Normalizing Glutamine Concentration Causes Mitochondrial Uncoupling in an In Vitro Model of Human Skeletal Muscle

Krajčová, Adéla; Žiak, Jakub; Jiroutková, Kateřina; Patková, Jana; Elkalaf, Moustafa; Džupa, Valér; Trnka, Ján; Duška, František

doi:10.1177/0148607113513801

Cited by 13 publications

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“…The effect of hyper glutamine levels on inflammatory mediator and HSP70 release during sepsis is also unknown. In in vitro models, high levels of glutamine supplementation (5 mM) resulted in respiratory uncoupling and energy wasting within the mitochondria and were associated with cell death [55]. Therefore, caution is required when translating results from experimental work particularly in septic shock models to inform clinical practice, especially as results from recent large clinical trials questioned the benefits of glutamine supplementation in adults with septic shock with multiorgan failure [53].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2016

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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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2016

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“…In addition, Krajcova et al have suggested the hypoglutaminaemia seen during critical illness may be a protective mechanism. They show that supra physiological levels of glutamine decrease the efficiency of the mitochondrial respiratory chain contributing to the bioenergetics failure of the mitochondria (a feature of critical illness), where engorged mitochondria are not able to meet adenosine tri phosphate demands of tissues, resulting in cell death [61]. As such the hypoglutaminaemia described by us [23] and others during critical illness [62], may be a protective mechanism decreasing mitochondrial size allowing for restoration and repair during the recovery phase [61].…”

Section: LV Marino Et Al / Clinical Nutrition Xxx (2014) 1e7mentioning

confidence: 99%

“…The mechanism for the effect of supra physiological glutamine levels on HSP70 release and inflammatory mediators in critical illness is not known. However, an in vitro model of human skeletal muscle linked supraphysiological levels of glutamine to respiratory uncoupling and energy wasting within the mitochondria which was associated with cell death [61]. In addition, Krajcova et al have suggested the hypoglutaminaemia seen during critical illness may be a protective mechanism.…”

Section: LV Marino Et Al / Clinical Nutrition Xxx (2014) 1e7mentioning

confidence: 99%

The effect of 2 mMol glutamine supplementation on HSP70 and TNF-α release by LPS stimulated blood from healthy children

et al. 2015

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s u m m a r yObjective: Glutamine has been shown to promote heat shock protein 70 (HSP70) release both within experimental in vitro models of sepsis (2e10 mM) and in adults post trauma (0.5 g/kg), although the efficacy varies and is dependent on the model used. The effect of glutamine supplementation on HSP70 release in children is less clear. Therefore, the aim of this study was to investigate the effect of 2 mM glutamine added to incubation media on HSP70 and inflammatory mediator release in an in vitro model of paediatric sepsis using whole blood from healthy paediatric volunteers.Methods: An in vitro whole blood endotoxin stimulation model using 1 mg/ml lipopolysaccharide (LPS) over a 24 h time period was used to investigate the effects of 2 mM glutamine on HSP70 and inflammatory mediator release in healthy children. Results: The addition of 2 mM glutamine to the incubation media significantly increased HSP70 release over time (p < 0.05). This was associated with an early pro-inflammatory effect on TNF-a release at 4 h (p < 0.005) which was not seen at 24 h. There was a non significant trend towards higher levels of IL-6 and IL-10 following the addition of 2 mM glutamine, which appears to differ from the response reported in adult and animal models.Conclusion: Glutamine supplementation of incubation media promotes HSP70 and early TNF-a release in an in vitro model using blood samples from healthy children.

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“…Such small samples allow for the measurement of activities of individual complexes of respiratory chain by spectrophotometry [Pecinová et al, 2011;Votion et al, 2012;Pesta and Gnaiger, 2012] or their concentration by blotting techniques [Wittig et al, 2007;Nabben et al, 2011;Smith et al, 2014]. Protocols for functional assessment of mitochondrial function based on measurement of O 2 consumption are developed for isolated mitochondria [Chance and Williams, 1956], permeabilised muscle fibres [Saks et al, 1998 Jul] or for the use of human cultured myoblasts [Krajcova et al, 2015]. The yield during mitochondria isolation is low (20-40% [Tonkonogi and Sahlin, 1997]) and isolated mitochondria may not represent the whole mitochondrial population in a cell, potentially introducing bias [Palmer et al, 1985;Ferreira et al, 2010;Picard et al, 2010].…”

Section: Introductionmentioning

confidence: 99%

Assessing the function of mitochondria in cytosolic context in human skeletal muscle: Adopting high-resolution respirometry to homogenate of needle biopsy tissue samples

et al. 2015

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Normalizing Glutamine Concentration Causes Mitochondrial Uncoupling in an In Vitro Model of Human Skeletal Muscle

Cited by 13 publications

References 44 publications

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

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

The effect of 2 mMol glutamine supplementation on HSP70 and TNF-α release by LPS stimulated blood from healthy children

Assessing the function of mitochondria in cytosolic context in human skeletal muscle: Adopting high-resolution respirometry to homogenate of needle biopsy tissue samples

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