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Keywords:

  • fatty acid;
  • hypoxia;
  • mitochondria;
  • PC12 cells;
  • reactive oxygen species

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

J. Neurochem. (2011) 10.1111/j.1471-4159.2011.07286.x

Abstract

There is an intense discussion about the subcellular origin of the generation of reactive oxygen species (ROS) under hypoxia. Since this fundamental question can be addressed only in a cellular system, the O2-sensing rat pheochromocytoma (PC12) cells were used. Severe hypoxia is known to elevate non-esterified fatty acids. Therefore, the site(s) of ROS generation were studied in cells which we simultaneously exposed to hypoxia (1% oxygen) and free fatty acids (FFA). We obtained the following results: (i) at hypoxia, ROS generation increases in PC12 cells but not in mitochondria isolated therefrom. (ii) Non-esterified polyunsaturated fatty acids (PUFA) enhance the ROS release from PC12 cells as well as from mitochondria, both in normoxia and in hypoxia. (iii) PUFA-induced ROS generation by PC12 cells is not decreased either by inhibitors of the cell membrane NAD(P)H oxidase or inhibitors impairing the PUFA metabolism. (iv) PUFA-induced ROS generation of mitochondria is paralleled by a decline of the NADH-cytochrome c reductase activity (reflecting combined enzymatic activity of complex I plus III). (v) Mitochondrial superoxide indicator (MitoSOXred)-loaded cells exposed to PUFA exhibit increased fluorescence indicating mitochondrial ROS generation. In conclusion, elevated PUFA levels enhance cellular ROS level in hypoxia, most likely by impairing the electron flux within the respiratory chain. Thus, we propose that PUFAs are likely to act as important extrinsic factor to enhance the mitochondria-associated intracellular ROS signaling in hypoxia.

Abbreviations used
Apo

apocynin

AR

Amplex Red

ASA

acetylsalicylic acid

CAT

carboxyatractyloside

COX

cyclooxygenase

DMEM

Dulbecco’s modified Eagle’s medium

Doco

docosahexaenoic acid

DPI

diphenylene iodonium

ETC

electron transport chain

FCCP

carbonyl cyanide 4-trifluoro-methoxyphenylhydrazone

FFA

free fatty acids

HRP

horse radish peroxidase

KCN

potassium cyanide

LDH

lactate dehydrogenase

MitoSOXred

mitochondrial superoxide indicator

O2−˙

superoxide

PBS

phosphate-buffered saline

PC12

pheochromocytoma cells

PC12mito

mitochondria isolated from PC12 cells

PUFA

polyunsaturated non-esterified fatty acids

ROS

reactive oxygen species

Uptake of oxygen by cells is crucial for supporting energy-linked mitochondrial functions. However, mitochondria generate also to a minor extent superoxide (O2−˙) by one-electron-transfer reactions to molecular oxygen, mainly at sites of complexes I and III of the electron transport chain (ETC; see for a review, Murphy 2009). The formed O2−˙ is mostly converted in the mitochondrial matrix into hydrogen peroxide (catalyzed by the manganese superoxide dismutase), which escapes mitochondria or becomes inactivated to water inside the mitochondria (catalyzed by the glutathione peroxidase). Generally, the mitochondrial production of ROS is modulated by several factors, such as the electrochemical proton gradient, the redox state of redox centers of the ETC, or the activity of the ROS scavenging systems (Murphy 2009; Aon et al. 2010).

Moreover, studies with different cell types, such as cardiomyocytes (Duranteau et al. 1998), hepatocytes (Lluis et al. 2005), myocytes (Gong et al. 2010), or neural PC12 cells (Höhler et al. 1999) revealed that cells respond to severe hypoxia with increased cellular ROS generation (also reviewed in Chandel 2009). This surprising phenomenon is observed at hypoxia, corresponding to an oxygen tension below 10%. Furthermore, there is evidence that this ROS generation stems from mitochondria (Kwast et al. 1999; Guzy et al. 2005). The role of mitochondria as a source of hypoxia-dependent ROS generation was originally derived from the observation that ρ° cells (cells depleted from mitochondrial DNA) do not display enhanced ROS generation during hypoxia (Kwast et al. 1999). Because ρ° cells are lacking critical subunits of the complexes I, III, and IV, they are unable to carry out functional electron transport. Nevertheless, it is still controversially discussed whether mitochondria are the site of hypoxia-induced cellular ROS generation (Hoffman et al. 2007; Brookes et al. 2008; Korge et al. 2008; Hoffman and Brookes 2009; Srinivasan et al. 2010). Since the ROS release by isolated mitochondria declines at low oxygen pressure (Hoffman et al. 2007), it has been concluded that the hypoxia-induced cellular ROS generation is not intrinsic to the mitochondrial ETC alone.

In this respect, it is worth to recall that hypoxia significantly raises the cellular level of non-esterified FFA (Bazan 1970; Gardiner et al. 1981; Ueno et al. 1988; Van der Vusse et al. 1992; Wetzels et al. 1993; Sun and Gilboe 1994; Michiels et al. 2002). Furthermore, mitochondria prepared from hypoxic tissue are functionally impaired because of enriched FFA (Feldkamp et al. 2006). In addition, it has been reported that isolated mitochondria respond to FFA with a concentration-dependent increase of ROS release, which could be explained by an interference of FFA with the electron transport in the ETC (Takeuchi et al. 1991; Cocco et al. 1999; Loskovich et al. 2005; Schönfeld and Reiser 2006; Schönfeld and Wojtczak 2007), and reviewed recently (Schönfeld and Wojtczak 2008). Consequently, it is conceivable that the interaction of FFA with the ETC contributes to the hypoxia-induced ROS generation. Indeed, stimulation of the cellular ROS generation by FFA has been reported in studies with various cell types in normoxia (e.g., see Chan et al. 1988; Kahlert et al. 2005; Hatanaka et al. 2006; Aitken et al. 2006; Lambertucci et al. 2008; Koppers et al. 2010). However, FFA are also potential candidates to enhance the ROS generation at non-mitochondrial sites, such as the cell plasma membrane-associated NADPH oxidases (Corey and Rosoff 1991; Cury-Boaventura and Curi 2005), the oxidative metabolism of arachidonic acid (Adibhatla and Hatcher 2006), or the peroxisomal enzymes (Mueller et al. 2002).

To get deeper insight into the role of mitochondria in the hypoxia-induced ROS generation, here we examined the effect of elevated FFA levels on the cellular and mitochondrial ROS generation in normoxia (21% O2) and severe hypoxia (1% O2). For this purpose, we employed rat PC12 cells which represent an established model of O2-sensitive cells (Greene and Tischler 1976; Höhler et al. 1999; Spicer and Millhorn 2003; for review, Conrad et al. 2001). We report here that PC12 cells respond with enhanced ROS generation to an elevated FFA level in normoxia and hypoxia. In conclusion, we propose that elevated levels of FFA in normoxia and hypoxia can increase cellular ROS generation, most likely by an interaction with the mitochondrial ETC.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

PC12 cells and mitochondria

Rat PC12 cells (DSMZ Nr. Acc 159) were grown in plastic culture flasks at 37°C and 6% CO2. Culture medium was Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4.5 g/L glucose, 950 mg/L glutamine, 10% (v/v) fetal calf serum, 17.5 mm Hepes, and 0.25% ciprofloxacin, a gyrase inhibitor with broad activity. Adherent cells were removed with Hank’s balanced salt solution medium plus 0.02% EDTA. Finally, cells were suspended in phosphate-buffered saline (PBS).

To have well-defined experimental conditions for measuring the response of cellular ROS generation to FFA, PC12 cells were exposed to a bolus addition of 50 μm of various saturated and unsaturated fatty acids. FFA concentrations ranging from 25 to 100 μm are usually applied in serum-free incubations with isolated cells (Chan et al. 1988; Aitken et al. 2006). A concentration of 50 μm FFA does not affect the cellular integrity of PC12 cells [measured as release of lactate dehydrogenase (LDH)] during long-term incubation. Stock solutions of fatty acids (10 mm) were made in ethanol.

To isolate mitochondria from PC12 cells, pellets of PC12 cells (2 × 107 cells) were suspended in 0.8 mL of ice-cold sucrose-based medium (250 mm sucrose, 5 mm Hepes, 0.1 mm EDTA, pH 7.2) and, homogenized using a Dounce homogeniser. After the addition of 0.8 mL sucrose-based medium to the homogenate, the mixture was centrifuged (700 g, 10 min, 4°C). The resulting supernatant was again centrifuged (12 000 g, 15 min, at 4°C). The mitochondrial pellet was washed once in sucrose-based medium, and was resuspended in 0.5 mL sucrose-based medium. The protein content of this stock suspension was measured by BioRad Protein Kit (Hercules, CA, USA).

Characterization of mitochondria

Functional intactness of mitochondria isolated from PC12 cells was estimated by measuring the respiratory control ratio using pyruvate plus malate as respiratory substrates. Oxygen uptake by mitochondria (0.4 mg of protein per 1.0 mL incubation medium) was measured using an oxygraph (Oroboros Oxygraph®; Bioenergetics and Biomedical Instruments, Innsbruck, Austria) at 37°C. The incubation medium contained 110 mm mannitol, 60 mm KCl, 60 mm Tris-HCl, 10 mm KH2PO4, 0.5 mm EGTA (pH 7.4), 5 mm pyruvate plus 5 mm malate, or 5 mm succinate.

Membrane polarization was monitored using safranine, which is accumulated as a permeant cation in energized mitochondria. Mitochondria (1 mg of protein/mL) suspended in 1 mL of incubation medium were equilibrated with 5 μm of safranine. Fluorescence was monitored at 37°C using a PerkinElmer Life Sciences LS-50B fluorescence spectrometer LS-50B (Beaconsfield, UK; excitation at 495 nm; emission at 586 nm).

The activity of the NADH-cytochrome c reductase (complexes I and III; EC 1.6.99.3) was measured photometrically at 25°C as described in Hatefi and Rieske (1967).

Lactate production, release of lactate dehydrogenase

Aliquots of PC12 stock suspension (0.15 mL, corresponding to about 3 × 106 cells) were added to 0.35 mL of PBS medium supplemented with 50 μm of FFA. The incubation mixture was kept at 37°C for 30 min. Lactate production was assessed in the incubation medium photometrically using a glycine-hydrazine buffer (pH 9.2) supplemented with 2.25 mm NAD+.

The release of LDH from PC12 cells was measured using a cytotoxicity detection kit.

Determination of ROS production

ROS generation by mitochondria

Mitochondrial ROS generation was estimated as release of H2O2 using the Amplex Red (AR)/horse radish peroxidase (HRP) system. Briefly, mitochondria (0.4 mg mitochondrial protein/mL) were incubated with 5 μm AR plus HRP (2 U/mL) to detect H2O2 release. Resorufin fluorescence was fluorimetrically monitored (excitation at 560 nm, emission at 590 nm). O2−˙ level in the mitochondrial compartment in intact cells was assessed with mitochondrial matrix-targeted MitoSOXred. For this purpose, PBS medium was supplemented with 0.2 μm MitoSOXred. ROS generation was monitored at 25°C. ROS-dependent fluorescence changes were measured using a microplate reader (Tecan Austria GmbH, Salzburg, Austria).

Imaging of mitochondrial ROS generation in situ

PC12 cells grown on glass cover slips were labeled with MitoSOXred (30 min, 5 μm, 37°C, 5% CO2 in DMEM without fetal calf serum, washed once, and incubated for further 30 min at 37°C in DMEM. Cover slips were transferred to single 35 mm diameter petri dishes filled with 2 mL DMEM supplemented either with 5 μL/mL EtOH (control), arachidonic acid, or antimycin A. After 30 min of incubation (37°C, 5% CO2), cells were fixed with 4% paraformaldehyde in PBS. Specimens were examined by a Leica TCS SPE 4000 confocal laser scanning microscope (Leica Mikrosysteme, Wetzlar, Germany), using 488 nm excitation and a 540–600 nm emission windows.

ROS generation by PC12 cells

Cellular ROS generation was estimated as release of H2O2 using the AR/HRP detection system. In short, aliquots of 200 μL of a PC12 cells suspension in PBS medium (2 μg of protein) were added to a 96-well plate. PBS medium contained 5 μm AR plus 2 U/mL HRP.

ROS release under hypoxia

Microplates (96-wells) were supplied with an aliquot of 0.2 mL of a PC12 cell suspension in PBS medium. PC12 cells were either loaded with MitoSOXred or suspended in PBS medium containing AR and HRP. Thereafter, microplates were put into a CO2-incubator C200 (Labor-Technik-Göttingen, Göttingen, Germany) and kept under 6% CO2, 93% N2, and 1% O2 at 37°C for 4 h. Formed resorufin or oxidized MitoSOXred was measured fluorimetrically.

Mitochondria were suspended in nitrogen-saturated incubation medium (0.05 mg/mL) supplemented with AR plus HRP. Microplates (96-wells) were supplied with an aliquot of 0.2 mL of the mitochondria-containing incubation medium and substrates under nitrogen-atmosphere, and thereafter were kept under 6% CO2, 93% N2, and 1% O2 at 37°C, for 1 h. Finally, at the end of the incubation period, aliquots (50 μL) of a solution of potassium cyanide (KCN; 100 mm) were added to the wells containing the incubation mixture. This addition immediately inactivates the AR/HRP detection system by an irreversible inhibition of the hem protein HRP. Control incubations were performed at normoxia.

Chemicals

DMEM was from Invitrogen (Carlsbad, CA, USA); PBS, Hank’s balanced salt solution, fatty acids, carbonyl cyanide 4-trifluoro-methoxyphenylhydrazone (FCCP), rotenone, antimycin A, Cu,Zn-superoxide dismutase, HRP, diphenylene iodonium (DPI), acetylsalicylic acid (ASA), NS328, lactate dehydrogenase, NAD+, ADP, and carboxyatractyloside (CAT) were from Sigma-Aldrich Chemie GmbH (Sternheim, Germany). AR and MitoSOXred were from Invitrogen (Eugene, OR, USA). Apocynin (Apo) was from Roth (Karlsruhe, Germany). LDH-cytotoxicity detection kit was from Roche Applied Science (Indianapolis, IN, USA).

Data analysis

Data are given as mean values ± SD. Significance of changes was examined by the paired t-test or by the one way anova combined with the Tukey’s post hoc test. Statistical calculations were carried out using SigmaPlot 11.2 software (Systat Software, Erkrath, Germany).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Free fatty acids and cellular ROS generation

To examine the effect of FFA on cellular ROS generation, the release of H2O2 from PC12 cells into the medium was recorded over a 6 h incubation period. The polyunsaturated arachidonic (C20:4) or docosahexaenoic acid (Doco; C22:6) strongly enhance the H2O2 release, whereas the monounsaturated oleic acid (C18:1) or the saturated palmitic acid (C16:0) show negligible activity or are even completely inactive (Fig. 1). It has to be taken into consideration that at the incubation conditions used (glucose- or serum-deprivation), apoptotic cell death of PC12 cells is initiated (Greene 1978). In addition, there is evidence that the apoptotic cell death is associated with enhanced ROS generation (Hansson et al. 2008). Nevertheless, from the low ROS release by PC12 seen in the absence of polyunsaturated fatty acids (PUFA) (Fig. 1), we conclude that there is no significant contribution of apoptosis of PC12 cells to the ROS release during the 6-h incubation period.

image

Figure 1.  Time-dependent H2O2 release by PC12 cells exposed to fatty acids. PC12 cells suspended in PBS (cell content corresponds to 10 μg protein/mL) were treated with various free fatty acids (50 μm) at 25°C. H2O2 release into the PBS was detected by the AR/HRP detection system (see Materials and methods). Resorufin fluorescence was recorded over a 6-h incubation period using a microplate reader (TECAN). Traces show one of three representative experiments.

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For getting insight into the cellular sources of ROS generation, PC12 cells were treated simultaneously with arachidonic acid plus inhibitors of the cell membrane-associated NADPH oxidase (apocynin, Apo; DPI) or inhibitors of arachidonic acid metabolism (ASA; NS328). Apo prevents the assembly of the active NADPH oxidase from its cytosolic and membrane subunits, whereas DPI acts as a broad-range inhibitor of flavoproteins (Hart et al. 1990; Qian et al. 2007). DPI did not significantly affect the H2O2 release in the presence of arachidonic acid (Fig. 2) suggesting NADPH oxidase-independent mechanisms. The decrease of cellular H2O2 release seen with Apo is because of a concentration-dependent desensization of the AR/HRP detection system by Apo (not shown). Thus, the NADPH oxidase is unlikely to be activated by arachidonic acid. Furthermore, we also examined a contribution of a conversion of arachidonic acid by the cyclooxygenase 1 (COX1; using the inhibitor ASA, affecting COX1 and COX2) or the inducible COX2 (using the sulfonanilide-type inhibitor NS328, affecting more selectively COX2) as potential source of the cellular ROS generation (Simmons et al. 2004). Also with these inhibitors, no decline of the arachidonic acid-induced ROS generation was seen (Fig. 2). The cause for the apparent, slight stimulation of the ROS release in the presence of ASA or NS328 is not clear.

image

Figure 2.  Analysis of non-mitochondrial sources in the arachidonic acid-stimulated H2O2 release from PC12 cells. PC12 cells suspended in PBS (cell content corresponds to 10 μg protein/mL) were treated with arachidonic acid (Ara; 50 μm) at 25°C. During incubation, the following inhibitors were added: 50 μm Apo (cell membrane-associated NADPH oxidase), 50 μm DPI (cell membrane-associated NADPH oxidase); 100 μm ASA, and 100 μm NS328. Data represent mean values obtained from several cell preparations (n = 3–6) and reflect released H2O2 during a 5 h incubation period. H2O2 release is expressed as percent of the inhibitor-free incubation caused by Ara (corresponding to 15 502 ± 1638 a.u./5 h/2 μg of protein). *Significantly different from the Ara-containing, inhibitor-free incubation (anova, Tukey’s post hoc test, < 0.05).

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As shown in Fig. 3, FFA-induced ROS generation was found to be paralleled by an enhanced lactate release. This suggests a partial inactivation of the enzymatic activity of the mitochondrial ETC, thereby increasing in compensation the glycolytic lactate generation.

image

Figure 3.  Free fatty acids stimulate lactate release from PC12 cells. PC12 cells suspended in PBS (supplemented with 5 mm pyruvate plus 5 mm malate) were treated with various FFA (50 μm) or antimycin A (AA; 5 μm) for 1 h at 37°C. The cell content corresponds to about 0.9 mg protein/mL. Released lactate was measured in the supernatant. Lactate release in the absence of FFA (100%) was 2.4 ± 0.3 μmol/mL PBS/30 min. Data represent mean values ± SD obtained from four separate incubations. *Significantly different from the fatty acid free-control incubation (anova, Tukey’s post hoc test, p < 0.05).

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Free fatty acids and mitochondrial ROS generation

To directly examine whether FFA stimulate mitochondrial ROS generation, mitochondria were isolated from PC12 cells. First, the response of the oxygen uptake of mitochondria isolated from PC12 cells (PC12mito; oxidizing pyruvate plus malate) to various effectors of the oxidative phosphorylation was measured. A typical experiment is shown in Fig. 4 A. This indicates that mitochondria respond to ADP with stimulation of the oxygen uptake, whereas CAT, known as potent inhibitor of the adenine nucleotide translocase, blunted the ADP-dependent oxygen uptake. Oxygen uptake was reactivated by several additions of the protonophoric uncoupler FCCP, whereas the inhibitor of cytochrome c oxidase KCN completely blocked oxygen uptake. Since mitochondrial ROS generation depends on the electrochemical proton gradient, the membrane potential was estimated by determining the accumulation of the cationic safranine by energized PC12mito (oxidizing pyruvate plus malate). PC12mito rapidly accumulate safranine, as indicated by the quenching of the safranine-linked fluorescence (Fig. 4b). Furthermore, the stimulation of the oxidative phosphorylation by addition of ADP partially depolarized the inner membrane. CAT reversed this depolarization. In addition, FCCP or KCN completely depolarized PC12mito. These findings taken together reveal that mitochondria isolated from PC12 cells were functionally intact.

image

Figure 4.  Response of the oxygen uptake and of the Δψ-dependent safranine accumulation of mitochondria to effectors of the oxidative phosphorylation. (a) Oxygen uptake. Mitochondria (0.4 mg of protein) were added to 1.5 mL of the incubation medium. Rate of the oxygen uptake with ADP and with ADP plus CAT was 67 and 21 nmol O2/min/mg of protein, respectively. (b) Δψ-dependent safranine accumulation. Mitochondria (0.2 mg of protein) were added to 860 μL incubation medium. Addition of ADP moderately depolarizes the inner mitochondrial membrane (indicated by the increase of safranine fluorescence). A subsequent addition of CAT reverses this depolarization. Both, FCCP (protonophoric uncoupler) and KCN (cytochrome c oxidase inhibitor) cause collapse of the membrane polarization. Fluorescence change by unspecific, Δψ-independent binding of safranine is indicated. Unspecific binding prevents the complete release of safranine from mitochondria after total depolarization. Substrate and added compounds: pyruvate plus malate (5 mm, 5 mm), ADP (1 mm), CAT (5 μm), FCCP (0.05 μm per addition), KCN (10 mm).

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As a next step, the mitochondrial H2O2 release in response to FFA was examined in mitochondria-oxidizing pyruvate plus malate. We used the AR/HRP detection system (Fig. 5). H2O2 release is dose-dependently enhanced when mitochondria were exposed to Doco (Fig. 5a). For mimicking the uncoupling effect of fatty acids, FCCP was applied. In contrast to Doco, FCCP does slightly decrease the basal H2O2 release. Furthermore, mitochondrial ROS release was strongly increased by both PUFA tested (arachidonic acid and Doco), whereas palmitic and oleic acid were nearly inactive (Fig. 5b). PUFA-linked increase of the ROS release might be caused by a partial inactivation of the ETC (Cocco et al. 1999; Schönfeld and Reiser 2006; Schönfeld and Wojtczak 2007). To examine this possibility, the enzymatic activity of the NADH-cytochrome c reductase (complexes I and III) was assessed. Indeed, in comparison with untreated control mitochondria, the activity of the NADH-cytochrome c reductase is significantly lowered in the presence of PUFA (Fig. 6).

image

Figure 5.  Polyunsaturated free fatty acids enhance mitochondrial H2O2 release. Mitochondria suspended in incubation medium (0.4 mg of protein/mL) supplemented with pyruvate plus malate (5 mm/5 mm). (a) Effect of indicated concentrations of Doco on the H2O2 release, recorded with the AR/HRP detection system. The concentration of 40 μm corresponds to 100 nmol per mg of protein. Concentration of FCCP was 0.1 μm. Numbers at the traces indicate rates of H2O2 release expressed as pmol H2O2/min/mg of protein. (b) Rates of the H2O2 release, obtained with FFA at a concentration of 40 μm, corresponding to 100 nmol per mg of protein. Data represent mean values ± SD obtained from 6 to 8 separate mitochondrial preparations. *Significantly different from the fatty acid free-control incubation (anova, Tukey’s post hoc test, p < 0.05).

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image

Figure 6.  Interdependence of the H2O2 release and the NADH-cytochrome c reductase activity. Data of the H2O2 release (Fig. 5) are plotted versus the NADH-cytochrome c reductase activity. Mitochondria were incubated for 2 min in incubation medium without and with 100 nmol FFA/mg of protein at 37°C. Enzymatic activity (mean ± SD) was measured using mitochondria, which were permeabilized by a threefold freezing/thawing cycle (five separated preparations). *Significantly different from the fatty acid free-control incubation (anova, Tukey’s post hoc test, p < 0.05) are rates of ROS generation and enzymatic activities measured in the presence of Ara and Doco.

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Finally, we examined the effect of arachidonic acid on the mitochondrial ROS generation in situ. For this purpose, PC12 cells were loaded with the O2−˙-sensitive mitochondria-targeted dye MitoSOXred, and the mitochondrial O2−˙ generation was microscopically monitored (Figure S1). In the absence of arachidonic acid, MitoSOXred fluorescence was only marginal, indicating that there was only minor mitochondrial ROS production. However, when MitoSOXred-loaded PC12 cells were treated with arachidonic acid (20 μm) or the complex III-inhibitor antimycin A, fluorescence of the MitoSOXred probe was markedly enhanced.

ROS generation and hypoxia

To study the effect of severe hypoxia (1% of O2) on the mitochondrial ROS generation, mitochondria isolated from PC12 cells were suspended in incubation medium equilibrated with a low oxygen pressure. The medium was supplemented with AR/HRP as detection system of H2O2. Under this condition, mitochondria were energized either with pyruvate plus malate or succinate (complex II-substrate). With pyruvate plus malate alone, generally, low basal O2−˙ production is observed. To stimulate O2−˙ production by mitochondria-oxidizing pyruvate plus malate, the incubation mixture was supplemented with antimycin A. Succinate, in contrast, is known to initiate high, complex I-associated O2−˙ production in mitochondria from rat tissues, caused by the reversed electron transport from complex II to complex I (Murphy 2009). All incubations were carried out for 1 h at hypoxia at 37°C and, for comparison, at normoxia (21% of O2). After inactivation of the H2O2 detection system (AR/HRP) with cyanide, the formed resorufin was measured fluorimetrically. Figure 7 shows that similar amounts of resorufin were formed by mitochondria oxidizing pyruvate plus malate at normoxia as well as hypoxia. With pyruvate/malate, the resorufin fluorescence increased considerably in the presence of antimycin A, but only at normoxia. The absence of stimulation at hypoxia might be attributed to the fact that the O2 concentration is limiting in hypoxia. Furthermore, unexpectedly, succinate-oxidizing mitochondria from PC12 cells generate also low resorufin fluorescence, both at normoxia as well as hypoxia. Taken together, these observations clearly indicate that isolated mitochondria do not respond with a change in the ROS generation to a low oxygen pressure.

image

Figure 7.  H2O2 release from PC12mito at normoxia and hypoxia. PC12mito were suspended in incubation medium (0.05 mg protein/mL) supplemented with AR/HRP. PC12mito were energized with pyruvate (P; 5 mm) plus malate (M; 5 mm), or succinate (Succ, 10 mm). Antimycin A (AA) was applied at 5 μm. Incubation mixtures were kept for 1 h at normoxia (21% O2) or hypoxia (1% O2) at 37°C. Thereafter, the formed resorufin was measured after inactivation of HRP by addition of KCN solution (final concentration 5 mm). The data shown are mean values ± SD from four experiments. *Significantly different from H2O2 release at normoxic incubation (with P/M) without antimycin A (anova, Tukey’s post hoc test, p < 0.05).

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To examine whether there is a modulatory effect of FFA at hypoxia (1% of O2) on the cellular ROS generation, PC12 cells were exposed to FFA (50 μm) for 4 h at 37°C. Control incubations were carried out at normoxia. ROS generation was measured as H2O2 release. With respect to normoxia, basal control H2O2 release by PC12 cells (without FFA) was about 30% higher in hypoxia (Fig. 8). A comparable increase was seen in the presence of the FFA.

image

Figure 8.  Stimulation of H2O2 release from PC12 cells by free fatty acids at normoxia and hypoxia. Suspensions of PC12 cells (cell content corresponds to 10 μg/mL) in PBS supplemented with AR/HRP were kept at 37°C under normoxia (21% O2) or hypoxia (1% O2). Additions were 50 μm FFA or 5 μm antimycin A (AA). After an incubation period of 4 h, the fluorescence of formed resorufin was measured. Data show mean values ± SD from four experiments. *Significantly different from the normoxic control incubation (one-way anova, Tukey’s post hoc test, p < 0.05). **Significantly different from hypoxic control incubation (one-way anova, Tukey’s post hoc test, p < 0.05). ***Significant difference between the normoxic and hypoxic control incubations (paired t-test; p < 0.001).

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Finally, we determined the in situ basal mitochondrial ROS level in control conditions and in response to oleic, arachidonic acid and Doco in normoxia and hypoxia. In these experiments, mitochondrial matrix O2−˙ was assayed using MitoSOXred. The basal control matrix O2−˙ levels are not significantly different when measured either in hypoxia or in normoxia (Fig. 9). This finding is in contrast with the H2O2 release by PC12 cells, as shown above in Fig. 8. A possible explanation for this discrepancy is given when the topology and sidedness of ETC-linked O2−˙ release is considered (see for recent review Brand 2010). Complex III releases O2−˙ to both sides of the inner membrane, and complex III is probably the main site of the O2−˙ generation in PUFA-impaired mitochondria (Stewart et al. 2000; Schönfeld and Wojtczak 2007). The latter view is supported by the reciprocal correlation between complex III-linked ROS generation and the enzymatic activity of complex III that we found with PUFA-treated mitochondria (Schönfeld and Wojtczak 2007). Furthermore, long-chain FFA interact strongly with cytochrome c (Stewart et al. 2000), which might reduce its ability to shuttle electrons from complex III to complex IV. Consequently, there is good reason to speculate that O2−˙ is mostly released from complex III into the intermembrane space in PUFA-impaired mitochondria. In this case, MitoSOXred has diminished access to O2−˙ and, consequently, mitochondrial ROS generation is only partly detected with MitoSOXred.

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Figure 9.  Effect of free fatty acids on the superoxide level in the mitochondrial matrix of PC12 cells at normoxia and hypoxia. Suspensions of PC12 cells (cell content corresponds to 10 μg/mL) in PBS supplemented with MitoSOXred, were kept at 37°C under normoxia (21% O2) or hypoxia (1% O2). Additions were 50 μm FFA, 5 μm AA. After an incubation period of 4 h, the fluorescence of oxidized MitoSOXred was measured. Data show mean values ± SD obtained from four experiments. *Significantly different from the normoxic control incubation (one-way anova, Tukey’s post hoc test, p < 0.05). **Significantly different from the hypoxic normoxic control incubation (one-way anova, Tukey’s post hoc test, p < 0.05).

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Moreover, importantly, both arachidonic acid and Doco induce not only in normoxia but even under low oxygen pressure (1%), a significant increase in the mitochondrial ROS generation as compared with the control levels (Fig. 9).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This study addresses the following fundamental question: Is the hypoxia-associated cellular ROS generation caused by an increased mitochondrial ROS generation? Here, we report that PC12 cells equilibrated with a low oxygen concentration of 1% O2, corresponding to approximately 10 μm of O2 in the incubation medium (Reynafarje et al. 1985), show an increased H2O2 release (Fig. 8). According to a widely accepted view of hypoxic cell signaling, mitochondrial ROS generation is increased in hypoxia (see for review Hoffman et al. 2007; and references therein). It is tempting to speculate that this enhanced ROS generation is connected to the low O2 concentration at hypoxia, because this condition is likely to limit the electron flux within the ETC and thereby facilitate the electron transfer reactions from complex I and/or complex III to molecular O2. However, the half-maximal O2 concentration for phosphorylating mitochondrial respiration is about 1 μm (Wilson et al. 1988). Thus, at 1% O2, respiration proceeds at 91% of the maximal rate. Consequently, a severe limitation of the electron flux within the ETC by this O2 concentration is unlikely to be the reason for the enhanced cellular ROS generation. Indeed, in mitochondria, which were isolated from PC12 cells, kept under 1% of O2 pressure, the mitochondrial H2O2 release was not changed as compared with an O2 concentration of 21% (Fig. 7). Thus, our observation agrees with the view that the higher ROS generation by cells at severe hypoxia is not intrinsic to the mitochondrial respiratory chain alone (Hoffman et al. 2007; Hoffman and Brookes 2009). Hence, there is good reason to take into consideration additional factors acting on mitochondrial and/or non-mitochondrial ROS generating sites in hypoxia (see also discussion in Hoffman et al. 2007; Hoffman and Brookes 2009).

Notably, hypoxic or ischemic episodes are characterized by an increase in the FFA tissue levels, particularly those of arachidonic acid and Doco (Van der Vusse et al. 1992; Sun and Gilboe 1994; Michiels et al. 2002). That is the reason why we particularly evaluated the effect of FFA as potential stimulators of mitochondrial and non-mitochondrial ROS production. Indeed, the exposure of PC12 cells to arachidonic acid and Doco significantly enhances the H2O2 release from PC12 cells under normoxia (Fig. 1). The very pronounced stimulation found with PUFA supports the idea that non-mitochondrial sources of O2−˙ generation, such as the membrane-associated NADPH oxidase and enzymes of the arachidonic acid metabolism (COX1 and 2), are activated by PUFA. However, the FFA-dependent ROS generation does obviously not seem to be related to sources of the non-mitochondrial O2−˙ generation. The overall H2O2 release is not affected by inhibitors of these enzymes (Fig. 2). Interestingly, these enzymes were also disregarded as O2−˙ source in the arachidonic acid-induced ROS generation in other cells, for example, spermatozoa (Aitken et al. 2006).

FFA-induced H2O2 release by PC12 cells was paralleled by increased release of lactate (Fig. 3). This increase indicates activation of anaerobic glycolysis, which compensates for reduced respiration and oxidative phosphorylation. This finding could suggest that FFA stimulate cellular ROS generation because of impairing the ETC. Therefore, in mitochondria isolated from PC12 cells, we examined the effect of FFA on the H2O2 release as well as on the enzymatic activity of the ETC. We show that PUFA significantly enhance the mitochondrial H2O2 release at normoxia (Fig. 5). However, mimicking the uncoupling activity of FFA by FCCP had no significant effect on the basal H2O2 release. This observation further substantiates our concept that, depending on the direction of the electron flux within the ETC, FFA decrease ROS generation by uncoupling or increase ROS generation by an impairment of the ETC (Korshunov et al. 1998; Cocco et al. 1999; Schönfeld and Wojtczak 2007). Thus, FFA decrease ROS generation during the reversed electron transport, but increase ROS generation during the forward electron transport. In addition, the increase of the ROS generation by PUFA is paralleled by a partial inactivation of the NADH–ubiquinone oxidoreductase (Fig. 6). This parallelism suggests that the increase of the mitochondrial H2O2 release initiated by PUFA is because of a partial impairment of ETC. Inactivation of the enzymatic activity of the NADH–ubiquinone oxidoreductase results in an increased reduction of electron carriers, a condition which facilitates one-electron transfer reactions to O2. Indeed, FFA applied at concentrations used in this study are able to enhance the reduction of the matrix-NAD(P) pool (Schönfeld and Reiser 2006). Moreover, the oxidation of the mitochondria-targeted O2−˙ probe MitoSOXred in PC12 cells by PUFA (Fig. 9 and Fig. S1) confirms that elevated concentrations of these FFA stimulate the mitochondrial O2−˙ production in situ.

The key question is whether externally added FFA increase the ROS production by PC12 cells kept at low oxygen pressure. Indeed, PC12 cells kept under hypoxia (1% O2) respond to an exposure with the polyunsaturated Doco with a further increase in H2O2 release (Fig. 8). In addition, PUFA-associated ROS generation was also found with PC12 cells loaded with MitoSOXred (Fig. 9). This observation further supports the view that PUFA-induced mitochondrial ROS generation contributes to the ROS generation of PC12 cells exposed to hypoxia plus PUFA. Taken together, we attribute the PUFA-induced release of H2O2 from PC12 cells to an impaired ETC

Moreover, a crucial question is whether PUFA levels increase to such an extent under tissue hypoxia that the ETC becomes impaired. In normoxia, the tissue content of saturated and unsaturated fatty acids is low, but is dramatically enhanced at hypoxia. We can give the following examples: the FFA content increased from 250 to 4000 nmol/g dry weigth of heart tissue at the transition from normoxia to hypoxia (Van der Vusse et al. 1992). In addition, for brain tissue, it has been reported that the liberated FFA are highly enriched in mitochondria (≈ 12 μg/mg of protein; corresponding to ≈ 40 nmol arachidonic acid/mg of protein; Sun and Gilboe 1994). Finally, all reported values for liberation of fatty acids are underestimates, since liberated non-esterified fatty acids are partly converted into esterified derivatives, such as acyl-CoAs, acylcarnitines, and N-acylethanolamines (Hütter et al. 1990; Hansen et al. 2000). These fatty acid derivatives are also known to modulate mitochondrial ROS generation (Wasilewski and Wojtczak 2005; Tominaga et al. 2008).

Furthermore, it should be noted that obtaining rigorous comparative dose–response relationships in experiments with either isolated mitochondria or cells incubated with FFA is a very complex task. In incubations with mitochondria, the added FFA mostly accumulate in mitochondria. In contrast, in experiments with cells, FFA distribute between medium, plasma membrane, non-mitochondrial organelles, and mitochondria. Nevertheless, we can conclude that the exposure of PC12 cells to 50 μm of PUFA initiates an increased release of H2O2 (Fig. 8), and this release is associated with an increased mitochondrial O2−˙ generation (Fig. 9). This supports a corresponding cell-linked and mitochondria-linked ROS generation at a given PUFA concentration.

In summary, PC12 cells and their mitochondria kept under hypoxia plus an enhanced PUFA level respond with an increase in ROS release. This observation encourages us to postulate that an enrichment of PUFA in the inner mitochondrial membrane facilitates the escape of electrons from the ETC, thereby enhancing the release of ROS. Consequently, we hypothesize that PUFA can operate as an important extrinsic factor, which amplifies the ETC-linked ROS generation in cells under hypoxia.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The work was supported by a financial support of the Kultusministerium of Sachsen-Anhalt. We thank Heidi Goldammer for her excellent technical assistance. The authors declare no conflict of interest.

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  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Figure S1. Imaging of mitochondrial ROS production in situ. PC12 cells grown on glass cover slips were stained with MitoSOXred (5 μM) for 30 min. After washing (twice), cells were treated either with 20 μM arachidonic acid (Ara) or 5 μM antimycin A (AA; inhibitor of complex III) for 30 min. Thereafter, cells were washed again, and finally fixed with 1% paraformaldehyde in PBS. Left column: pseudo color image representing the red fluorescence of the oxidized MitoSOXred probe. Right column: overlay of the red fluorescence channel with the respective differential interference contrast images showing the cells.

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