The Tricarboxylic Acid Cycle, An Ancient Metabolic Network ... - PLOS

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P. fluorescens and HepG2 cells were challenged respectively with menadione (O2− producer), hydrogen peroxide, and various toxic metals known to promote oxidative stress. Following treatment of these cells with these toxicants, oxidized lipids and proteins in P. fluorescens and HepG2 cells was analyzed. Marked increments in oxidized lipids and proteins were evident in P.fluorescens and HepG2 cells exposed to Al, Ga, Zn, and menadione compared to controls (Table 1 and Table 2). The oxidative properties of these metals were also confirmed by measuring H2O2 formation. Indeed the cell free extract (CFE) isolated from P.fluorescens exposed to Al, Ga, or menadione produced more H2O2 when incubated in 5 mM citrate (Table 3). Similar experiments were performed with HepG2 cells exposed to Al. Dichlorofluorescein-diacetate analysis revealed a higher level of intracellular ROS in HepG2 cells exposed to Al (data not shown). Following the establishment of the oxidative damage suffered by the cells exposed to these toxicants, it was important to evaluate how cellular metabolism was affected under these conditions. P. fluorescens and HepG2 cells exposed to metal toxicants and ROS-producing molecules accumulated succinate in the media, a biomarker for oxidative stress (data not shown) [13]. To probe the disparate metabolic profiles observed during oxidative stress further, cell-free extracts from P. fluorescens and HepG2 cells exposed to labelled citrate were analyzed by 13C-NMR and HPLC.

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Table 1. Oxidized lipid and protein profile in P. fluorescens exposed to oxidative and metal stress

https://doi.org/10.1371/journal.pone.0000690.t001

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Table 2. Oxidized lipid and protein profile in HepG2 cells exposed to metal stress

https://doi.org/10.1371/journal.pone.0000690.t002

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Table 3. H202 concentrations in P. fluorescens exposed to oxidative and metal stress

https://doi.org/10.1371/journal.pone.0000690.t003

The first evidence for the intriguing role of the TCA cycle in modulating oxidative tension was obtained when Ga-citrate was incubated with the CFE (cell-free extract) from P. fluorescens. 13C-NMR chemical shifts at 32 ppm and 181 ppm attributable to the CH2 and COO− of succinate were evident (Figure 1A). On the other hand, the diagnostic fingerprints indicative of KG were present in the CFE with citrate as the substrate (Figure 1A). No succinate peaks were evident. As NAD was the only exogenous cofactor utilized, KG was an important metabolite generated via the enzyme ICDH. However, in the presence of either Al or Ga, two metals known to generate ROS [14]–[16], succinate was also produced. The inclusion of catalase prior to the addition of the metal-citrate complex provided KG peaks only. The labelling pattern of 13C peaks would eliminate the production of succinate via isocitrate lyase (ICL). If this enzyme was involved, only a peak at 32 ppm indicative of the CH2 would have been present. Furthermore, the same diagnostic peaks were obtained in the presence of malonate (5 mM), a potent inhibitor of ICL (Figure S1). Thus, it appears that succinate was a product of the decomposition of KG by the ROS generated by Ga. Similarly, cells obtained from the Al and menadione media respectively did readily yield the succinate signal upon incubation with labelled citrate (Figure S2). Hence, the 13C-NMR data pointed to a metabolic shift promoting the detoxification of ROS in P. fluorescens subjected to Al, Ga or menadione. Studies performed with HepG2 cells exposed to Al, a pro-oxidant, also revealed the accumulation of KG and succinate. HPLC analyses of the control and Al-stressed HepG2 cells revealed the marked accumulation of both metabolites in cytosol and mitochondria of the Al-treated cells (Figure 1, Panel B). Treatment of control cells with Al-citrate for 24h confirmed the observed accumulation of KG and succinate during oxidative tension. In addition treatment of Al-stressed HepG2 cells with 5 mM KG for 24h encouraged the cytosolic and mitochondrial accumulation of succinate (Figure 1, Panel B). Thus these observations indicate that the oxidative insult provoked by Al toxicity encouraged the accumulation of KG and succinate, an end product of KG-mediated detoxification of ROS. To further confirm the mitochondrial accumulation of KG and succinate in Al-treated cells, mitochondria were treated for 1h with citrate and NAD. The mitochondria from the Al-stressed cells accumulated more KG and succinate following citrate treatment as opposed to control mitochondria (Figure 1, Panel C). In addition exposure of Al-stressed HepG2 cells with 10 mM 13C-labelled citrate confirmed the observed accumulation of succinate (Figure 1, Panel D). To confirm the antioxidant properties of KG, membrane fractions from control and Al-stress P. fluorescens were incubated in KG and H2O2. In contrast to the control fractions KG was poorly metabolized in the reaction mixture containing Al-treated membranes and the KG was strictly dedicated to the detoxification of H2O2 as indicated by the presence of a succinate peak (Figure 2). The inclusion of catalase in the Al-stressed reaction mixture seemed to ablate the antioxidant properties of KG as indicated by the lowered succinate signal (Figure 2). Thus, it became obvious that KG was an important component of the ROS detoxification strategy in these systems.

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Figure 1. Metabolite analyses in Pseudomonas fluorescens and in HepG2 cells.

Panel A: 13C-NMR analyses of CFE from Pseudomonas fluorescens grown in a defined medium with citrate as the sole carbon source.

I) Ga-13C2-2,4-citrate and NAD as substrates

II)13C2-2,4-citrate and NAD as substrates

Panel B: Analysis of α-ketoglutarate (open bars) and succinate (crossed bars) in the cytosol and mitochondria of HepG2 cells exposed to control (A), Al-citrate (B), Al-treated cells exposed to 5 mM KG (C), and control cells exposed to Al-citrate (D). Samples were treated treated with perchloric acid and injected into the HPLC. The peaks were manually quantified using EMPOWER software. 100% corresponds to the absorbance value for KG and succinate peaks in the control cytosol and mitochondria (cytosol: 100% for KG and succinate is equivalent to an absorbance value of 0.0015 and 0.0012. Mitochondria: 100% for KG and succinate is equivalent to an absorbance value of 0.002 and 0.003). n = 3, mean±S.D., p≤0.05. Panel C: Representative chromatographs showing the consumption of citrate in HepG2 mitochondria. Mitochondria were isolated following a 24 h exposure to I) citrate and II) Al-citrate. Mitochondria were incubated for 1h at 37°C in a phosphate reaction buffer consisting of 1 mM citrate and 0.1 mM NAD. Top right corner: the purity of the mitochondrial fraction was assessed by the immunodetection of VDAC, F-actin, and H2A (note: for all NMR and HPLC experiments the purity of the fractions was assessed prior to the experiment). Panel D: Proton-decoupled 13C-NMR spectra obtained from the incubation of whole HepG2 cells with 10 mM 13C2-2,4-citrate for 24 h. HepG2 cells were exposed to I) citrate and II) Al-citrate for 24h and isolated for the 13C-NMR analysis.

https://doi.org/10.1371/journal.pone.0000690.g001

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Figure 2. Analysis of the KG mediated detoxification of H2O2.

1 mg/ml equivalent of membraneous protein from control and Al-stressed P. fluorescens was reacted for 15min in the following conditions: I) control protein+5 mM KG+5 mM H2O2, II) Al-stressed protein+5 mM KG+5 mM H2O2, III) Al-stressed protein+5 mM KG+5 mM H2O2+10units of catalase, and IV) control protein+5 mM KG+5 mM H2O2+50 µM malonate+50 µM NaN3. The reaction mixtures were then treated accordingly for HPLC. Time zero measurements and reactions in the absence of protein were performed to assure the peak specificity.

https://doi.org/10.1371/journal.pone.0000690.g002

These findings prompted us to probe the activity and expression of the key enzymes involved in the homeostasis of this keto acid, namely KGDH, NADP-ICDH, and NAD-ICDH. When P. fluorescens was exposed to Ga, Al, Fe, H2O2 or menadione, all known to create an oxidative environment, the activity of NADP-ICDH was increased while the activities of KGDH and NAD-ICDH were markedly decreased. Compared to the controls, a 3-fold reduction in KGDH activity was observed in a Ga-stressed medium. However in a Ca-citrate culture, a metal not known to perturb the redox environment, the activity of this enzyme was similar to that observed in the control cultures (Figure 3). Similarly, NADP-ICDH activity was higher in a menadione medium. At least a 2-fold increase compared to the control was recorded (Figure 3). This situation was reversed when these cells were transferred to a control medium (Figure S3). Irrespective of the source of carbon, this NADPH-generating enzyme was more active while the NADH producing counterpart and KGDH were less active in the cells subjected to an oxidative stress.

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Figure 3. Activities of KGDH, NADP-ICDH, and NAD-ICDH in P. fluorescens grown in various media.

KGDH activity was determined by incubating the membrane CFE (0.2 mg/mL protein equivalents) with 0.3 mM KG, 0.5 mM Coenzyme A and 0.5 mM NAD. Dinitrophenylhydrazone was monitored [38]. NADP-ICDH activity was determined by incubating soluble CFE (0.2 mg/mL protein equivalents) with 2 mM isocitrate, 4 mM malonate and 0.5 mM NADP. Formation of NADPH was monitored at 340 nm (ε = 6220 M-1 cm-1). For NAD-ICDH activity, the membrane CFE was utilized and NAD was the cofactor. In some instances, activities of these enzymes were also obtained by quantifying activity bands using Scion Imaging Software for Windows (SCION corperation, Frederick, MD). All activities are expressed as a percent of the control (100% corresponds to corresponds to 40 nmol.min-1.mg protein-1 for KGDH, 55 nmol.min-1.mg protein-1 for NAD-ICDH, and 55 nmol.min-1.mg protein-1 for NADP-ICDH). Crossed bar: KGDH, open bar: NADP-ICDH, and solid bar: NAD-ICDH. Values are mean±SD, n = 3, p≤0.05.

https://doi.org/10.1371/journal.pone.0000690.g003

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE), 2D SDS-PAGE and immunoblot assays helped establish the relationship between activity and protein expressed. P. fluorescens grown in control, metal stress, and pro-oxidant media revealed the negative impact of the metal/oxidative stress on KGDH activity (Figure 4, Panel A). To establish if the TCA cycle was indeed an integral component of the cellular machinery involved in defending the organism against ROS, glucose and malate were utilized as the sole carbon sources respectively (Figure 4, Panel A). And, when the cells were exposed to oxidants like H2O2 and menadione, a significant decrease in KGDH activity was observed. The ability of a pro-oxidative environment to inhibit KGDH was further confirmed by two dimensional and immunoblot analysis of P. fluorescens grown in citrate or Ga-citrate containing media (Figure 4, Panel B and C). When Ga-stressed cells were introduced into citrate control media a significant increase in KGDH activity was observed (Figure 4, Panel D). Similarly a decrease in KGDH activity was evident upon the introduction of control cells into the Al containing media (Figure 4, Panel D). As KGDH is known to be a producer of ROS, its diminished activity will lead to a marked reduction of these oxidants [17]. These results strongly suggest that the TCA cycle is an important metabolic network utilized by organisms to survive an oxidative environment. In HepG2 cells, a decrease in KGDH activity was also observed, however there did not appear to be a significant change in expression of this dehydrogenase (Figure 4, Panels E and F). Since oxidative stress diminished the ability of KGDH to produce NADH, we decided to probe the activity and expression of other NADH producing enzymes in particular NAD-ICDH. NAD-ICDH displayed a marked decrease in P. fluorescens and HepG2 cells exposed to metal and oxidative stress (Figure 5). HPLC studies confirmed the alterations in nicotinamide dinucleotide metabolism as a result of oxidative stress. Bacteria and HepG2 cells exposed to menadione, Al, and Ga displayed higher NADP(H) and lower NAD(H) levels when compared to control cells (data not shown). The net impact of this concerted metabolic reconfiguration led to a dramatic decrease in NADH, a major contributor to the production of ROS in vivo. On the other hand, the overexpression of NADP-ICDH resulted in increased NADPH, a critical modulator of the cellular redox potential and KG for ROS detoxification (Figure 6). Indeed, increased activity and expression of the NADP-dependent enzyme was recorded in the soluble fraction from the bacterial cells (Figure 6, Panel A). The increased activity was attributed to the emergence of an isozyme at the upper part of the gel (Figure 6, Panel A represented by I). In addition increased NADP-ICDH activity was also recorded in the membrane components from P. fluorescens (Figure 6, Panel B). The mitochondrial extracts from the HepG2 cells exposed to oxidative stress also exhibited higher activity and expression of the NADP ICDH (Figure 6, Panel C-E). Immunofluorescence experiments confirmed the increased expression of the NADP-dependent mitochondrial ICDH in the HepG2 cells exposed to Al (Figure 7). Hence, elevated levels of NADPH, KG and decreased levels of NADH may contribute to the survival of an organism in an oxidative environment. Furthermore, the downstream enzyme succinate dehydrogenase (SDH) also displayed lowered activity and expression in the stressed conditions while malate dehydrogenase (MDH) did not show any significant change in activity (Figure 8).

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Figure 4. In-gel activity staining of KGDH.

Panel A: Membrane CFE from P. fluorescens grown in various conditions (1–9) were analyzed. Lane 1: citrate medium, Lane 2: Ga-stressed medium, Lane 3: Al-stressed medium, Lane 4: malate medium; Lane 5: Al-malate medium; Lane 6: H2O2-stressed medium, Lane 7: O2−-stressed medium, Lane 8: glucose medium, Lane 9: glucose/H2O2-stressed medium. Panel B: 2D BN-PAGE activity staining of KGDH. Lane 1: membrane CFE from P. fluorescens grown in citrate medium. Lane 2: in Ga-citrate medium. Panel C: The bands from lanes 1 and 2 in Panel A were excised and analyzed by 2D immunoblot. Lane 1: citrate medium. Lane 2: Ga-citrate. Panel D: Modulation of KGDH activity in membrane CFE isolated from P. fluorescens grown in different media. Lane 1: P. fluorescens grown in citrate medium. Lane 2: Ga-citrate medium, Lane 3: Al-stressed cells introduced into citrate medium, Lane 4: citrate cells introduced into Al-citrate medium. Panel E: In-gel detection of mitochondrial KGDH in HepG2 cells. Lane 1: citrate, Lane 2: Al-citrate, Lane 3: Zn-citrate, and Lane 4: citrate and H2O2. Panel F: Immunoblot analysis of KGDH expression in HepG2 mitochondria. Lane 1: citrate, Lane 2: Al-citrate, Lane 3: Zn-citrate, and Lane 4: citrate and H2O2.

https://doi.org/10.1371/journal.pone.0000690.g004

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Figure 5. Activity and expression of NAD-ICDH in P. fluorescens membrane CFE and HepG2 mitochondria.

Panel A: I) 2D PAGE activity stain for NAD-ICDH in P. fluorescens membrane CFE from Lane 1: citrate medium and Lane 2: Al-citrate medium. II) Activity bands were excised and detected by silver staining. Lane 1: citrate and Lane 2: Al-citrate. Panel B: In-gel NAD-ICDH activity in membrane CFE isolated from P. fluorescens grown in different media. Lane 1: citrate medium, Lane 2: Fe-citrate medium, Lanes 3-7: citrate media containing 0.1, 1.0, 5.0, 10.0 and 15.0 mM Al, respectively. Panel C: NAD-ICDH activity in the mitochondrial extract from HepG2 cells grown in Lane 1: citrate, Lane 2: Al-citrate, Lane 3: Zn-citrate, and Lane 4: H2O2 and citrate conditions.

https://doi.org/10.1371/journal.pone.0000690.g005

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Figure 6. The detection of NADP-ICDH/NAD-ICDH in the soluble and the membrane CFE from P. fluorescens and the mitochondria from HepG2 cells.

Panel A: BN-PAGE analysis of NADP-ICDH activity in soluble CFE isolated from P. fluorescens grown in various media. Lane 1: citrate, Lane 2: cells grown in Ga-citrate, Lane 3: control cells transferred into Ga-citrate media for 6 h, Lane 4: control cells transferred to medium with 1 mM menadione for 6h, and Lane 5: control cells transferred to medium with H2O2 (15 mM). I and II correspond to two bands with ICDH activities. Panel B: The membrane CFE isolated P. fluorescens grown in Lane 1: citrate, Lane 2: Al-citrate, and Lane 3: Ga-citrate media were tested for membrane NADP-ICDH and NAD-ICDH activity. Following the visualization of the NAD-ICDH activity band, the gel was treated with 2 mM isocitrate and 0.1 mM NADP. Panel C: HepG2 cells grown in Lane 1: citrate, Lane 2: Al-citrate, Lane 3: Zn-citrate, and Lane 4: citrate and H2O2 were analyzed for the presence of a mitochondrial NADP-ICDH. Following the detection of NAD-ICDH, NADP-ICDH bands were detected as in Panel A. Panel D: The NADP-ICDH activity bands from Panel C were excised and subject to 2D SDS-PAGE. Lane 1: citrate, Lane 2: Al-citrate, Lane 3: Zn-citrate, and Lane 4: citrate and H2O2. Panel E: 2D immunodetection of NADP-ICDH in HepG2 mitochondria isolated from HepG2 cells grown in Lane 1: citrate, Lane 2: Al-citrate, Lane 3: Zn-citrate, and Lane 4: citrate and H2O2 conditions.

https://doi.org/10.1371/journal.pone.0000690.g006

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Figure 7. Immunofluorescent studies of mitochondrial NADP-ICDH in HepG2 cells subjected to oxidative stress.

HepG2 cells were grown to a minimal density on coverslips and exposed to I) citrate and II) 0.5 mM Al-citrate for 24 h. The fluorescein isothiocyanate tagged secondary antibody (green) was used to visualize NADP-ICDH. The yellow fluorescence indicates the presence of NADP-ICDH in the mitochondria. Scale bar: 10 µm.

https://doi.org/10.1371/journal.pone.0000690.g007

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Figure 8. Determination of the activity and expression of SDH and MDH in P. fluorescens and the mitochondria of HepG2 cells.

Panel A: In-gel detection of SDH activity and expression in P. fluorescens grown in Lane 1: citrate and Lane 2-6: citrate media containing 0.1, 0.5, 5, 10, and 15 mM Al. Panel B: 2D immunodetection of SDH in P. fluorescens grown in Lane 1: citrate and Lane 2: Al-citrate. Lanes 1 and 6 from panel A were excised and analyzed. Panel C: In-gel detection of SDH activity in mitochondria of HepG2 grown in Lane 1: citrate and Lane 2: Al-citrate conditions. Panel D: Immunodetection of SDH in the mitochondria of HepG2 cells grown in Lane 1: citrate and Lane 2: Al-citrate conditions. Panel E: In-gel detection of MDH activity in the mitochondria of HepG2 cells grown in Lane 1: citrate and Lane 2: Al-citrate conditions (I = activity stain, II = 2D electrophoresis).

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