Zhi-Yong Peng, MD, PhD*; Christine R. Hamiel, BS*; Anirban Banerjee, PhD[dagger]; and Paul E. Wischmeyer, MD*
From the * Department of Anesthesiology, University of Colorado Health Science Center, Denver, Colorado; and the [dagger] Department of Surgery, University of Colorado Health Science Center, Denver, Colorado
ABSTRACT. Background: Glutamine (GLN) has been shown to improve outcome after experimental and clinical models of critical illness. Enhanced expression of heat shock protein (HSP) has been hypothesized to be responsible for this protection. The heat shock response has been shown to inhibit inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production. This study tested the hypothesis that GLN-mediated activation of the HSP pathway is responsible for improved survival and attenuation of iNOS expression after an inflammatory cytokine-induced injury. Methods: Heat shock factor-1 (HSF-1) wild-type and knockout mouse embryonic fibroblasts (HSF-1+/+ and HSF-1-/-) were used in all experiments. Cells were treated with 0 mmol/L or 8 mmol/L GLN and cytomix (tumor necrosis factor-α, lipopolysaccharide, and interferon-γ) in a concurrent treatment model once they had reached confluence. Cell viability was assayed with MTS/PMS mixture. Apoptosis and necrosis were assayed via immunohistochemistry. iNOS and HSP-70 expression were detected via Western blotting. NO production was measured using the Griess reagent. Results: GLN treatment significantly attenuated inflammatory cytokineinduced cell death and apoptosis in HSF-1+/+ cells vs 0 mmol/L GLN treatment; however, GLN's cellular protection was lost in HSF-1-/- cells. GLN supplementation attenuated cytomix-induced iNOS expression and NO production only in HSF-1+/+ cells. Further, GLN induced HSP-70 expression only in HSF-1+/+ cells. Conclusions: This is the first demonstration that GLN-mediated cellular protection after inflammatory cytokine injury is due to HSF-1 expression and cellular capacity to activate an HSP response. (Journal of Parenteral and Enteral Nutrition 30:400-407, 2006)
Critical illness and injury are often manifested by the occurrence of sepsis and inflammation. Sepsis is the leading cause of death in critical illness in the United States, where 500,000-750,000 patients develop sepsis annually and 230,000 patients die of this disease each year.1-4 Further, although age-adjusted death rates are decreasing, the death rate from sepsis continues to increase.5 The death rate from sepsis has increased >90% in the last 20 years, and the Centers for Disease Control and Prevention indicates that septic death rates have increased at a rate greater then any other common cause of mortality in the last year for which data were available.5 Sepsis and inflammation commonly lead to the occurrence of multiple organ dysfunction syndrome (MODS).3 MODS is often the ultimate cause of death in the intensive care unit. A key molecule that can lead to inflammatory injury is inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO).6-8 In response to systemic inflammation, iNOS is expressed in several diverse cell types, resulting in increased production of NO. NO is thought to have dual functionality (ie, it is beneficial in host defense in lower concentration ranges) but may also be pathogenic when overproduction of NO leads to the formation of peroxynitrite, which can initiate oxidant injury.9,10 Attenuation of NO production results in significant reduction of local tissue damage caused by ischemia and reperfusion.6,11 Mice deficient in iNOS also demonstrated decreased morbidity after hemorrhagic shock.7 Therefore, attenuating the overactivation of iNOS and overproduction of NO may be an important target and prove useful in the treatment of sepsis and critical illness in humans.11
Glutamine (GLN), traditionally considered a nonessential amino acid, now appears to be a conditionally essential nutrient during serious injury or illness.12-14 An update of a recent meta-analysis of all clinical trials using GLN as a sole agent indicate GLN treatment shows a strong trend toward reduction of infectious complication rates in postsurgical patients and a reduction in complication and mortality rates in critically ill patients.15 We and others have demonstrated that GLN is protective against multiple forms of cellular stress both in vivo and in vitro.16-23 We have hypothesized the mechanism of this protection is related to the enhanced expression of heat shock protein (HSP). Despite previous data indicating a correlative relationship between GLN-mediated cellular protection and HSP expression after injury, no conclusive studies using gene-knockout cells or animals have been conducted to clearly establish the relationship. The heat shock response is also known to inhibit iNOS gene expression and NO production.24-26 A pharmacologie intervention enhancing HSP70 expression could attenuate iNOS expression and be therapeutically useful for reducing inflammatory cytokine-induced injury.27,28
According to the aforementioned findings, we hypothesized that GLN treatment given at the onset of an inflammatory cytokine-induced injury (concurrent treatment) may improve survival via activation of the HSP pathway. We compare the effects of GLN in heat shock transcription factor-1 (HSF-1) wild-type and knockout cells. We believe GLN's effect on survival after inflammatory cytokine injury will be lost in cells with a specific gene-deletion of HSF-1.
MATERIALS AND METHODS
Cell Culture
All experiments were performed in mouse embryonic fibroblasts. The HSF-1 wild-type (HSF-1+/+) and HSF-1 null mutant (HSF-1-/-) mouse embryonic fibroblasts were obtained as a gift from Dr Hector Wong (Cincinnati, OH) and Dr Ivor Benjamin (Salt Lake City, UT). These cells were previously demonstrated to be a useful model for studying the role of HSP pathway activation in inflammation.29,30 Cells were grown and maintained in a room air/5% carbon dioxide incubator at 37
Experimental Conditions
All experiments are grouped as follows: HSF-1+/+ /control (no injury), HSF-1+/+/cytomix, HSF1+/+/cytomix/GLN, HSF-1-/-/control (no injury), HSF-1-/-/cytomix, and HSF-1-/-/cytomix/GLN. For cytomix groups, cells were treated with a combination of murine cytokines: 10 ng/mL of tumor necrosis factor-α (TNF-α; Roche, Indianapolis, IN), 200 ng/mL of lipopolysaccharide from E coli (LPS; Sigma) and 10 ng/mL of interferon-y (IF-γ; Sigma). For convenience, this cytokine combination is referred to as "cytomix." In the cytomix/GLN group, cytomix and 8 mmol/L GLN were added simultaneously to cells. In control groups, cytomix and media containing 0 mmol/L GLN were added concurrently. Before experimental injury, routine growth media (containing 2 mmol/L GLN and 10% fetal bovine serum [FBS]) was replaced by minimal media for 24 hours (only containing Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum and mercaptoethanol, no mercaptoethanol in nitrate and nitrite measurement experiments). Each experiment was repeated 4 times.
Cell Survival Studies
Cell viability was assayed with MTS/PMS mixture (Promega, Madison, WI). For cell viability preparations, 4000/mL cells were seeded in 96-well plates. After 24 hours, cells underwent experimental injury. Forty-eight hours after injury, cells were examined and 20 µL of the MTS/PMS mixture was pipetted into each well of the 96-well assay plates. Plates were incubated for 1-4 hours at 37°C in a humidified, 5% carbon dioxide atmosphere. The absorbance at 492 and 650 nm was performed using an ELISA plate reader (Thermo Electro Corporation, San Jose, CA) was recorded. The readings at 650 nm were subtracted from corresponding reading at 492 nm. The values for the same 3 wells were averaged.
Cell Apoptosis and Necrosis Immunohistochemistry
Vybrant Apoptosis Assay Kit #2 (Invitrogen/Molecular Probes) was used to visualize annexin V and propidium iodide positive cells. Cells were grown on 4-well chamber slides (Nunc). All chemicals were purchased from Sigma-Aldrich unless otherwise specified. Images were acquired with a Ziess Axiovert microscope fitted with a Cooke CCD SensiCam camera and Intelligent Imaging Innovations SlideBook software.
HSF-1+/+ and HSF-1-/- cells were seeded in 4-well chamber slides at a density of 10,000 cells per well and treated as previously described. The cells were then exposed to either 0 mmol/L or 8 mmol/L GLN with and without cytomix for 24 hours. Alexa Fluor 488-conjugated annexin 5 and propidium iodide (PI) were added for 15 minutes at room temperature (in the dark) as per kit directions. After the annexin/PI incubation, cells were rinsed 3 times in phosphate-buffered saline (PBS), pH 7.4, and then fixed in 4% paraformaldehyde PBS for 15 minutes at room temperature. Cells were washed 3 times (10 minutes each) with PBS. Cell membranes were made porous in ice-cold acetone methanol (70%/30% respectively) for 10 minutes and then allowed to air dry. Nuclei were stained with a 1 mg/100 mL bis-benzimide (Hoeschst No. 33342) solution for 30 seconds and then washed again in PBS (3 more times, 10 minutes each). Slides were mounted in antiquenching media and examined for annexin/propidium iodide positive cells. All procedures and incubations were performed in the dark.
Western Blotting Analysis
For Western blot preparations, 5 × 10^sup 6^ cells were seeded in 10-cm dishes. After 48 hours, cells underwent experimental injury. Cells were harvested for Western blot analysis after 24-hour treatments. The harvested cells were lysed in an ice-cold buffer containing 1% Triton X-100, 1 M tris (pH 7.2-8.0), 1 M magnesium sulfate, 1 complete protease inhibitor tablet, RNAase and DNAase. Protein concentrations were determined using the Fluoroskan Ascent FL (Thermo Electro Corporation). Western blotting was performed with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis using 1× treatment buffer (0.125 M tris, pH 6.8, 4% sodium dodecylsulfate, 20% glycerol, and 10% β-mercaptoethanol). Cytoplasmic lysates containing 30 µg of protein were loaded onto NuPAGE 4%-12% Bis-TrisGel (Invitrogen). Proteins were separated electrophoretically and transferred to nitrocellulose membranes using the minigel system. Membranes were blocked with 5% nonfat milk in PBS-Tween for 2 hours. Primary antibodies against either the iNOS (Transduction Lab, Lexington, KY) or HSP-70 (catalog no. SPA-810; StressGen, Victoria, BC, Canada) were applied at dilution of 1:500 and 1:3000, respectively, overnight. After washing 3 times with PBS-Tween over 30 minutes, secondary antibody (peroxidase-conjugated goat antimouse IgG; Sigma) was applied at a 1:2000 dilution for 2 hours. Blots were washed 3 times with PBS-Tween over 30 minutes, incubated in commercial enhanced chemiluminescence reagents (Pierce, Rockford, IL), and exposed to photographic film. Densitometry was performed to quantify the protein express level.
NO Production Assay. In order to determine NO production, total nitrate and nitrite accumulation in the media of cells was measured using the Griess reagent (Cayman Chemical, Ann Arbor, MI).31 Cells (2 × 10^sup 5^/mL) were seeded in 6-well plates for 24 hours, then cells were treated for another 24 hours. Eighty microliters of media was used for nitrate and nitrite detections. First, a nitrate standard curve was performed in order to quantify sample nitrate and nitrite concentrations. The enzyme cofactor mixture and the nitrate reductase mixture were added into the samples and incubated for 2 hours. The nitrate and nitrite accumulations (µM) were determined via a colorimetric reaction based on the Griess reagent and normalized to cellular protein. Total nitrate and nitrite contents are expressed as NOx (µM).
Statistical Analysis
iNOS and HSP-70 expressions were quantified with optical density (OD). All data are expressed as mean ± SD. Differences among experimental groups were evaluated by one-way ANOVA. A p < .05 was considered statistically significant. SPSS software package (version 10.07; SPSS Inc, Chicago, IL) was used for all statistical analyses.
RESULTS
Effects of Cytomix and GLN on Cell Viability.
In these experiments, we exposed HSF-1+/+ cells and HSF-1-/- cells to either cytomix/0 mmol/L GLN or cytomix/8 mmol/L GLN for 48 hours, and survival was measured via MTS assay. After the exposure, about 44% of HSF-1+/+ cells and 49% of HSF-1-/cells survived when compared with the control cells. When 8 mmol/L GLN was concomitantly given with cytomix, approximately 100% of the HSF-1+/+ cells and 51% of the HSF-1-/- cells survived (Figure 1, p < .05). These data indicate that cells lacking the HSF-1 gene have decreased survival when exposed to inflammation. GLN treatment improves survival in HSF-1+/+ cells, but not HSF-1-/- cells.
Effects of Cytomix and GLN on Cellular Apoptosis and Necrosis
In these experiments, we exposed HSF-1+/+ cells and HSF-1-/- cells to either cytomix/0 mmol/L GLN or cytomix/8 mmol/L GLN for 24 hours. Cellular apoptosis and necrosis was measured via annexin 5/PI staining at 24 hours postinjury. After the injury, the HSF-1+/+ and HSF-1-/- cells that did not receive GLN were found to be in early apoptosis and/or late apoptosis/necrosis vs control cells. When 8 mmol/L GLN was concomitantly given with cytomix, a significant reduction of apoptotic and necrotic cells was observed in the HSF-1+/+ cells, but no protection was observed in the HSF-1-/- cells (Figure 2). These data further support that cells lacking the HSF-1 gene have decreased survival when exposed to inflammation. GLN treatment attenuates apoptosis and necrosis in HSF-1+/+ cells, but not HSF-1-/- cells.
Effects of Cytomix and GLN on iNOS Expression
To further understand the mechanisms by which deletion of HSF-1 renders cells more susceptible to cytomix and to examine potential mechanisms by which GLN protects cells from an inflammatory stimulus, we measured iNOS expression in HSF-1+/+ cells and HSF-1-/- cells treated with cytomix and GLN. Cytomix treatment induced iNOS expression both in HSF-1+/+ and HSF-1-/- cells. GLN supplementation inhibited cytomix-induced iNOS expression (p < .05) in HSF-1+/+ cells, but not in HSF-1-/- cells (Figure 3). These data demonstrated that GLN treatment attenuates cytomix-induced iNOS expression via effects on HSF-1 and HSP pathway activation.
Effects of Cytomix and GLN on HSP-70 Gene Expressions
Having demonstrated that GLN attenuated cytomix-induced iNOS expression only in HSF-1 wild-type cells, we hypothesized that it was related to the HSP-70 expression. GLN induced HSP-70 expression only in the HSF-1+/+ cells (p < .05, Figure 4). The results showed that attenuated iNOS expression may be related to increased HSP-70 expression induced by GLN.
Effects of Cytomix and GLN on NO Production
Cytomix treatment induced total nitrate and nitrite accumulation both in HSF-1+/+ and HSF-1-/- cells. GLN supplementation attenuated cytomix-induced total nitrate and nitrite accumulation (p < .05) in HSF-1+/+ cells, but not in HSF-1-/- cells (Figure 5). These data demonstrated that NO production was highly correlated to the iNOS expression and GLN treatment inhibited the cytomix-induced NO accumulation in HSF-1+/+ cells, but not in HSF-1-/- cells.
DISCUSSION
These data are the first demonstration that GLN-mediated protection against inflammatory cytokine-induced injury is dependent on HSF-1 expression and the ability of the cell to activate an HSP response. Our results show that GLN supplementation attenuates inflammation cytokine-induced cell injury and apoptosis, iNOS gene expression, and NO production in HSF-1 wild-type cells, but not in HSF-1 knockout cells. These findings provide the first mechanistic support of previous work from our laboratory indicating a relationship of enhanced HSP expression and GLN-mediated protection in gut epithelial cells22 and cardiomyocytes32 in vitro from oxidant injury, lethal heat stimulus, and ischemia-reperfusion injury. These results also support the role of HSP expression in previously observed studies correlating enhanced HSP expression and GLN-mediated protection against endorgan injury in vivo and improvement of survival from sepsis.17,18,21 These results also may indicate that improved outcome in critically ill patients may be due to enhanced HSP expression.33 To this point, it has been unclear how enhanced HSP expression may prevent inflammatory cytokine-induced injury in sepsis, nor was it clear that the HSP pathway was vital in GLN's protective effect against shock and sepsis. To our knowledge, our study is also the first study indicating that GLN can attenuate inflammatory cytokineinduced iNOS expression and NO production via manipulation of the HSP pathway.
We chose HSF-1 wild-type and HSF-1 null mutant mouse embryonic fibroblasts as our target cells because these cells provided the best opportunity to determine if the HSP pathway plays a role in GLN-mediated protection.29,30 These cells are the only readily available cell line from the HSF-1 knockout mouse model.34 HSF-1 is a transcription factor that regulates the expression of various HSP, including HSP-70. At steady state, HSF-1 is located in the cytoplasm in an inactive monomeric form. After exposure to environmental stress, HSF-1 is activated by phosphorylation and trimerization and translocates into the nucleus, where it binds to the regulatory heat shock elements in the promoter regions of HSP.35 We focused our studies in iNOS and NO because they are important mediators in the pathophysiology of sepsis and other inflammatory conditions and mechanisms of their regulation are well described.6,10 A key known effect of NO overexpression is the formation of peroxynitrite.9 Immunohistochemical and biochemical evidence demonstrates the production of peroxynitrite in shock states. Peroxynitrite can initiate a wide range of toxic oxidative reactions. These include initiation of tyrosine nitration, lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane sodium/potassium ATP-ase activity, inactivation of membrane sodium channels, and other oxidative modifications of proteins. All these toxicities are likely to play a role in the pathophysiology of shock.9 A recent study using an iNOS inhibitor did not show benefit in sepsis.36 This study may have failed to show benefit as the inhibitor caused a near-complete inhibition of the iNOS enzyme, which likely leads to a nonphysiologic derangement of NO production. GLN treatment led to an attenuation, but not complete blockade, of INOS activity.
Specific to the fibroblast cell type, recent reports indicate iNOS expression occurs in fibroblasts.37,38 Treatment with cytomix is known to lead to cytotoxicity in mouse fibroblasts.39 In previous data, maximum cytotoxicity occurred after 48 hours of cytomix treatment40; thus, we measured the cell viability after 48 hours of injury. To assay NO production, we measured the final products of NO, nitrate (NO^sub 3^^sup -^) and nitrite (NO^sub 2^^sup -^). The relative proportion of nitrate and nitrite is variable and cannot be predicted with certainty. Thus, the best index of total NO production is the sum of both nitrate and nitrite. The dose of GLN chosen for this experiment is based on previous ira vitro data indicating that the maximal HSP-70 expression occurs at a concentration of between 4 and 8 mmol/L,16,22 and no adverse effects have been observed from ira vivo doses leading to plasma concentrations in this range.21
HSP, especially HSP-70, has been shown to protect cells, tissues, and organs from injury ira vivo and in vitro.35 The heat shock response is a primitive and highly conserved cellular defense response, which protects against a wide range of environmental stressors. It has been reported that a deficit in HSP-70 expression occurs after endotoxemia and sepsis, and inflammation-induced organ injury and metabolic dysfunction is improved if HSP-70 expression is enhanced.17,18,41,42 Previous data reveal that enhanced HSP-70 inhibits iNOS expression.27,43 This may be due to a known interaction between HSP-70 and iNOS.27,43 Geldanamycin has been shown to inhibit iNOS expression and improve organ function via induction of HSP-70.27,28 Using immunoprecipitation and immunoblotting analysis, previous data reveal that HSP-70 forms a complex with iNOS and its transcription factor Kruppel-like factor 6 (KLF-6), probably by iNOS and KLF-6 binding to the peptide-binding domain of HSP-70. No complex formation is detected between HSP-70 and p53 or Bcl-2 proteins, suggesting that HSP-70 specially couples to iNOS and KLF-6. This complex formation between HSP-70 and iNOS was observed after treatment with geldanamycin.27 It is possible that the complexes formed between HSP-70 and iNOS might decrease the enzymatic activity of iNOS and subsequently decrease NO production. A great deal of recent research has been shown that GLN may preserve cell and organ function via the induction of HSP -70.17,18,21,22 Therefore, we hypothesized that GLN might attenuate cytokine-induced iNOS expression and prevent cell apoptosis and injury via the induction of the HSP pathway. Our findings support that GLN'S protective effect against inflammatory cytokine-induced injury appears to be due to enhanced HSP pathway activation and thus confirmed our hypothesis.
There were some limitations in this study. First, these studies do not indicate what effect GLN-mediated HSP expression may have in other cell lines after inflammatory injury (such as immune cells [ie, macrophages]). However, as fibroblasts and other endothelial cells are often the unintended target of inflammatory cytokine injury, we felt that this cell line was a reasonable model for study of the toxic effects of cytokine-induced injury. Further, although the mouse fibroblasts are active and respond to inflammatory stimulus, they do not necessarily represent the full spectrum of inflammation-related effector cells. It is not known whether other cell types might behave differently when subjected to such stimulus. Our study is further limited in scope as to mechanism by only examining iNOS gene expression. We did not detect the complex of iNOS and HSP-70, which is the direct evidence to show the interaction between iNOS and HSP-70. Moreover, all studies were carried out ira vitro, and it is difficult to directly extrapolate the effect of GLN in vivo.
Our current data indicate that GLN can protect cells from inflammatory cytokine-induced injury and apoptosis. This effect appears to be dependent on HSF-1 expression and the ability of the cell to activate the HSP pathway. These data may be clinically important because many previous studies have demonstrated the benefit of enhanced HSP-70 expression after experimental sepsis. Recently we have demonstrated that GLN can enhance HSP-70 in critically ill patients, and this enhancement of HSP-70 expression was correlated with decreased ICU length of stay.33 It is possible that an important mechanism of GLN's improvement in outcome after critical illness and injury is due to modulation of the HSP pathway.
ACKNOWLEDGMENT
Dr Paul Wischmeyer receives funding through NIH grant K23 RR01379-01 for the Department of Anesthesiology, University of Colorado, Denver, Colorado.
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Received for publication February 27, 2006.
Accepted for publication June 20, 2006.
Correspondence: Paul Wischmeyer, MD, Director of Nutrition Support Service, Associate Professor of Anesthesiology, University of Colorado Health Sciences Center, Department of Anesthesiology, 4200 E. Ninth Avenue, Campus Box B113, Denver, CO 80262. Electronic mail may be sent to Paul.Wischmeyer@UCHSC.edu.
Presented in the Premier Paper Session at the American Society for Parenteral and Enterai Nutrition Clinical Nutrition Week, February 12-15, 2006.
Discussant
Randall S. Friese, MD
University of Texas Southwestern Medical Center
First, I would like to congratulate the authors on furthering their elucidation of the mechanisms by which glutamine attenuates inflammatory cytokine release, reduces organ dysfunction and damage, and improves survival in animal models.
In prior work, these authors have demonstrated that glutamine supplementation results in suppression of NFκB transcriptional activation and translocation to the nucleus, as well as inhibition of IκB phosphorylation and degradation in septic rat lung. These findings suggest that the anti-inflammatory effects of glutamine act, at least in part, through the modulation of the NFκB signal transduction pathway.
In the current study presented today, the authors further elucidate the mechanism by which glutamine provides cytoprotection in the face of an inflammatory stimulus by examining the effects of glutamine on heat shock protein expression. They successfully demonstrate that glutamine induced HSP expression in heat shock factor-1 wild-type mouse embryonic fibroblasts. Additionally, they demonstrated that this increased expression of the molecular chaperone heat shock protein 70 was associated with increased cell survival after an inflammatory stress.
To further support their hypothesis that glutamine exerts its effect via increasing heat shock protein expression, the authors demonstrated that knockout mouse embryonic fibroblasts that lack heat shock factor-1, and therefore are unable to produce the molecular chaperone HSP-70, have survival equal to controls after an inflammatory stress in the presence of supplemental glutamine.
As if this was not enough, the authors took their investigation a step further by examining the effect of glutamine supplementation on iNOS expression. They successfully demonstrated that in heat shock factor-1 wild-type fibroblasts, glutamine attenuated iNOS expression after an inflammatory stress. However, in the heat shock factor-1 knockout fibroblasts, glutamine had no effect on heat shock protein expression after an inflammatory stress. The authors further described that inflammatory stress induced total nitrate and nitrite accumulation in both heat shock factor-1 wild-type and knockout cell lines; however, glutamine supplementation attenuated the total nitrate and nitrite accumulation in the wild-type cell line only. This indicates that glutamine attenuates nitric oxide production only in the presence of the heat shock protein molecular chaperone.
Again, I would like to congratulate the authors on their excellent, high-caliber investigation into the mechanisms of glutamine-mediated cellular protection.
I have only 3 questions, and I warn you, they may not be entirely fair.
1. Other than an effect on iNOS, where else are the heat shock proteins exerting their effect? Typically, these molecular chaperones are expressed at times of cellular stress and aid in the folding and assembly of polypeptide chains into a functional conformation, as well as assist in the transport of the new protein to the site where it will carry out its function.
Your prior work suggested an effect on the NFκB pathway. Does the induced heat shock protein affect, augment, or stabilize the inhibitory-κB protein complex, thereby preventing the translocation of NFκB into the nucleus, and thereby decreasing the inflammatory cascade?
2. As a follow-up to the first question, does the effect of glutamine on the attenuation of iNOS induction and nitric oxide production reflect only 1 small portion of its anti-inflammatory properties? Can you speculate on other possible mechanisms?
3. Lastly, how do we translate your findings with the mouse embryonic fibroblast to the effects of glutamine on our own patients' enterocytes, hepatocytes, cardiomyocytes, macrophages, etc.
I would like to thank the society for the opportunity to discuss this extremely well-performed and well-designed study.
Author's Response
1. Heat shock proteins (HSPs) exert their effects in a variety of cellular processes; the particular effect exerted by the protein is dependent on which HSP is being discussed (ie, HSP-70, HSP-90 excedra). As you state, HSP-70 primarily acts as a molecular chaperone during times of stress. This protein binds to denaturing polypeptides and allows for either proper reconfiguration of the peptide or proper disposal of an irreversibly damaged peptide. The effect on iNOS is probably an indirect one, as HSP-70 and glutamine (GLN) are known to attenuate the overall inflammatory response and the iNOS effect is likely a result of this global effect. As we have recently published,1 GLN does appear to stabilize the IκB inhibitor complex and prevent nuclear translocation of NFκB. Recently presented data from our laboratory2 indicates GLN does not attenuate NFκB activation in HSP-70 knockout mice.
2. Yes, as stated previously the effect of GLN on iNOS is only part of a global anti-inflammatory effect of GLN. Our laboratory has shown this effect is mediated via the NFκB pathway and the MAP Kinase pathway.1 There is also data showing that there are Heat Shock Factor-1 (HSF-1) binding sites in the IL-1 β gene that directly lead to the repression of IL-1 beta expression. We have recently shown that GLN can directly activate HSF-1 binding,3 and this may also play a role in GLN's anti-inflammatory effect.
3. I believe this data reinforces other data from our laboratory that GLN exerts a direct pharmacologie signaling effect on multiple cells in the body. This effect directs the cell to go into a defensive mode to protect itself against stress. This is particularly relevant in various states of critical illness and malnutrition when GLN levels are known to be low. In these patients low GLN levels likely lead to an inability to make heat shock proteins and regulate inflammation properly. It is possible that GLN could be used as a pharmacologie agent to attenuate inflammation in critical illness and auto-immune diseases (ie, ulcerative colitis, Crohn's disease, arthritis).
Thank you for your kind comments and questions.
Paul Wischmeyer M.D.
University of Colorado
Health Sciences Center
REFERENCES
1. Singleton KD, Beckey V, Wischmeyer PE. Glutamine prevents activation of NF-kB and stress kinase pathways, attenuates inflammatory cytokine release and prevents Acute Respiratory Distress Syndrome (ARDS) following sepsis. Shock. 2005;24:583589.
2. Singleton K, Wischmeyer PE. Effect of glutamine on inflammation and survival from sepsis in HSP-70.1.3 knock out mice. Presented at: 2006 Shock Society Meeting; June 3-6, 2006; Broomfield, CO.
3. Morrison A, Dinges M, Singleton KD, Odoms K, Wong H, Wischmeyer PE. Glutamine's protection against injury is dependent on heat shock factor-1 expression. Am J Physiol Cell Physiol. 2006; 290:C1625-C1632. Epub. Jan 25. 2006.
