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Glutamine-Mediated Attenuation of Cellular Metabolic Dysfunction and Cell Death After Injury Is Dependent on Heat Shock Factor-1 Expression

From the Department of Anesthesiology, University of Colorado Health Sciences Center, Denver, Colorado

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{at}UCHSC.edu.

Background: Cellular metabolic dysfunction is associated with occurrence of multiple-organ failure after critical illness. Glutamine (GLN) attenuates cellular metabolic dysfunction in critical illness models. The mechanism of this protection is unclear. We previously demonstrated that GLN's benefit in critical illness might be due to enhanced heat shock protein (HSP) expression. We hypothesize that GLN's attenuation of cellular metabolic dysfunction is dependent on presence of heat shock factor-1 (HSF-1). Methods: HSF-1 wild-type and knockout mouse embryonic fibroblasts (HSF-1+/+ and HSF-1–/–) were used in all experiments. Cells were not treated, or were treated with 8 mmol/L GLN and immediately exposed to heat stress injury (45°C for 45 minutes). Cells were harvested for metabolic analysis by nuclear magnetic resonance (NMR) at 24 hours postinjury. Cell survival was assessed using the MTS assay. Results: GLN treatment in HSF-1+/+ cells led to significant attenuation of decreases in adenosine triphosphate (ATP)/adenosine diphosphate (ADP) ratio, phosphomonoester/phosphodiester (PME/PDE) ratio, and cell survival observed in non-GLN-treated HSF-1+/+ cells. In HSF-1–/– cells, the beneficial effect of GLN on preservation of ATP/ADP ratio, PME/PDE proliferation, and cell survival was lost. GLN-treated HSF-1–/– cells had a significant increase in extracellular lactate concentrations vs GLN-treated HSF+/+ cells. Conclusions: GLN treatment attenuated cellular metabolic dysfunction and improved cell membrane recovery only in HSF-1+/+ cells. Cellular injury, as measured by lactate release and cell survival assay, was improved by GLN treatment in HSF-1+/+ cells alone. Thus, GLN's beneficial effect on cellular metabolic dysfunction and cell survival appears to be dependent on HSF-1 expression.

Critical illness and injury commonly lead to the occurrence of multiple organ dysfunction syndrome (MODS).¹ MODS is often the ultimate cause of death in the intensive care unit. It has been hypothesized that the cause of multiple-organ failure is cellular metabolic dysfunction.² In sepsis and systemic inflammatory response syndrome (SIRS), recent data suggest that the predominant defect might lie in cellular oxygen use rather than in oxygen delivery per se.² Mitochondrial dysfunction exists in ischemia/reperfusion and sepsis.^3,4 Severe injury causes an intrinsic derangement in mitochondrial activity and depletion of high-energy phosphates in muscle and red cells.⁵ A recent clinical trial found an association between nitric oxide overproduction, antioxidant depletion, mitochondrial dysfunction, and decreased adenosine triphosphate (ATP) that appears to relate to organ failure and eventual outcome. This report implicated bioenergetic failure as an important pathophysiological mechanism underlying MODS.⁶

Some novel methods of protecting the structural and metabolic components of the cell against injury include up-regulation of intrinsic protective mechanisms.⁷ These mechanisms include an increase in the levels of heat shock proteins (HSP), which are a class of highly conserved proteins that act as molecular chaperones during periods of cell stress.⁸ Evidence exists that enhanced HSP-70 expression in heart and lung can preserve tissue metabolism and ATP contents in ischemia/reperfusion and sepsis.^8–13

Glutamine (GLN), traditionally considered a nonessential amino acid, now appears to be a conditionally essential nutrient during serious injury or illness.¹⁴ Numerous clinical trials are now emerging that show intervention with GLN affects reduction of infectious complication rates in postsurgical patients and a reduction in complication and mortality rates in critically ill patients.¹⁵ This mechanism may be related to the enhanced expression of HSP, in particular HSP-70 induced by GLN.^16–19 Specific to cell metabolism, we have showed that GLN can attenuate cellular metabolic dysfunction in sepsis, endotoxin-induced lung injury, and myocardial ischemia and reperfusion.^11–13 However, the mechanism of this metabolic protection is unclear.

Heat shock transcription factor-1 (HSF-1) is a transcription factor that regulates HSP expression, including HSP-70.²⁰ We hypothesize that GLN's attenuation of cellular metabolic dysfunction and cell death after injury is dependent on the presence of HSF-1 and the subsequent expression of HSPs. In this study, we examined the role of GLN in preventing cellular metabolic dysfunction and cell injury in HSF-1 wild-type and gene knockout cells. We hypothesized GLN's protective effect on metabolic dysfunction and cell injury would be lost in cells with a specific gene-deletion of HSF-1.

	MATERIALS AND METHODS

Top MATERIALS AND METHODS RESULTS DISCUSSION

 
Cell Culture
All experiments used 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's laboratory (Cincinnati, OH). These cells were previously demonstrated to be a useful model for studying the role of HSP expression.^21,22 Cells were grown and maintained in a room air/5% carbon dioxide incubator at 37°C using Dulbecco's modified Engle's medium (DMEM, Cellgro Mediatech Inc, Herndon, VA) containing 10% fetal bovine serum (Cellgro Mediatech Inc), 55 µM β-mercaptoethanol (Invitrogen, Carlsbad, CA), 0.1 mmol/L nonessential amino acids solution (Cellgro Mediatech Inc), 2 mmol/L D-GLN (Sigma, St. Louis, MO), and 10 mL/L of antibiotic solution containing penicillin G (10,000 U/mL) and streptomycin (10,000 µg/mL; Cellgro Mediatech Inc).

Experimental Conditions
All experiments were grouped as follows: HSF-1+/+ untreated (WT/(–) GLN), HSF-1+/+ treated with 8 mmol/L GLN (WT/(+) GLN), HSF-1–/– treated with 8 mmol/L GLN (KO/(+) GLN), and HSF-1–/– untreated (KO/(–) GLN). Each group of cells was divided into 2 subgroups, the control and the heat shock (HS) subgroups. For the HS treatment cells, the dishes of cells were sealed with Parafilm and seated within a water-tight bag and then exposed to a water bath at 45°C for 45 minutes, whereas the control cells were exposed to 37°C for the same period.

Cell Survival Studies
Cell viability was assayed with MTS/PMS mixture (Promega, Madison, WI). Cells were seeded in 96-well plates. After 24 hours, cells received treatments (media without GLN or 8 mmol/L GLN, HS or control). After another 24 hours, cell survival was analyzed. Briefly, 1 part phenazine methosulfate (PMS) was added to 20 parts tetrazolium salt (MTS) immediately before the solution was diluted 1:5 in phenol red–free DMEM and was then added to PBS-washed cells. MTS was bioreduced by cells into a colored, soluble formazan product. Absorbance values were read every 60 minutes for 4 hours at 490 nm; references included readings at 650 nm and no-cell blank wells. Higher absorbance values reflect greater cell proliferation/viability. HS cell readings were ratioed to the uninjured control cell readings to account for baseline variability of the 2 cell types.

Western Blot Analyses
For the Western blot preparations, 5 x 10⁶ cells were seeded in individual 10-cm dishes. After 48 hours, cells received aforementioned treatments (media without GLN or 8 mmol/L GLN, HS or control). Cells were harvested for Western blot analysis after 24-hour treatments (media without GLN or 8 mmol/L GLN, HS or control). Western blotting was performed as previously described.²³ Primary antibodies against HSP-70 (catalog No. SPA-810; StressGen, Victoria, BC, Canada) were applied at dilution of 1:3000 overnight. After washing 3 times with PBS-Tween over 30 minutes, the secondary antibody (peroxidase-conjugated goat anti-mouse IgG; Sigma) was applied at a 1:2000 dilution for 2 hours. The 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 on an immunoblot to quantify the protein level.

Quantification of Cell Metabolism
The cells were extracted with 2 mL ice-cold perchloric acid (12%) in the presence of liquid nitrogen, as described in detail by Serkova et al.²⁴ The extracted samples were centrifuged and neutralized using KOH and centrifuged once again. The aqueous extract and media from the cells were lyophilized overnight. The lyophilisates if the aqueous extracts were redissolved in 0.5 mL deuterium oxide (D₂O), adjusted to pH 7.0–7.3 using deuterium chloride (DCI) and deuteroxide (NaOD). The media samples were dissolved in 1.0 mL D₂O after centrifugation. All the samples were analyzed by magnetic resonance spectroscopy (MRS).

Assessment of cell metabolism using ¹H- and ³¹P-nuclear magnetic resonance (NMR) spectroscopy was carried out as described previously.²⁵ In brief, all one-dimensional magnetic resonance (MR) spectra of tissue aqueous PCA extracts were recorded on a Bruker AMX 360 spectrometer and processed using WINNMR software (Bruker, Karlsruhe, Germany). A 5 mm ¹HX-inverse probe was used for all experiments. For proton MRS, the operating frequency was 360 MHz, and a standard presaturation pulse program was used for water suppression. Other parameters were 40 accumulations, 90° pulse angle, 0-dB power level, 7.35-µs pulse width, 10 parts per million spectral width, and 12.85-second repletion time. Trimethylsilyl propionic-2,2,3,3,-d₄ acid (TSP; 0.6 mmol/L) was used as external standard for the quantification of metabolites according to ¹H-MR spectra. ¹H chemical shifts of spectra were referenced to TSP at 0 ppm. For ³¹P-MRS analysis of PCA extracts, 100 mmol/L EDTA was added for complexing of divalent ions, resulting in significantly narrower line width of ³¹P peaks. The pH was adjusted to about 7 using KOH and HCI. The following NMR parameters with a composite pulse decoupling program were used: 145.7-MHz operating ³¹P frequency, 800 accumulations, 90° pulse angle, 12-dB power level for ³¹P channel, 9-µs pulse width, 35 ppm spectral width, and 2.0-second repetition time. The chemical shift of -ATP at 10 ppm was used as shift reference. The absolute concentrations of phosphocreatine (PCr) or phosphomonoesters (PMEs) calculated from ¹H-MRS were used for metabolite quantification of ³¹P-MR spectra.

View larger version (8K): [in this window] [in a new window]   FIGURE 1. Effect of GLN on cellular ATP/ADP ratio after heat shock injury (45°C for 45 minutes; mean ± SE, n = 4/group). Cells harvested at 24 hours for evaluation of ATP/ADP ratio via NMR. Groups as defined in Materials and Methods (*p < .05 vs all other groups).

	RESULTS

Top MATERIALS AND METHODS RESULTS DISCUSSION

 
Effects of GLN on HS-Induced Cell Metabolism Dysfunction
To examine the effects of GLN on HS-induced cellular metabolic dysfunction, all cells were analyzed via ¹H- and ³¹P-MRS. Specifically, ATP/ADP, lactate, phosphomonoesters/phosphodiesters (PME/PDE), and reduced glutathione (rGSH) were measured.

Cellular ATP/ADP Ratio
After HS, GLN pretreatment in wild-type HSF-1+/+ cells significantly attenuated the fall of ATP/ADP ratio observed in non-GLN-treated HSF-1+/+ cells (0.70 ± 0.041 [WT/(+) GLN] vs 0.44 ± 0.097 [WT/(–) GLN, p < .05]. No effect of GLN treatment was observed in HSF-1–/–cells after GLN treatment (0.48 ± 0.12 [KO/(–) GLN] vs 0.50 ± 0.034 [KO (+) GLN], Figure 1). GLN's treatment benefit on ATP/ADP expression was lost in the HSF-1 KO cells (0.70 ± 0.041 [WT/(+) GLN] vs 0.50 ± 0.034 [KO (+) GLN], Figure 1).

View larger version (8K): [in this window] [in a new window]   FIGURE 2. Effect of GLN on cellular PME/PDE ratio after heat shock injury (45°C for 45 minutes; mean ± SE, n = 4/group). Cells harvested at 24 hours for evaluation of PME/PDE ratio via NMR. Groups as defined in Materials and Methods (*p < .01 vs both KO groups; #p < .05 vs WT/(–) GLN).

GLN pretreatment in HSF-1+/+ cells significantly increased the PME/PDE ratio (0.65 ± 0.059 [WT/(+) GLN] vs 0.35 ± 0.097 [WT/(–) GLN], p < .05), but GLN had no effect on PME/PDE ratio in HSF-1–/– cells (0.15 ± 0.037 [KO (+) GLN] vs 0.18 ± 0.045 [KO/(–) GLN], Figure 2).

Extracellular Lactate Concentration
To further assess cellular injury after GLN treatment, extracellular lactate concentrations were measured. A nonstatistically significant decrease in extracellular lactate was observed in GLN-treated HSF-1+/+ cells vs no GLN treatment in HSF-1+/+ cells (1.12 ± 0.21 mmol/L [WT/(+) GLN] vs 1.80 ± 0.59 [WT/(–) GLN], p < .05). In the absence of HSF-1, GLN treatment exerted no beneficial effect on extracellular lactate concentrations (2.67 ± 0.11 [KO (+) GLN] vs 1.50 ± 0.67 [KO/(–) GLN]). Deletion of HSF-1 led to a significant increase in lactate concentrations cells treated with GLN vs HSF-1 WT cells treated with GLN (1.12 ± 0.21 mmol/L [WT/(+) GLN] vs 2.67 ± 0.11 [KO (+) GLN], p < .05).

Cellular rGSH Level
GLN is known to be an essential precursor of glutathione, a vital cellular antioxidant. To assess the effect of GLN treatment in HSF-1+/+ and HSF-1–/– cells after heat injury, reduced GSH (rGSH) was measured. GLN treatment in HSF-1+/+ cells led to a significant increase in rGSH concentrations (2.63 ± 0.87 µM/G [WT/(+) GLN] vs 0.091 ± 0.090 µM/G [WT/(–) GLN], p < .05). GLN treatment in HSF-1–/– cells also led to a significant increase in rGSH concentrations (1.46 ± 0.20 µM/G [KO(+) GLN] vs 0.026 ± 0.03 µM/G [KO/(–) GLN], Figure 3). There were no significant differences between GLN-treated HSF+/+ vs HSF–/– cells.

View larger version (6K): [in this window] [in a new window]   FIGURE 3. Effect of GLN on cellular reduced glutathione (rGSH) after heat shock injury (45°C for 45 minutes; mean ± SE, n = 4/group). Cells harvested at 24 hours for evaluation of rGSH via NMR. Groups as defined in Materials and Methods.

View larger version (8K): [in this window] [in a new window]   FIGURE 4. Effects of glutamine treatment on cell viability after heat shock injury (45°C for 45 minutes; mean ± SE, n = 12/group). Cell survival analyzed via MTS assay. Groups as defined in Materials and Methods (#p < .01 vs WT/(–)GLN, *p < .05 vs KO/(+)GLN).

View larger version (13K): [in this window] [in a new window]   FIGURE 5. Effects of glutamine treatment on HSP-70 expression after heat shock injury (45°C for 45 minutes; mean ± SE, n = 3/group). HSP-70 analyzed via Western blot. Groups as defined in Materials and Methods (#p < .01 vs WT/(–)GLN/Heated, *p < .01 vs WT/(+)GLN/Unheated and all KO groups).

Effects of GLN on HSP70 Expression
Having demonstrated that GLN attenuated the HS-induced cell death only in the HSF-1 wild-type cells, we hypothesized that this effect may be related to enhanced HSP-70 expression. HSP-70 is the primary protective inducible HSP.²⁸ We measured the HSP-70 expression via Western blot. GLN enhanced HSP-70 expression only in the HSF-1+/+ cells (p < .05, Figure 5). No HSP70 expression was observed in HSF-1–/– cells.

	DISCUSSION

Top MATERIALS AND METHODS RESULTS DISCUSSION

 
This study demonstrates that GLN administration decreases HS-induced metabolic dysfunction as measured by an increased ATP/ADP ratio, improved PME/PDE ratio, and decreased extracellular lactate accumulation. This effect on energy and cellular membrane metabolism was associated with attenuation of HS-induced cell death. The improvement in ATP/ADP ratio, PME/PDE ratio, extracellular lactate accumulation, and survival was only observed in the HSF-1+/+ cells; no beneficial effect of GLN was observed in the HSF-1–/– cells. As expected, HSP-70 was only expressed in HSF-1+/+ cells. Thus, these results indicate that GLN's beneficial effects on cell metabolism after HS seem to be related to HSF-1 expression and activation of the HSP pathway. This study may have significant clinical importance because it provides further evidence to confirm that GLN may improve survival in critically ill or injured patients via enhanced HSP expression.

A great deal of recent research has been focused on the importance of tissue metabolic dysfunction in critical illness.² Fink² has recently suggested that the central defect in sepsis is an uncoupling of oxidative phosphorylation. In this hypothesis, septic cells are unable to use oxygen and energy-producing substrates to make high-energy phosphate compounds. Moreover, recent clinical data show that there is an association between metabolic dysfunction and severity and outcome of septic shock.⁶

Increased levels of HSP-70 have previously been shown to be beneficial in improving cellular metabolism.^4,8–10 Sammut et al²⁹ reported that heat stress preconditioning contributed to the enhancement of cardiac mitochondrial complex activity. In HSP-70 gene-transfected rats, the cardiac metabolic and ventricular function were improved during myocardial ischemia reperfusion.⁸ We have found that GLN could attenuate the sepsis-induced and ischemia-reperfusion–induced metabolic dysfunction.^11–13 As GLN is a vital nutrient source in stressed cells, it is unknown if GLN's ability to prevent metabolic dysfunction is related to its role as a metabolic substrate or GLN's known ability to enhance HSP-70 expression. In sepsis and endotoxin-induced lung injury, we found that GLN's ability to increase the ATP/ADP ratio was associated with enhanced expression of HSP-70.^11,12 However, in myocardial ischemia-reperfusion injury, GLN attenuated myocardial tissue metabolic defects, but no changes in HSP levels were observed immediately after injury.¹³

To elucidate the role of HSP-70 expression in the manifestation of GLN's beneficial effects on cellular metabolism, we used HSF-1 wild-type and HSF-1 null mutant mouse embryonic fibroblasts as the HSF-1 KO cells are unable to express inducible HSPs.^21,22 HSF-1 is a transcription factor that regulates the expression of HSPs, including HSP-70. In unstressed cells, HSF-1 is located in the cytoplasm in an inactive, monomeric form.³⁰ After exposure to environmental stress, HSF-1 is activated by phosphorylation and trimerization and translocates into the nucleus, where it binds to the regulatory HS elements in the promoter regions of HSP genes.²⁸ The dose of GLN chosen for the present study is based on previous in vitro data indicating that maximal HSP-70 expression occurs at a concentration between 4 and 8 mmol/L,^31,32 and no adverse effects from plasma levels in this range have been observed in in vivo models.²³ The temperature and heating time period used in the present study was based upon preliminary studies in our laboratory as this experimental condition was found to result in approximately 70% cell death.

Our results indicate that HS injury results in a decrease in ATP/ADP, and that GLN attenuates this decrease in HSF-1+/+ cells, a result that is consistent with previous reports.^11–13 ATP is produced in mitochondria, and mitochondrial damage results in an intracellular decrease in ATP concentration, as well as generation of superoxide anions via the electron transport chain, leading to cell death. Overexpression of HSP-70 provides significant mitochondrial protection against injury.^4,9,10,29 It has been suggested that there is a specific mechanism involved in the HSP-70–mediated mitochondrial protection pathway. The specific mechanism involved may be HSP-70 chaperone activity involved with mitochondrial membrane proteins or other mitochondrial proteins, including respiratory chain enzymes.^33,34 HSP-70 appears to play an important role in the transport of newly synthesized mitochondrial proteins into mitochondria.^33,34 GLN exerted no beneficial effect on the ATP/ADP ratio in HSF-1–/– cells. This result indicates that GLN appears to attenuate cellular metabolic dysfunction as a direct result of enhanced HSP expression.

To our knowledge, this report is the first to describe that HS injury leads to a decrease in PME/PDE ratio and GLN pretreatment can attenuate this effect. The phospholipid metabolites measured are strictly associated with cell membrane synthesis and degradation. The phospholipids examined here were represented in the ³¹P-MR spectrum by 2 peaks, the PME peak and the PDE peak, with each peak found to contain several individual compounds. The PME metabolites phosphorylcholine and phosphorylethanolamine are precursors of phosphatidylcholine and phosphatidylethanolamine, which are major components of the phospholipids of the cell membrane. In contrast, the PDE glycerophosphorylcholine and glycerophosphorylethanolamine are formed as intermediate metabolites during cell membrane degradation.³⁵ Thus, the PME/PDE ratio reflects the cell membrane regeneration process. A significant correlation between the value PME/PDE ratio and the time after renal transplantation has been reported.³⁵ A decreased PME/PDE ratio in patients with delayed graft function due to acute tubular necrosis was also found by Heindel et al.³⁶ GLN's ability to increase the PME/PDE ratio indicates that GLN may allow injured cells to survive and regenerate their cell membranes. In the cells not receiving GLN, an increase in irreversible cell injury leading to cell death appears to occur. GLN's beneficial effect on cell membrane regeneration was not observed in HSF-1–/– cells. This result indicates that GLN's effect on cell membrane regeneration is dependent on activation of the HSP pathway.

Our results additionally indicate that GLN attenuated HS-induced rGSH depletion. Glutathione is an important cellular antioxidant molecule. It has been reported that the depletion of glutathione in states of myocardial ischemia and reperfusion injury can worsen metabolic and overall organ function.³⁷ GLN is an important precursor of glutathione. GLN supplementation can increase the concentration of rGSH after injury.³⁸ GLN enhanced rGSH levels in both HSF-1+/+ and HSF-1–/– cells after HS. This result indicates that GLN's effect on rGSH is not dependent on HSF-1 or HSP-70. GLN-mediated enhancement of rGSH in HSF-1–/– cells did not lead to improved survival. In this study, there were no significant changes in rGSH levels between HSF-1 wild-type and knockout cells. These results indicate that GLN-mediated enhancement of rGSH did not play a significant role in GLN's protective effect on cell metabolism or survival.

In summary, these data provide further evidence confirming that GLN can attenuate cellular metabolic dysfunction and improve cell survival after injury. GLN's beneficial effects are dependent on HSF-1 expression and presumably enhanced HSP expression. The present findings offer further evidence that GLN's pharmacologic modulation of HSP may play a central role in the future treatment of critically ill and injured patients.

Dr Paul Wischmeyer receives funding through NIH grant K23 RR01379-01 for the Department of Anesthesiology, University of Colorado, Denver, Colorado.

Top MATERIALS AND METHODS RESULTS DISCUSSION

 
Presented in the Premier Paper Session at the American Society for Parenteral and Enteral Nutrition Clinical Nutrition Week, February 12–15, 2006.

Received for publication February 27, 2006. Accepted for publication April 17, 2006.

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Discussant

Dr Peng and his collaborators are to be congratulated on contributing a novel mechanistic approach that examines the relationship between metabolic stress, glutamine and heat shock factor-1 expression in an embryonic fibroblast cell culture model. Although glutamine supplementation in critically ill models has shown benefit, it remains unclear how glutamine may provide protection from cellular metabolic dysfunction. The authors clearly demonstrate that the beneficial metabolic effects provided with supplemental glutamine are dependent on the expression of heat shock protein-1. Consequently, the information presented by Peng and coworkers significantly contributes to the growing body of literature that examines the impact of glutamine modulation in critical illness.

1. You mentioned within your manuscript's Discussion that the heat shock protein, HSP-70, plays an important role in the maintenance of mitochondrial health—through HSP-70's chaperone function that assists delivery of newly synthesized mitochondrial proteins to mitochondria. Consequently, the continuous presence of HSP-70 appears to be generally important to overall cellular health, whether or not the cell experiences injury. My question is, therefore, the following: since the absence of HSF-1 prevents expression of HSP-70, was there any evidence that HSF-1 knockout cells behaved differently metabolically relative to wild-type cells—in the absence of injury?
   
2. As you describe, HSF-1 is the transcription factor that is critical to expression of HSP-70. As you additionally discuss, elimination of the HSF-1 gene prevents the expression of not only HSP-70, but all heat shock proteins. Do you plan to study the effect of injury upon cells that have had individual heat shock genes knocked out in order to investigate a possible hierarchal or combinatorial role of individual heat shock proteins?
   
3. Do you plan to examine the specific intracellular role of glutamine in this protective process; ie, would you please speculate as to how you think glutamine may specifically be involved in the heat shock protein regulation of cellular injury?
   

Author's Response

To address your second question, we have currently begun this exact research. Utilizing an HSP-70 knockout mouse, our preliminary data indicates this specific heat shock protein is necessary for GLN's protection of the whole animal against sepsis and sepsis-induced lung injury. However, with advances in small interfering RNA (siRNA) we plan to perform cellular (and hopefully in vivo) experiments knocking down specific HSPs, including HSP-70 and HSP-25/27.

Finally, to answer your third question, we have recently published data showing that GLN can directly lead to the nuclear translocation, promoter binding, and phosphorylation event required for HSF-1 activation that leads to HSP expression in a cellular model of injury. In fact, in nonstressed cells GLN can increase the nuclear translocation of nonactivated HSF-1. It appears to "prime" the cell for stress or injury resistance. We currently believe we have determined how GLN induces HSF-1 activation and HSP expression. We are currently performing the experiments to prove it, and to this point, the data support our new hypothesis. We hope to have data to present at the upcoming A.S.P.E.N. meeting. We do not believe it is a GLN receptor and we feel strongly it is a metabolic pathway that leads to activation of the HSP response. Stay tuned.

Journal of Parenteral and Enteral Nutrition, Vol. 30, No. 5, 373-379 (2006)
DOI: 10.1177/0148607106030005373


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