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Dose response of endotoxin on hepatocyte and muscle mitochondrial respiration in vitro.

Jeger V, Brandt S, Porta F, Jakob SM, Takala J, Djafarzadeh S - Biomed Res Int (2015)

Bottom Line: In HepG2 cells, LPS reduced mitochondrial membrane potential and cellular ATP content but did not modify basal respiration.LPS reduced cellular ATP content of HepG2 cells, most likely as a result of the induced decrease in membrane potential.LPS decreased cellular and isolated mitochondrial respiration in a time-dependent, dose-dependent and complex-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Intensive Care Medicine, Inselspital, Bern University Hospital and University of Bern, Freiburgstraße 10, 3010 Bern, Switzerland ; Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland.

ABSTRACT

Introduction: Results on mitochondrial dysfunction in sepsis are controversial. We aimed to assess effects of LPS at wide dose and time ranges on hepatocytes and isolated skeletal muscle mitochondria.

Methods: Human hepatocellular carcinoma cells (HepG2) were exposed to placebo or LPS (0.1, 1, and 10 μg/mL) for 4, 8, 16, and 24 hours and primary human hepatocytes to 1 μg/mL LPS or placebo (4, 8, and 16 hours). Mitochondria from porcine skeletal muscle samples were exposed to increasing doses of LPS (0.1-100 μg/mg) for 2 and 4 hours. Respiration rates of intact and permeabilized cells and isolated mitochondria were measured by high-resolution respirometry.

Results: In HepG2 cells, LPS reduced mitochondrial membrane potential and cellular ATP content but did not modify basal respiration. Stimulated complex II respiration was reduced time-dependently using 1 μg/mL LPS. In primary human hepatocytes, stimulated mitochondrial complex II respiration was reduced time-dependently using 1 μg/mL LPS. In isolated porcine skeletal muscle mitochondria, stimulated respiration decreased at high doses (50 and 100 μg/mL LPS).

Conclusion: LPS reduced cellular ATP content of HepG2 cells, most likely as a result of the induced decrease in membrane potential. LPS decreased cellular and isolated mitochondrial respiration in a time-dependent, dose-dependent and complex-dependent manner.

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Related in: MedlinePlus

Tracings from the OROBOROS-high-resolution respirometry of permeabilized cells. Legend: the upper tracing represents a cell sample incubated with placebo (control) for 24 hours; the lower tracing represents a cell sample at the same passage number incubated with 1 μg/mL LPS for 24 hours. The experiments were recorded simultaneously. The blue line represents oxygen concentration; the red line represents the oxygen flow (slope of oxgygen concentration). GM: Glutamate + malate, ADP: adenosine diphosphate, ROT: rotenone, SUC: succinate, AA: antimycin A, ASC TMPD: ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine, NaN3: sodium azide, and S3: state 3. State 4 cannot be measured due to the saturating concentration of ADP during the experiment.
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fig4: Tracings from the OROBOROS-high-resolution respirometry of permeabilized cells. Legend: the upper tracing represents a cell sample incubated with placebo (control) for 24 hours; the lower tracing represents a cell sample at the same passage number incubated with 1 μg/mL LPS for 24 hours. The experiments were recorded simultaneously. The blue line represents oxygen concentration; the red line represents the oxygen flow (slope of oxgygen concentration). GM: Glutamate + malate, ADP: adenosine diphosphate, ROT: rotenone, SUC: succinate, AA: antimycin A, ASC TMPD: ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine, NaN3: sodium azide, and S3: state 3. State 4 cannot be measured due to the saturating concentration of ADP during the experiment.

Mentions: Representative respiration rates of permeabilized HepG2 cells using high-resolution respirometry are shown in Figure 4 (n = 10 for each experiment). 24 hours of incubation with LPS (1 μg/mL) induced a significant reduction in maximal complex II-dependent and IV-dependent respiration of HepG2 cells but did not affect cell viability (Table 1). 0.1 and 10 μg LPS/mL as well as 4, 8, and 16 hours of incubation induced no significant changes.


Dose response of endotoxin on hepatocyte and muscle mitochondrial respiration in vitro.

Jeger V, Brandt S, Porta F, Jakob SM, Takala J, Djafarzadeh S - Biomed Res Int (2015)

Tracings from the OROBOROS-high-resolution respirometry of permeabilized cells. Legend: the upper tracing represents a cell sample incubated with placebo (control) for 24 hours; the lower tracing represents a cell sample at the same passage number incubated with 1 μg/mL LPS for 24 hours. The experiments were recorded simultaneously. The blue line represents oxygen concentration; the red line represents the oxygen flow (slope of oxgygen concentration). GM: Glutamate + malate, ADP: adenosine diphosphate, ROT: rotenone, SUC: succinate, AA: antimycin A, ASC TMPD: ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine, NaN3: sodium azide, and S3: state 3. State 4 cannot be measured due to the saturating concentration of ADP during the experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4306363&req=5

fig4: Tracings from the OROBOROS-high-resolution respirometry of permeabilized cells. Legend: the upper tracing represents a cell sample incubated with placebo (control) for 24 hours; the lower tracing represents a cell sample at the same passage number incubated with 1 μg/mL LPS for 24 hours. The experiments were recorded simultaneously. The blue line represents oxygen concentration; the red line represents the oxygen flow (slope of oxgygen concentration). GM: Glutamate + malate, ADP: adenosine diphosphate, ROT: rotenone, SUC: succinate, AA: antimycin A, ASC TMPD: ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine, NaN3: sodium azide, and S3: state 3. State 4 cannot be measured due to the saturating concentration of ADP during the experiment.
Mentions: Representative respiration rates of permeabilized HepG2 cells using high-resolution respirometry are shown in Figure 4 (n = 10 for each experiment). 24 hours of incubation with LPS (1 μg/mL) induced a significant reduction in maximal complex II-dependent and IV-dependent respiration of HepG2 cells but did not affect cell viability (Table 1). 0.1 and 10 μg LPS/mL as well as 4, 8, and 16 hours of incubation induced no significant changes.

Bottom Line: In HepG2 cells, LPS reduced mitochondrial membrane potential and cellular ATP content but did not modify basal respiration.LPS reduced cellular ATP content of HepG2 cells, most likely as a result of the induced decrease in membrane potential.LPS decreased cellular and isolated mitochondrial respiration in a time-dependent, dose-dependent and complex-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Intensive Care Medicine, Inselspital, Bern University Hospital and University of Bern, Freiburgstraße 10, 3010 Bern, Switzerland ; Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland.

ABSTRACT

Introduction: Results on mitochondrial dysfunction in sepsis are controversial. We aimed to assess effects of LPS at wide dose and time ranges on hepatocytes and isolated skeletal muscle mitochondria.

Methods: Human hepatocellular carcinoma cells (HepG2) were exposed to placebo or LPS (0.1, 1, and 10 μg/mL) for 4, 8, 16, and 24 hours and primary human hepatocytes to 1 μg/mL LPS or placebo (4, 8, and 16 hours). Mitochondria from porcine skeletal muscle samples were exposed to increasing doses of LPS (0.1-100 μg/mg) for 2 and 4 hours. Respiration rates of intact and permeabilized cells and isolated mitochondria were measured by high-resolution respirometry.

Results: In HepG2 cells, LPS reduced mitochondrial membrane potential and cellular ATP content but did not modify basal respiration. Stimulated complex II respiration was reduced time-dependently using 1 μg/mL LPS. In primary human hepatocytes, stimulated mitochondrial complex II respiration was reduced time-dependently using 1 μg/mL LPS. In isolated porcine skeletal muscle mitochondria, stimulated respiration decreased at high doses (50 and 100 μg/mL LPS).

Conclusion: LPS reduced cellular ATP content of HepG2 cells, most likely as a result of the induced decrease in membrane potential. LPS decreased cellular and isolated mitochondrial respiration in a time-dependent, dose-dependent and complex-dependent manner.

Show MeSH
Related in: MedlinePlus