An in vitro model to consider the effect of 2 mM glutamine and KNK437 on endotoxin-stimulated release of heat shock protein 70 and inflammatory mediators

Abstract

Please cite this article as: Marino LV, Pathan N, Meyer R, Wright VJ, Habibi P, An in vitro model to consider the effect of 2mM glutamine and KNK437 on endotoxin stimulated release of HSP70 and inflammatory mediators, Nutrition (2015), doi: 10.1016/j.nut.2015.09.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Objective: Glutamine has been shown to promote heat shock protein 70 (HSP70) release both within 2 experimental in vitro models of sepsis and in adults with septic shock. 3Methods: An in vitro whole blood endotoxin stimulation assay was used to investigate the effects of 4 2mM glutamine and an inhibitor of HSP70 (KNK437) on the release of HSP70 and inflammatory 5 mediators in healthy adult volunteers. 6Results: 2mM glutamine significantly increased HSP70 levels over time (p<0.05). HSP70 release had a 7 positive correlation at 4 hours with IL-1β (r=0.51, p=0.03), an inverse correlation with TNF-α (r=-8 0.56, p=0.02) and IL-8 levels (r=-0.52, p=0.03), and there were no significant correlations between 9 HSP70 and IL6 or IL-10 or glutamine. Glutamine supplementation significantly (p<0.05) attenuated 10 the release of IL-10 at 4 hours and IL-8 at 24 hours, compared to conditions without glutamine. In 11 endotoxin stimulated blood there were no significant differences in the release of IL-6, TNF-α and IL-12 1β with glutamine supplementation at 4 and 24 hours. However, glutamine supplementation (2mM) 13 appeared to attenuate the release of inflammatory mediators (IL-1β, IL-6, TNF-α), although this 14 effect was not statistically significant. The addition of KNK 437, an HSP70 inhibitor significantly 15 diminished HSP70 release which resulted in lower levels of inflammatory mediators (p<0.05). 16Conclusion: Glutamine supplementation promotes HSP70 release in an experimental model of 17 sepsis. Following the addition of KNK437 the effects of glutamine on HSP70 and inflammatory 18 mediator release appear to be lost, suggesting the HSP70 in part orchestrates the inflammatory 19 mediator response to sepsis. The clinical implications require further investigation.

Ex Vivo Evaluation of Glutamine Treatment in Sepsis and Trauma in a Human Peripheral Blood Mononuclear Cells Model

“…In a randomized, double-blind, interventional clinical trial in critically ill children with sepsis or surgery, glutamine supplementation maintained HSP70 levels for longer but did not increase glutamine levels and did not influence IL-10 or IL-6 levels and outcome [ 55 ]. Addition of 2 mM glutamine to the incubation media enhanced extracellular HSP70, TNF-α, and IL-10 release at 4 h and IL-8 at 24 h [ 56 , 57 ]. Increased release of extracellular HSPs was associated with repressed intracellular expression of HSP70 and HSP90α in sepsis [ 58 , 59 , 60 ].…”

“…Increased release of extracellular HSPs was associated with repressed intracellular expression of HSP70 and HSP90α in sepsis [ 58 , 59 , 60 ]. As plasma from whole blood has been used in previous studies, discrimination of HSP70 released from granulocytes, lymphocytes, or peripheral mononuclear cells could not be established [ 56 , 57 ].…”

Glutamine: A Conditionally Essential Amino Acid with Multiple Biological Functions

“…Interestingly, administration of Gln enhanced the HSP70 expression through the crosstalk with the hexosamine pathway ( Figure 10). An in vitro study demonstrated that the promotion in HSP70 synthesis by Gln is accompanied by a favorable inflammatory response mediated by IL-8 and IL-10 [17].…”