Kristen D. Singleton

GLUTAMINE PREVENTS ACTIVATION OF NF-kappaB AND STRESS KINASE PATHWAYS, ATTENUATES INFLAMMATORY CYTOKINE RELEASE, AND PREVENTS ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) FOLLOWING SEPSIS.

Glutamine (GLN) has been shown to attenuate cytokine release from LPS-stimulated human peripheral blood mononuclear cells; however, the in vivo antiinflammatory effect of GLN in polymicrobial sepsis and ARDS is unknown. This study evaluates the effect of GLN on inflammatory cytokine release and the pathways that may mediate antiinflammatory effects of GLN in the lung. Either 0.75 g/kg of GLN or saline placebo (SP) was administered to male rats 1 h after cecal ligation and puncture (CLP). NF-κB activation, IKBα degradation, phosphorylation of p38 MAPK, ERK, and MKP-1 expression were evaluated in lung tissue 6 h post-CLP. Lung tissue iNOS and eNOS, TNF-α, IL-6, and IL-18 cytokines were assayed. Last, lung histopathology for occurrence of ARDS and survival were examined. GLN given 1 h postsepsis led to inhibition of lung tissue NF-κB activation (P < 0.001 vs. SP), attenuated degradation of IKBα, and inhibited phosphorylation of p38 MAPK, and ERK, pathways critical for cytokine release. GLN treatment increased MKP-1 peptide expression and significantly attenuated TNF-α and IL-6 6 h after CLP. IL-18 was attenuated by GLN at multiple time points post-CLP. Further, GLN abrogated increases in lung iNOS expression and enhanced lung eNOS postsepsis. Finally, GLN prevented the histopathologic appearance of ARDS after sepsis and significantly improved survival. These data reveal that GLN exerts an antiinflammatory effect in sepsis that may be mediated via attenuation of multiple pathways of inflammation such as NF-κB, p38 MAPK, ERK, and MKP-1. GLN also showed an inhibition of increases in iNOS expression. The antiinflammatory effect of GLN was associated with attenuation of ARDS and mortality.

Glutamine attenuates tumor necrosis factor-α release and enhances heat shock protein 72 in human peripheral blood mononuclear cells

OBJECTIVE Overexpression of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) can contribute to multiple organ dysfunction syndrome and septic shock in critically ill patients. We previously found that glutamine (GLN) can attenuate cytokine expression, induce heat shock protein 72 (HSP 72), and protect against endotoxin-induced mortality and organ injury in an in vivo rat model. However, data on the effect of GLN on direct attenuation of cytokine release and HSP 72 expression in human peripheral blood polymorphonuclear cells (PBMCs) is lacking. METHODS In this study, we assessed the effect of GLN on TNF-alpha and HSP 72 expression in human PBMCs. After treating with various doses of GLN, human PBMCs were stimulated with lipopolysaccharide (LPS). TNF-alpha release was analyzed via enzyme-linked immunosorbent assay and HSP 72 via western blot. RESULTS GLN at doses greater than 4 mM decreased TNF-alpha release at 4 and 24 h after LPS stimulation. Sublethal heating of PBMCs before LPS also markedly decreased TNF-alpha after LPS. Doses of GLN greater than 2 to 4 mM led to an increase in HSP 72 expression after LPS. CONCLUSION These results indicate that GLN, which may improve outcomes in critically ill patients, can directly attenuate pro-inflammatory cytokine release in PBMCs. This effect may be related to enhanced HSP 72 expression.

Distance of Cecum Ligated Influences Mortality, Tumor Necrosis Factor-Alpha and Interleukin-6 Expression following Cecal Ligation and Puncture in the Rat

Background/Aim: A mainstay of laboratory research into new therapies for sepsis has been the cecal ligation and puncture (CLP) model in rodents. Previous data indicate that the number of punctures made in the cecum and needle size utilized are primary determinants of mortality. Despite this, variability exists in mortality from this model, even when needle size is held constant. The aim of the present study was to evaluate the influence of the length of cecum ligated, independent of needle size, as a determinant of mortality. Materials and Methods: We evaluated this by ligating various cecal lengths in male Sprague-Dawley rats. A double puncture was then made with a 20-gauge needle, and mortality was analyzed. Plasma TNF-α and IL-6 expression was assessed at 6 h. Animals received no antibiotics, were not fasted, and fluid resuscitation was administered. Results: We determined that mortality does not begin to occur until a distance of >5% of cecal length is ligated. Further, our findings indicate that in this model, 90–100% mortality occurs 4 days following CLP when a distance of >30% is ligated. TNF-α and IL-6 expression is markedly increased with increasing length of cecum ligated. Conclusions: Our data demonstrate that the length of cecum ligated is a major determinant of mortality in the CLP model of sepsis. These findings indicate that investigators must rigorously control the distance of the cecum ligated in order to generate consistent mortality and inflammation data when utilizing the CLP model in rats. Further, the mortality from this model can be adjusted to fit the individual needs of a particular experiment.

Oral glutamine enhances heat shock protein expression and improves survival following hyperthermia.

ABSTRACT No pharmacologic agent has shown benefit in treating heatstroke. Previous data indicate that enhanced heat shock protein 70 (HSP-70) expression can improve survival postexperimental heatstroke. Glutamine (GLN) can enhance HSP-70 expression in other injury models. This study assessed if orally administered GLN could enhance tissue HSP expression and could improve survival following whole body hyperthermia. Intestinal permeability and plasma endotoxin were assayed to determine if enhanced HSP expression correlated with improved organ function. GLN (0.65 g/kg) or an iso-nitrogenous control (Travasol; T) was given to rats via gavage twice daily for 5 days pre-heatstroke. Hyperthermia was performed in anesthetized rats by heating animals to 42°C (rectal temperature) for 30 min. HSP-70 analyzed via Western blot. Gut permeability was measured 6 and 24 h post-hyperthermia. Plasma endotoxin was measured 24 h post-hyperthermia. Survival was analyzed for 5 days post-hyperthermia. GLN administration enhanced gut and lung HSP-70 post-hyperthermia. GLN administration led to significantly enhanced gut heat shock factor 1 (HSF-1) activation before heatstroke and at 1 h postheat stress. GLN decreased gut permeability at 6 and 24 h post-hyperthermia versus T. Plasma endotoxin also decreased in GLN-treated rats 24 h post-hyperthermia. Oral GLN therapy significantly improved survival (P < 0.05). Our results indicate that oral GLN can enhance tissue HSP-70 and HSF-1 activation post-hyperthermia. These results also indicate that enhanced HSP-70 may have functional significance as GLN-treated animals had decreased gut permeability, plasma endotoxin, and improve survival following lethal hyperthermia. Enhanced expression of HSP-70 may be an important mechanism leading to enhanced survival via GLN. These data indicate that oral GLN may useful in prevention of mortality from heatstroke in at risk populations.

Single dose of glutamine enhances myocardial tissue metabolism, glutathione content, and improves myocardial function after ischemia-reperfusion injury

BACKGROUND Myocardial ischemia and reperfusion (I/R) injury causes significant morbidity and mortality. Protection against I/R injury may occur via preservation of tissue metabolism and ATP content, preservation of reduced glutathione, and stimulation of heat shock protein (HSP) synthesis. Supplementation with glutamine (GLN) has been reported to have beneficial effects on all of these protective pathways. Thus, we hypothesized that GLN pretreatment given to the rat in vivo would protect the myocardium against I/R-induced dysfunction. METHODS GLN (0.52 g/kg, intraperitoneally, given as alanine-glutamine dipeptide), alanine alone (0.23 g/kg), or a Ringers lactate solution (control) was administered to Sprague-Dawley rats 18 hours before heart excision, perfusion, exposure to global ischemia (15 minutes) and reperfusion (1 hour). Tissue metabolites were analyzed via magnetic resonance spectroscopy. RESULTS In control and alanine-treated animals, I/R injury resulted in cardiac dysfunction, indicated by a decrease in cardiac output. Administration of GLN 18 hours before I/R injury preserved cardiac output after reperfusion. Metabolic analysis of the myocardial tissue revealed that [/R injury led to significant diminution of myocardial tissue glutamate, ATP content, accumulation of myocardial lactate, and a reduction in reduced glutathione content in control animals. GLN significantly reduced the deleterious changes in myocardial metabolism and improved reduced glutathione content. No changes in pre- or post-I/R injury HSP expression were observed after GLN administration. CONCLUSIONS These observations demonstrate that remote in vivo administration of GLN before cardiac I/R injury can improve post-I/R cardiac function. This effect may be mediated via improved myocardial metabolism and enhanced reduced glutathione content.

Glutamine induces heat shock protein expression via O-glycosylation and phosphorylation of HSF-1 and Sp1.

BACKGROUND Glutamine (GLN) improves outcome in experimental and clinical states of illness and injury. The authors hypothesized GLN-mediated enhancement of O-glycosylation and subsequent phosphorylation of key transcription factors in the HSP70 pathway would lead to increased HSP70 expression following experimental sepsis. METHODS Mice underwent cecal ligation and puncture (CLP)-induced sepsis and were treated with GLN (0.75 g/kg) or a saline placebo 30 minutes after CLP. A separate group of mice was treated with mithramycin, an Sp1 inhibitor. Lung tissue was harvested at 1, 2, 6, and 24 hours after CLP and was analyzed for HSF-1 and Sp1 O-GlcNAc modification, alpha-p-threonine modification, and HSP70. RESULTS GLN increased O-GlcNAc modification of HSF-1 and Sp1 at 1 and 2 hours after sepsis (P < .001 vs saline). Samples immunoprecipitated for Sp1 and probed for subsequent phosphorylation showed a significant increase in nuclear alpha-p-threonine-modified Sp1 at 2 and 6 hours after sepsis (P < .001 vs saline). GLN increased phosphorylated nuclear HSF-1 at 1 and 2 hours after CLP (P < .001). Finally, GLN treatment increased HSP70 4-fold (P < .01), but when treated with mithramycin, this increase was attenuated at 2, 6, and 24 hours (P < .001 vs no mithramycin treatment). CONCLUSIONS These results indicate that GLN induces HSF-1 and Sp1, which is known to lead to their nuclear translocation. The molecular mechanism of GLN-mediated HSP70 expression appears to be dependent on O-GlcNAc pathway activation and subsequent O-glycosylation and phosphorylation of key transcription factors required for HSP70 induction.

Glutamine attenuates inflammation and NF-κB activation via Cullin-1 deneddylation

Glutamine (GLN) can inhibit NF-kBeta activation and cytokine expression following sepsis. NF-kappaB activation and inflammatory cytokine expression, depend on neddylation of Cullin-1 (Cul-1) to proceed. Our aim was to evaluate whether GLN inhibits Cul-1 neddylation, and further determine if GLN-mediated Cul-1 deneddylation attenuates NF-kappaB activation and subsequent cytokine expression following experimental sepsis in the mouse. Sepsis-induced via cecal ligation and puncture (CLP) led to a significant increase in lung Cul-1 neddylation. GLN administration post-sepsis led to enhanced lung Cul-1 deneddylation and attenuated NEDD8 expression (p<0.01 vs. saline). Cul-1 deneddylation was associated with decreased NF-kappaB activation and IkappaB alpha degradation in GLN treated mice (( *)p<0.01 vs. saline). Lastly, GLN treatment led to a significant decrease in lung TNF-alpha and IL-6 post-sepsis. These are the first data describing a direct effect of GLN on Cul-1 deneddylation and provide a possible mechanistic explanation for GLNs anti-inflammatory effects.