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.
- Top of page
- Materials and methods
- 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.
- Top of page
- Materials and methods
- 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.
As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.