Endotoxin or lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria is a trigger of the systemic inflammatory response in sepsis [
LPS triggers macrophages via cell surface receptors (mainly TLR-4) to produce powerful inflammatory mediators including tumor necrosis factor-alpha (TNF-
Research on the effects of LPS on mitochondria has been confounded by the wide range of doses and exposure times. This has likely contributed to the heterogeneous and sometimes contradictory results [
LPS (TLR4 agonist, ultrapure
Human hepatocellular carcinoma cell line (HepG2, Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany, N°ACC 180) was cultured in 25 cm2 flasks (for respiration assays) or 96-well plates (for determination of cellular ATP content and mitochondrial membrane potential) in RPMI 1640 culture medium containing 10% heat-inactivated fetal calf serum (FCS), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37°C in a humid atmosphere (5% CO2, 95% air). Cells were passaged upon reaching confluence.
The primary human hepatocytes were kindly provided by D. Stroka (University of Bern). Primary human hepatocytes were isolated from wedges of resected liver tissues taken from patients undergoing liver surgery. Written informed consent was obtained prior to surgery in compliance with the local ethical committee [
Quiescent cells were obtained by total deprivation of FCS for 14 to 16 hours before the experiments. All experiments were performed when cells reached 90–95% confluency. On the day of experiment, HepG2 was exposed to placebo or LPS (0.1, 1, 10
Respiration was determined as described before [
To confirm that mitochondrial outer membrane integrity was not compromised in permeabilized cells, we performed digitonin titration to determine the optimal concentration for permeabilization of HepG2 cells and primary human hepatocytes. For these experiments, cells were suspended in respiration buffer (
After incubation with LPS (HepG2: 1
The mitochondrial electrochemical potential gradient (Δ
In some additional experiments, to evaluate the quality of the mitochondria, cytochrome c test was performed to verify outer mitochondrial membrane integrity. For these experiments, HepG2 and primary human hepatocytes were incubated with LPS and permeabilized with digitonin (8.1
For these experiments, the cells were grown in 96-well plates and treated with LPS for 24 hours (1
Cells were grown in 96-well plates and treated with LPS for 24 hours (1
Quadriceps muscle biopsy was taken from 7 anaesthetized pigs, from which mitochondria were isolated by differential centrifugation as described by Porta et al. [
The protocol started immediately after isolation of the mitochondria, and determination of respiration was finished within 6 hours in all groups. Mitochondria from each biopsy sample were divided into 12 groups and incubated on ice with vehicle or 0.1, 1, 10, 50, and 100
Mitochondrial respiration was performed as described before [
Statistical analysis was performed with the SPSS software package (SPSS 15.0, SPSS Inc., Chicago, IL) and Prism 6 (GraphPad Software, La Jolla, CA). Mitochondrial respiration of each complex at different doses of LPS was compared at each time point using the nonparametric Friedman test followed by the Dunn’s test for multiple comparisons. If significant, the Wilcoxon test was used to compare this dose with its control. For primary human hepatocytes, the Wilcoxon test was used as only one dose of LPS was explored, due to the limited availability of primary human hepatocytes. For unpaired data (ATP content, mitochondrial membrane potential) the Mann-Whitney test was used. Data are shown as median and interquartile range (IQR).
For the measurement of cellular respiration of intact cells, cells were exposed to LPS and respiration rates were measured in the absence of exogenous substrates and ADP. LPS at 1
Basal respiration of intact cells. HepG2 cells’ basal oxygen consumption, in the presence of oligomycin (leak respiration), and FCCP, respectively, in LPS treated and control cells ((a);
LPS at 1
ATP content and mitochondrial membrane potential of intact cells. (a) Cellular ATP content of HepG2 was determined after 24 hours exposure with 1 μg/mL LPS (
Measurements of total free radical ROS and RNS levels in supernatants of intact cells. Total reactive oxygen species (ROS) and reactive nitrogen species (RNS) were determined after reaction with DCFH to DCF (2′,7′-dichlorodihydrofluorescein) in supernatants of LPS treated and control cells (
Representative respiration rates of permeabilized HepG2 cells using high-resolution respirometry are shown in Figure
Effects of LPS exposure on cellular respiration and cell viability of cultured, digitonin-permeabilized human hepatoma cells (HepG2) for various time intervals and at different concentrations. For cellular respiration, all values are expressed as pmol O2/(sec ∗ Mill. Cells); ∗∗
HepG2 permeabilized | Control | 0.1 |
1 |
10 |
Friedman |
|
---|---|---|---|---|---|---|
Complex I | 4 h | 99 (86–112) | 107 (88–115) | 105 (101–111) | 106 (98–119) | 0.430 |
8 h | 90 (78–99) | 89 (77–105) | 89 (82–96) | 88 (76–95) | 0.392 | |
16 h | 95 (68–101) | 96 (83–106) | 95 (84–101) | 89 (81–99) | 0.472 | |
24 h | 96 (82–110) | 105 (92–111) | 87 (84–97) | 91 (77–110) | 0.339 | |
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Complex II | 4 h | 121 (107–126) | 115 (108–134) | 123 (109–127) | 124 (114–130) | 0.516 |
8 h | 126 (109–135) | 117 (95–133) | 131 (118–145) | 125 (99–135) | 0.339 | |
16 h | 104 (81–129) | 120 (91–132) | 115 (95–126) | 108 (95–126) | 0.669 | |
24 h | 94 (78–111) | 94 (84–105) |
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84 (67–99) |
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Complex IV | 4 h | 172 (166–187) | 179 (155–210) | 170 (166–181) | 174 (161–185) | 0.669 |
8 h | 166 (97–200) | 144 (118–166) | 145 (126–168) | 147 (129–173) | 0.840 | |
16 h | 139 (90–171) | 127 (108–165) | 123 (111–156) | 122 (109–174) | 0.840 | |
24 h | 98 (90–130) | 99 (84–118) |
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98 (89–133) |
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Viability (%) | 8 h | 84 (68–91) | 87 (72–95) | 88 (71–93) | 88 (70–92) | |
16 h | 95 (93–96) | 93 (90–94) | 94 (91–95) | 94 (90–95) | ||
24 h | 94 (92–95) | 92 (89–93) | 94 (93–95) | 93 (90–95) |
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.
Basal respiration rates were determined in intact primary human hepatocytes in the absence of exogenous substrates (Figure
Basal respiration of intact cells. Primary human hepatocytes’ basal oxygen consumption, in the presence of oligomycin (leak respiration), and FCCP, respectively, in LPS treated and control cells ((a);
1
Time course of 1
Primary hepatocytes permeabilized | Control | 1 |
|
| |
---|---|---|---|---|---|
Complex I | 4 h | 252 (91–351) | 287 (116–337) | 8 | 0.547 |
8 h | 248 (93–340) | 198 (88–293) | 7 | 0.156 | |
16 h | 191 (112–368) | 172 (113–329) | 8 | 0.641 | |
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Complex II | 4 h | 411 (198–474) | 342 (201–510) | 8 | 0.844 |
8 h |
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7 |
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|
16 h | 317 (239–530) | 274 (241–516) | 8 | 0.742 | |
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Complex IV | 4 h | 242 (192–308) | 256 (146–346) | 8 | 0.641 |
8 h | 257 (167–350) | 203 (143–383) | 7 | 0.375 | |
16 h | 287 (208–362) | 308 (179–388) | 8 | 0.844 | |
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Viability (%) | control | 98 (93–99) | 5 | ||
4 h | 91 (83–95) | 5 | |||
8 h | 99 (99-100) | 5 | |||
16 h | 97 (93–99) | 5 |
Cytochrome c did not enhance respiration of the HepG2 or primary human hepatocytes treated with placebo or LPS indicating that there was no loss of cytochrome c from mitochondrial outer membrane and mitochondrial integrity was preserved (data not shown).
Control mitochondria kept on ice for 4 hours were significantly lower compared to the same mitochondria analyzed 2 hours after isolation. To prevent the time effect, each exposed sample of mitochondria was compared to its time-matched control sample. Two-hour incubation with 50 and 100
Time and dose course of LPS on isolated pig skeletal muscle mitochondria. Data are expressed as median (IQR). All values are in pmol O2/(sec ∗ mg mitochondrial protein).
Isolated mitochondria | Control | 0.1 |
1 |
10 |
50 |
100 |
Friedman |
|
---|---|---|---|---|---|---|---|---|
Complex I state 3 | 2 h | 1676 (1171–1914) | 1512 (1093–1527) | 1365 (1110–1790) | 1396 (992–1591) |
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4 h |
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1250 (493–1742) | 1050 (657–1548) | 1207 (407–1814) | 1258 (584–1543) | 1081 (804–1424) | 0.576 | |
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Complex I state 4 | 2 h | 454 (306–556) | 420 (267–525) | 418 (259–558) | 419 (198–526) |
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4 h | 269 (191–445) | 309 (175–427) | 310 (205–346) | 361 (149–448) | 334 (204–532) | 332 (201–510) | 0.846 | |
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Complex I RCR | 2 h | 3.3 (2.7–4.5) | 3.6 (2.4–4.3) | 3.3 (2.9–5.7) | 3.4 (2.5–3.8) | 3.4 (1.9–4.6) | 4.3 (2.6–5.3) | 0.670 |
4 h | 3.6 (3.0–4.9) | 4.1 (2.8–4.2) | 4.1 (2.5–4.9) | 4.1 (2.3–4.6) | 3.9 (2.4–4.6) | 3.5 (2.4–5.0) | 0.946 | |
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Complex II state 3 | 2 h | 1753 (469–2340) | 1479 (449–2046) | 1042 (476–1671) | 1468 (388–2430) |
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4 h |
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1428 (459–1680) | 1181 (425–1594) | 1359 (326–1495) | 958 (386–1019) | 754 (479–1097) | 0.136 | |
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Complex II state 4 | 2 h | 631 (398–882) | 576 (363–902) | 578 (413–683) | 591 (323–866) | 627 (300–989) | 637 (293–890) | 0.600 |
4 h | 716 (322–888) | 732 (375–891) | 679 (337–803) | 599 (284–1086) | 618 (311–923) | 595 (323–656) | 0.227 | |
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Complex II RCR | 2 h | 2.2 (1.4–2.7) | 2.0 (1.2–2.6) | 2.0 (1.2–2.6) | 2.0 (1.2–2.5) | 1.2 (1.1–1.8) | 1.4 (1.2–2.1) | 0.123 |
4 h | 1.3 (1.1–2.3) | 1.9 (1.1–2.1) | 1.3 (1.1–2.3) | 1.3 (1.2–1.9) | 1.3 (1.2–1.7) | 1.4 (1.2–1.9) | 0.923 | |
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Complex IV state 3 | 2 h | 2338 (684–4335) | 1762 (415–4223) | 977 (690–2325) | 1912 (435–3679) | 1890 (379–2722) | 1553 (368–2379) | 0.084 |
4 h |
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1553 (399–3516) | 1356 (399–2320) | 1276 (422–3043) | 2145 (450–2344) | 979 (388–1643) | 0.068 |
LPS did not impair basal respiration in intact HepG2 cells. Despite maintained oxygen consumption, cellular ATP content was reduced, likely as a result of the induced reduction of the mitochondrial membrane potential. The effects were similar, albeit less strong, than those of valinomycin, an ionophore capable of depolarizing mitochondria [
Mitochondrial oxygen consumption was only affected by LPS when respiration was stimulated with complex specific substrates (e.g., succinate for complex II). In primary human hepatocytes LPS induced a reduction in complex II-dependent respiration rates. Due to the limited availability of primary human hepatocytes, we evaluated further effects of LPS in HepG2 cells, a human hepatoma cell line. Also in HepG2 cells, LPS reduced stimulated respiration. Finally, maximal respiration was also impaired in isolated skeletal muscle mitochondria.
Our data confirm the findings of a previous review on effects of sepsis on mitochondrial functions and may explain the heterogenous results in the literature: LPS effects seem to be time, dose, tissue, and method dependent. We found impaired respiration after 8 hrs (human hepatocytes) or only after 24 hrs of LPS incubation (HepG2 cells) and at 1
Transient effects of LPS on primary hepatocytes’ mitochondrial respiration have been reported previously [
LPS is recognized by TLR4, which is present not only on the surface of immune cells but also on the surface of hepatocytes [
In addition to a TLR4 mediated pathway, a mechanism of LPS endocytosis is postulated and it has been shown that LPS can reach mitochondria directly [
In HepG2 and primary human hepatocytes, LPS decreased cellular respiration in the presence of exogenous substrates (permeabilized cells) without impairing basal respiration of intact cells. A limitation of the present study is that most of the cellular experiments were performed in HepG2 cells, due to the limited availability of primary human hepatocytes. HepG2, a human hepatoma cell line, has a different phenotype compared to primary hepatocytes and is thought to be a mature epithelial cell line, which can form structures similar to bile canaliculi [
LPS decreased mitochondrial membrane potential and cellular ATP content in intact HepG2 cells without impairing basal respiration. However, maximal (stimulated) respiration was decreased in the presence of exogenous substrates (permeabilized cells) in a time-dependent and dose-dependent fashion. In primary hepatocytes, LPS decreased as well as cellular respiration in the presence of exogenous substrates (permeabilized cells) without impairing basal respiration.
Our data confirm the heterogeneous reaction of mitochondrial respiration to LPS.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Victor Jeger, Sebastian Brandt, and Francesca Porta all contributed equally to this paper.
The authors thank Sandra Nansoz and Tsilla Sunier for their technical assistance. This study was supported by the Swiss National Science Foundation (Grant no. 32003B_127619). Victor Jeger was supported by MD-PhD scholarship of the Swiss National Science Foundation (no. 133901).