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Full: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0050407
Glutamine Treatment Attenuates Endoplasmic Reticulum Stress and Apoptosis in TNBS-Induced Colitis
Irene Crespo,Beatriz San-Miguel,Carolina Prause,Norma Marroni,María J. Cevas,Javier González-Gallego,María J. Tuñón
Porto Alegre Clinical Hospital, Federal University of Rio Grande do Sul, Brazil
December 31, 2012
Introduction
Conventional IBD therapy typically involves pharmacological agents such as aminosalicylates, corticosteroids and immunosuppressive drugs. However, these treatments have demonstrated variable efficacy, adverse side effects and potential long-term toxicity [16]. Therefore, the need for alternative therapeutic strategies is of utmost importance. Amino acids are key regulators of metabolic pathways, and evidence has indicated additional roles for amino acids in maintaining gut health [17]. Glutamine, the most abundant amino acid in the bloodstream, plays a central role in nitrogen transport within the body, is a fuel for rapidly-dividing cells, and has many other essential metabolic functions. Lower levels of glutamine have been associated with immune dysfunction and increased mortality [18], and it has been reported that glutamine therapy improves outcome of in vitro and in vivo experimental colitis models [19]. A mechanism by which glutamine seems to exert its beneficial effects appear to be correlated with the decrease of oxidative stress [19], [20]. In addition, different studies have shown that glutamine supplementation delays human neutrophil apoptosis and reduced T-cell apoptosis [21]. Glutamine deprivation also induces apoptosis in rat intestinal epithelial cells [22] and renders premonocytic and HL-60 cells significantly more susceptible to Fas-mediated apoptosis [23].
In previous research we have demonstrated that treatment with glutamine markedly decreases the severity of macroscopic damage and the histopathological scores in several experimental animal models of colitis [1], [2]. Reduced myeloperoxidase activity and expression of inducible nitric oxide synthase, cyclooxygenase-2 and adhesion molecules confirmed the anti-inflammatory effect of glutamine. These protective effects are associated with changes in nuclear factor kappa B and signal transducers and activators of transcription (STAT) signaling pathways [1], [2]. Moreover, glutamine treatment not only attenuates the outcome of colitis by impairing the inflammatory response, but also by reducing the risk of fibrosis and stricture formation through down-regulation of several gene pathways that contribute to the accumulation of matrix proteins [24]. The purpose of our study was to investigate, using both in vitro and in vivo models, whether inhibition of ER stress and apoptosis contributes to the beneficial effects of glutamine. The present research provides evidence that reduction of colon damage by glutamine is associated with direct attenuation of ER stress through a modulation of the three arms of UPR signaling, and with a diminution of apoptotic cell death.
Abstract
Endoplasmic reticulum (ER) stress and apoptotic cell death play an important role in the pathogenesis and perpetuation of inflammatory bowel disease (IBD). We aimed to explore the potential of glutamine to reduce ER stress and apoptosis in a rat model of experimental IBD. Colitis was induced in male Wistar rats by intracolonic administration of 30 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Glutamine (25 mg/dL) was given by rectal route daily for 2 d or 7 d. Both oxidative stress (TBARS concentration and oxidised/reduced glutathione ratio) and ER stress markers (CHOP, BiP, calpain-1 and caspase-12 expression) increased significantly within 48 h of TNBS instillation, and glutamine attenuated the extent of the changes. Glutamine also inhibited the significant increases of ATF6, ATF4 and spliced XBP-1 mRNA levels induced by TNBS instillation. TNBS-colitis resulted in a significant increase in p53 and cytochrome c expression, and a reduced Bcl-xL expression and Bax/Bcl-2 ratio. These effects were significantly inhibited by glutamine. Treatment with the amino acid also resulted in significant decreases of caspase-9, caspase-8 and caspase-3 activities. Double immunofluorescence staining showed co-localization of CHOP and cleaved caspase-3 in colon sections. Phospho-JNK and PARP-1 expression was also significantly higher in TNBS-treated rats, and treatment with glutamine significantly decreased JNK phosphorylation and PARP-1 proteolysis. To directly address the effect of glutamine on ER stress and apoptosis in epithelial cells, the ER stress inducers brefeldin A and tunicamycin were added to Caco-2 cells that were treated with glutamine (5 mM and 10 mM). The significant enhancement in PERK, ATF6 phosphorylated IRE1, BiP and cleaved caspase-3 expression induced by brefeldin A and tunicamycin was partly prevented by glutamine. Data obtained indicated that modulation of ER stress signalling and anti-apoptotic effects contribute to protection by glutamine against damage in TNBS-induced colitis.
Figure 1. Glutamine reduces the ER stress induced by TNBS-colitis.
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(A–E). Protein from colonic extracts was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting for CHOP, BiP, calpain-1 and caspase-12. CHOP, BiP, calpain-1 and caspase-12 were markedly expressed in rats treated with TNBS alone. However, glutamine administration partially abolished CHOP, BiP, calpain-1 and caspase-12 expression induced by TNBS. Results are representative of four independent experiments. Equal loading of proteins is illustrated by β-actin bands. (A) Representative Western-blot photographs for CHOP, calpain-1, BiP, caspase-12, and β-actin. (B) Densitometric quantification of CHOP. (C) Densitometric quantification of calpain-1. (D) Densitometric quantification of BiP. (E) Densitometric quantification of caspase-12. Data are expressed as mean ± S.E.M. from 8 rats. *P<0.05 compared with control group. #P<0.05 compared with TNBS group. &P<0.05 compared with same group 2 d. (F) Photomicrographs of immunohistochemistry for BiP in sections of colonic samples. Paraffin-embedded sections were immunostained with a BiP antibody. Original magnification: 200X.
Competing Interests
The authors have declared that no competing interests exist.
Glutamine Treatment Attenuates Endoplasmic Reticulum Stress and Apoptosis in TNBS-Induced Colitis
Irene Crespo,Beatriz San-Miguel,Carolina Prause,Norma Marroni,María J. Cevas,Javier González-Gallego,María J. Tuñón
Porto Alegre Clinical Hospital, Federal University of Rio Grande do Sul, Brazil
December 31, 2012
Introduction
Conventional IBD therapy typically involves pharmacological agents such as aminosalicylates, corticosteroids and immunosuppressive drugs. However, these treatments have demonstrated variable efficacy, adverse side effects and potential long-term toxicity [16]. Therefore, the need for alternative therapeutic strategies is of utmost importance. Amino acids are key regulators of metabolic pathways, and evidence has indicated additional roles for amino acids in maintaining gut health [17]. Glutamine, the most abundant amino acid in the bloodstream, plays a central role in nitrogen transport within the body, is a fuel for rapidly-dividing cells, and has many other essential metabolic functions. Lower levels of glutamine have been associated with immune dysfunction and increased mortality [18], and it has been reported that glutamine therapy improves outcome of in vitro and in vivo experimental colitis models [19]. A mechanism by which glutamine seems to exert its beneficial effects appear to be correlated with the decrease of oxidative stress [19], [20]. In addition, different studies have shown that glutamine supplementation delays human neutrophil apoptosis and reduced T-cell apoptosis [21]. Glutamine deprivation also induces apoptosis in rat intestinal epithelial cells [22] and renders premonocytic and HL-60 cells significantly more susceptible to Fas-mediated apoptosis [23].
In previous research we have demonstrated that treatment with glutamine markedly decreases the severity of macroscopic damage and the histopathological scores in several experimental animal models of colitis [1], [2]. Reduced myeloperoxidase activity and expression of inducible nitric oxide synthase, cyclooxygenase-2 and adhesion molecules confirmed the anti-inflammatory effect of glutamine. These protective effects are associated with changes in nuclear factor kappa B and signal transducers and activators of transcription (STAT) signaling pathways [1], [2]. Moreover, glutamine treatment not only attenuates the outcome of colitis by impairing the inflammatory response, but also by reducing the risk of fibrosis and stricture formation through down-regulation of several gene pathways that contribute to the accumulation of matrix proteins [24]. The purpose of our study was to investigate, using both in vitro and in vivo models, whether inhibition of ER stress and apoptosis contributes to the beneficial effects of glutamine. The present research provides evidence that reduction of colon damage by glutamine is associated with direct attenuation of ER stress through a modulation of the three arms of UPR signaling, and with a diminution of apoptotic cell death.
Abstract
Endoplasmic reticulum (ER) stress and apoptotic cell death play an important role in the pathogenesis and perpetuation of inflammatory bowel disease (IBD). We aimed to explore the potential of glutamine to reduce ER stress and apoptosis in a rat model of experimental IBD. Colitis was induced in male Wistar rats by intracolonic administration of 30 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Glutamine (25 mg/dL) was given by rectal route daily for 2 d or 7 d. Both oxidative stress (TBARS concentration and oxidised/reduced glutathione ratio) and ER stress markers (CHOP, BiP, calpain-1 and caspase-12 expression) increased significantly within 48 h of TNBS instillation, and glutamine attenuated the extent of the changes. Glutamine also inhibited the significant increases of ATF6, ATF4 and spliced XBP-1 mRNA levels induced by TNBS instillation. TNBS-colitis resulted in a significant increase in p53 and cytochrome c expression, and a reduced Bcl-xL expression and Bax/Bcl-2 ratio. These effects were significantly inhibited by glutamine. Treatment with the amino acid also resulted in significant decreases of caspase-9, caspase-8 and caspase-3 activities. Double immunofluorescence staining showed co-localization of CHOP and cleaved caspase-3 in colon sections. Phospho-JNK and PARP-1 expression was also significantly higher in TNBS-treated rats, and treatment with glutamine significantly decreased JNK phosphorylation and PARP-1 proteolysis. To directly address the effect of glutamine on ER stress and apoptosis in epithelial cells, the ER stress inducers brefeldin A and tunicamycin were added to Caco-2 cells that were treated with glutamine (5 mM and 10 mM). The significant enhancement in PERK, ATF6 phosphorylated IRE1, BiP and cleaved caspase-3 expression induced by brefeldin A and tunicamycin was partly prevented by glutamine. Data obtained indicated that modulation of ER stress signalling and anti-apoptotic effects contribute to protection by glutamine against damage in TNBS-induced colitis.
Figure 1. Glutamine reduces the ER stress induced by TNBS-colitis.
show more
(A–E). Protein from colonic extracts was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting for CHOP, BiP, calpain-1 and caspase-12. CHOP, BiP, calpain-1 and caspase-12 were markedly expressed in rats treated with TNBS alone. However, glutamine administration partially abolished CHOP, BiP, calpain-1 and caspase-12 expression induced by TNBS. Results are representative of four independent experiments. Equal loading of proteins is illustrated by β-actin bands. (A) Representative Western-blot photographs for CHOP, calpain-1, BiP, caspase-12, and β-actin. (B) Densitometric quantification of CHOP. (C) Densitometric quantification of calpain-1. (D) Densitometric quantification of BiP. (E) Densitometric quantification of caspase-12. Data are expressed as mean ± S.E.M. from 8 rats. *P<0.05 compared with control group. #P<0.05 compared with TNBS group. &P<0.05 compared with same group 2 d. (F) Photomicrographs of immunohistochemistry for BiP in sections of colonic samples. Paraffin-embedded sections were immunostained with a BiP antibody. Original magnification: 200X.
Competing Interests
The authors have declared that no competing interests exist.
