plays a role in adult gut epithelial homeostasis by regulating epithelial cell migration from crypt to villus tip and by enhancing apoptosis.     

Regulation of intestinal protein metabolism by amino acids

Gut homeostasis plays a major role in health and may be regulated by quantitative and qualitative food intake. In the intestinal mucosa, an intense renewal of proteins occurs, at approximately 50 % per day in humans. In some pathophysiological conditions, protein turnover is altered and may contribute to intestinal or systemic diseases. Amino acids are key effectors of gut protein turnover, both as constituents of proteins and as regulatory molecules limiting intestinal injury and maintaining intestinal functions. Many studies have focused on two amino acids: glutamine, known as the preferential substrate of rapidly dividing cells, and arginine, another conditionally essential amino acid. The effects of glutamine and arginine on protein synthesis appear to be model and condition dependent, as are the involved signaling pathways. The regulation of gut protein degradation by amino acids has been minimally documented until now. This review will examine recent data, helping to better understand how amino acids regulate intestinal protein metabolism, and will explore perspectives for future studies.     

Milk fat globule-epidermal growth factor 8 is decreased in intestinal epithelium of ulcerative colitis patients and thereby causes increased apoptosis and impaired wound healing

Milk fat globule-epidermal growth factor 8 (MFG-E8) plays an important role in maintaining intestinal barrier homeostasis and accelerating intestinal restitution. However, studies of MFG-E8 expression in humans with ulcerative colitis are lacking. We examined MFG-E8 expression in colonic mucosal biopsies from ulcerative colitis patients and healthy controls (n = 26 each) by real-time quantitative polymerase chain reaction (PCR), Western blot analysis and immunohistochemistry. MFG-E8 mRNA and protein expression was lower in ulcerative colitis patients than in controls. MFG-E8 expression was inversely correlated with mucosal inflammatory activity and clinical disease activity in patients. MFG-E8 was present in human intestinal epithelial cells both in vivo and in vitro. Apoptosis induction was also detected in the intestinal epithelium of ulcerative colitis patients by terminal-deoxynucleoitidyl transferase mediated nick-end labeling assay. We used lentiviral vectors encoding human MFG-E8 targeting short hairpin RNA to obtain MFG-E8 knockdown intestinal epithelia cell clones. MFG-E8 knockdown could promote apoptosis in intestinal epithelial cell lines, accompanied by a decrease in level of the antiapoptotic protein B-cell lymphoma 2 (BCL-2) and induction of the proapoptotic protein BCL2-associated protein X (BAX). The addition of recombinant human MFG-E8 led to decreased BAX and cleaved caspase-3 levels and induction of BCL-2 level in intestinal epithelia cells. MFG-E8 knockdown also attenuated wound healing on scratch assay of intestinal epithelial cells. The mRNA level of intestinal trefoid factor 3, a pivotal factor in intestinal epithelial cell migration and restitution, was downregulated with MFG-E8 knockdown. In conclusion, we demonstrated that decreased colonic MFG-E8 expression in patients with ulcerative colitis may be associated with mucosal inflammatory activity and clinical disease activity through basal cell apoptosis and preventing tissue healing in the pathogenesis of ulcerative colitis.     

Glutamine regulates the expression of proteins with a potential health-promoting effect in human intestinal Caco-2 cells

Glutamine is an essential amino acid for the enterocytes with respect to maintaining the gut mucosal integrity and function. This study was conducted to explore a molecular basis for the beneficial effects of glutamine on intestinal cells by searching for glutamine-dependent changes in the proteome. Caco-2 cells were exposed to different concentrations of L-glutamine with or without L-methionine sulfoximine, an inhibitor of the glutamine synthetase activity. 2-DE combined with MALDI-TOF-MS was used to identify proteins whose expression is changed by glutamine. To assess the relative protein synthesis rate, incorporation of L-[2H5]glutamine into individual proteins was monitored. The expression levels of 14 proteins changed significantly with the glutamine availability. Examples of differentially expressed proteins with potential health-promoting effects on the intestine are plasma retinol-binding protein, ornithine aminotransferase, apolipoprotein A-I, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase, and acyl-CoA synthetase 5. Expression of these proteins was not changed by arginine deprivation. The differential change in the expression levels of the proteins was not correlated with their rate of synthesis, excluding an effect of glutamine depletion on general protein synthesis. Together, this study shows a gene-specific effect of glutamine on intestinal cells.