Lipoamide Acts as an Indirect Antioxidant by Simultaneously Stimulating Mitochondrial Biogenesis and Phase II Antioxidant Enzyme Systems in ARPE-19 Cells

In our previous study, we found that pretreatment with lipoamide (LM) more effectively than alpha-lipoic acid (LA) protected retinal pigment epithelial (RPE) cells from the acrolein-induced damage. However, the reasons and mechanisms for the greater effect of LM than LA are unclear. We hypothesize that LM, rather than the more direct antioxidant LA, may act more as an indirect antioxidant. In the present study, we treated ARPE-19 cells with LA and LM and compared their effects on activation of mitochondrial biogenesis and induction of phase II enzyme systems. It is found that LM is more effective than LA on increasing mitochondrial biogenesis and inducing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its translocation to the nucleus, leading to an increase in expression or activity of phase II antioxidant enzymes (NQO-1, GST, GCL, catalase and Cu/Zn SOD). Further study demonstrated that mitochondrial biogenesis and phase II enzyme induction are closely coupled via energy requirements. These results suggest that LM, compared with the direct antioxidant LA, plays its protective effect on oxidative damage more as an indirect antioxidant to simultaneously stimulate mitochondrial biogenesis and induction of phase II antioxidant enzymes.


Introduction
The dysfunction of retinal pigment epithelium (RPE) is strongly related to age-related macular degeneration (AMD) [1,2]. Oxidative stress has been proven as a key factor in AMD pathology by a number of studies and RPE is very susceptible to oxidative stress [3]. Since mitochondria IL). Polyclonal rabbit antibodies against PPARGC1a and Nrf2 were purchased from Santa Cruz Biotechnology Inc. (San Diego, CA); monoclonal antibodies for Complex I (NADH ubiquinone oxidoreductase 39-kDa subunit), Complex II (succinate-ubiquinone oxidoreductase 70-kDa subunit), Complex III (ubiquinol-cytochrome c oxidoreductase core II 50-kDa), Complex IV (cytochrome c oxidase 48-kDa) and Complex V (ATP synthase, 53-kDa), were from Molecular Probes (Eugene, OR); monoclonal antibodies for GCLc, catalase and GST were from Stressgen Biotechnologies Corporation (Victoria, British Columbia, Canada). TRIzol, primers and other reagents for cell culture were from Invitrogen (Carlsbad, USA). 2,3-Naphthalenedicarboxyaldehyde, buthionine sulfoximine (BSO), diethyl maleate and other common reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell culture and treatments
The human ARPE-19 cell line was obtained from Nancy J. Philp (Thomas Jefferson University, Philadelphia, PA) and was cultured according to her methods. ARPE-19 cells were cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. ARPE-19 cells were used within 10 generations. For all experiments, if not otherwise stated, cells were cultured to 60% density then treated with LM or LA for 48 h, and the cell density would grow to 90%-95% confluence. Then the cells were washed with PBS and collected for various assays.

Mitotracker staining for viable mitochondria
Cells were cultured in six-well plates. After LM treatment, cells were stained with 100 nmol/L Mitotracker Green in medium for 30 min; then the staining medium was discarded after centrifugation. Cells were resuspended with PBS, and fluorescence was detected with BD FACS Arisa [24].
Mitochondrial membrane potential (MMP) assay MMP changes in live ARPE-19 cells cultured in 96-well plates were determined by using the lipophilic cationic probe JC-1 [25] with a fluorescence spectrometer (Flex StationII 384, Molecular Devices). The fluorescence ratio (590 nm emission to 530 nm emission) was used for quantitative analysis.

Intracellular adenosine 5 0 -triphosphate (ATP)
Cells were cultured in six-well plates. After various treatments, cells were lysed by 0.5% Triton X-100 in 100 mM glycine buffer, pH 7.4. Intracellular ATP levels were assayed with an ATP bio-luminescence assay kit (Sigma) based on the luciferase-catalyzed oxidation of d-luciferin.

Oxygen consumption determination
Oxygen consumption was determined using the BD Oxygen Biosensor System (BD Biosciences). Generally, cells were cultured in 6-well plates, after indicated treatment cells were suspended in culture medium and subsequently transferred to the 96-well oxygen biosensor plate. The plate was tightly sealed to isolate outside atmosphere. The oxygen was consumed by the cells and the oxygen-sensitive fluorescence dye could emit fluorescence under excitation light. Levels of oxygen consumption were measured under baseline conditions. Fluorescence was recorded using a fluorescence microplate reader (Flex StationII 384, Molecular Devices) at 1-min intervals for 2 h at an excitation of 485 nm and emission of 630 nm [27][28][29]. Cell numbers were counted with a hemocytometer. The maximum slope of fluorescence (in fluorescence units/s) was measured and converted into percent of control (set to 100%).
Real time PCR for mitochondrial DNA quantification and mRNA expression assay Total DNA was isolated by a standard method with a DNA isolation kit (U-gene Biotech Co. Ltd, China). Total RNA was isolated with Trizol following a standard protocol. cDNA was obtained with Reverse Transcriptase from Toyobo Co. Ltd (Osaka, Japan) and oligo dT primers from Takara Co. (Dalian, China). Real-time PCR quantification was preformed using Master SYBR Green Premix and expressed relative to 18S rDNA or 18S rRNA level in the same sample [30]. For mitochondrial DNA copy numbers, D-LOOP region of the mitochondrial genome was selected for quantification and primers were designed as shown in Table 1. The ratio of D-LOOP to 18S rDNA was caculated as relative mitochondrial DNA copy number. Primers of Mn superoxide dismutase (MnSOD), Cu/ZnSOD, thioredoxin 2 (Trx 2), peroxiredoxin 3 (Prx 3), and peroxiredoxin 5 (Prx 5) were the same as described by Valle et al. [31]. The specificity of the primers was examined by both agarose gel electrophoresis and dissociation curve generation. All the primers and annealing conditions are shown in Table 1. All PCR amplification was performed using the Mx3000P system (Applied Biosystems, United States). Data were calculated using the formula 2 -ΔCt , where ΔCt = Ct target -Ct 18s [32].

Western blots for protein expression
Total protein or nuclear protein was isolated with a commercial lysis buffer (Pierce, Thermo Scientific) following standard instructions. Protein was frozen at -20°C before use. Protein concentrations were quantified by BCA assays (Pierce BCA Protein Assay Kit, Thermo Scientific), and samples were adjusted to the same concentration with lysis buffer. For each sample, the same quantity of protein (10 to 30 μg) was subjected to 10% or 12% SDS-PAGE, then proteins separated on the gel were transferred to a nitrocellulose membrane and blocked with 5% defatted milk/TBST for 1 h at room temperature. The membrane was incubated with primary antibodies at 4°C overnight. The primary antibodies were diluted in 5% defatted milk/TBST with After washing membranes with TBST three times, membranes were incubated with horseradish peroxidase-conjugated antibody for 1 h at room temperature. Western blots were developed using ECL (Roche Manheim, Germany) and quantitative analysis based on optical density was performed with Quantity One software.

Catalase activity assay
Catalase activity was measured with a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Generally, cells were lysed with lysis buffer and centrifuged at 13,000 g for 15 min at 4°C; the supernatant was collected for the test. The reaction system contained 50 mmol/L hydrogen peroxide; 5 min after adding 15 μg protein, the reaction was terminated; the remaining hydrogen peroxide was oxidized by peroxidase to N-4-(antipyryl)-3-chloro-5-sulfonate-benzoquinonemonoimine. The red color produced with an absorbance maximum at 520 nm was measured with a spectrometer.

NQO-1 activity assay
NQO-1 activity was measured as previously described [33] with minor modifications. Cells were washed three times with PBS and scraped from the dishes using a rubber, and then collected in TE buffer (20 mmol/L Tris HCl containing 2 mmol/L EDTA, pH 7.4). This was followed by sonic disruption on ice, 4 x 5 s with 10 s interval between sonications; after centrifugation at 13,000 g the supernatant was used for further testing. The reaction mixture (200 μl final volume) consisted of 25 mmol/L Tris-HCl (pH 7.4), 80 μmol/L 2,6-dichlorophenolindophenol (DCPIP), 0.2 mg/ml BSA and 0.01% (v/v) Tween-20, with or without 10 μmol/L dicoumarol, and 10 μg cell lysate protein. The reaction was started by the addition of 180 μmol/L NADPH. Reduction of DCPIP was measured at room temperature for 1-2 min at 600 nm (ε = 21×10 3 M −1 cm −1 ). NQO1 activity was considered to be the dicoumarol-inhibitable part of DCPIP reduction.

GST activity assay
Cells were lysed ultrasonically in 10 mmol/L sodium phosphate buffer, pH 6.5. The protein content of the cell lysate was quantified by the BCA method. GST activity was measured using 5 mg protein, 1 mM GSH, 1 mM chloro-2, 4-dinitrobenzene, 3 mg/ml BSA in 10 mM sodium phosphate buffer. The mixture was scanned at 340 nm for 5 min at 25°C [34].

GCL activity and GSH level assays
The methods used to assay GCL activity and GSH levels were as previously described [35]. Briefly, a fluorescent dye 2,3-naphthalenedicarboxaldehyde (NDA), reacts with both glutamylcysteine (GC) and GSH to form NDA-c-GC or NDA-GSH. For each sample, two tubes were prepared, one for the GSH test, the other for the GCL activity test. In both tubes, 50 μl GCL reaction cocktail (400 mmol/L Tris, 40 mmol/L ATP, 20 mmol/L L-glutamic acid, 2.0 mmol/L EDTA, 20 mmol/L sodium borate, 2 mmol/L serine, 40 mmol/L MgCl 2 ) was pre-incubated with 50 μl cell lysate (2000 μg/ml) for 5 min at 37°C; then the GCL reaction was initiated by adding 50 μl of 2 mmol/L cysteine (dissolved in TES/SB) to each GCL activity tube and incubated at 37°C for 1 h before terminating the reaction by adding 50 μl of 200 mmol/L 5-sulfosalicylic acid (SSA). The GSH tube was incubated at 37°C at the same time and otherwise treated the same, except that the same amount of cysteine was added after instead of before termination. Both tubes were then vortexed and held on ice for at least 20 min, then centrifuged for 5 min at 3000 rcf in a Beckman tabletop centrifuge. Following centrifugation, 20 μl aliquots of supernatant from each tube were transferred to a 96-well plate designed for fluorescence detection. NDA derivatization solution (50 mmol/L Tris, pH 10.0, 0.5 mol/L NaOH, and 10 mmol/L NDA in dimethylsulfoxide (DMSO), v/v/v 1.4/0.2/0.2) were added to each well of this plate. The plate was covered to protect the wells from room light and allowed to incubate at room temperature for 30 min. The plate was read on a fluorescence plate reader with wavelengths set to 472 nm excitation/528 nm emission. The GCL activity was considered to be the fluorescence value of the GSH tube subtracted from the fluorescence value of the GCL tube.

GSH/GSSG ratio assay
The GSH/GSSG ratio assay was performed by GSH/GSSG-Glo kit (Promega, WI) following provided protocol. Total glutathione (GSH+GSSG) and GSSG was quantified with standard curve, and GSH/GSSG ratio was calculated accordingly.

G6PD activity assay
Cells were lysed ultrasonically on ice in 0.1 mole/L Tris and 0.5 mmole/L EDTA, pH 8.0. To aliquots of lysate containing equal amounts of protein, 10 mmole/L MgCl 2 and 0.25 mmole/L NADP + were added. The reaction was started at 37°C by adding 0.6 mmole/L glucose 6-phosphate and absorbance was measured in a spectrophotometer. G6PD activity was measured as the increase in absorbance/min at 340 nm [36]. The enzymatic activity was expressed as milliOD/min/μg protein.

Statistical analysis
Statistical significance was established by student's t test for single comparisons, or by ANOVA followed by the Tukey test or the LSD test for multiple comparison analysis.

LM increased expression of mitochondrial electron transfer chain complexes
The electron transfer chain complexes located on the inner mitochondrial membrane are essential for mitochondrial function. We tested their protein expression after 48 hours of LM treatment, and found that LM stimulated the protein expression of all five complexes. For complex I, except at the low LM doses of 5 and 10 μmol/L, all other concentrations (20, 40 and 80 μmol/L) increased protein expression significantly ( Fig 1A); for complex II, both 40 and 80 μmol/L increased the protein expression significantly ( Fig 1B); for complex III, 10, 20, 40 and 80 μmol/L increased the protein expression significantly ( Fig 1C); for complex IV, 10, 20 and 40 μmol/L increased the protein expression significantly (Fig 1D), and for complex V, all concentrations (5, 10, 20, 40 and 80 μmol/L) increased the protein expression significantly ( Fig 1E).

LM increased viable mitochondria mass
Treating ARPE-19 cells for 48 hours with different concentrations of LM, showed an increase in the number of viable mitochondria. The fluorescence unit of Mitotracker, a widely used stain for mitochondrial abundance, significantly increased by 23% at 40 μmol/L LM treatment, compared with control ( Fig 1F and 1G). The mitochondrial DNA copy number, another

LM increased expression of mitochondrial biogenesis-related transcription factors
To confirm the effect of increasing mitochondrial mass, and to unveil the mechanism by which mitochondrial biogenesis is promoted by LM, we measured the expression of mitochondrial biogenesis transcription factors. PPARGC1a is the key transcriptional coactivator for inducing mitochondrial biogenesis. With LM treatment, the protein expression of PPARGC1a in ARPE-19 cells increased in a concentration-dependent manner, and both 40 and 80 μmol/L enhanced the protein expression significantly (Fig 2A). For the downstream transcription factors of PPARGC1a, we measured the mRNA expression of nuclear respiration factor 1 and 2 (NRF1 and NRF2), and Tfam with real time RT-PCR. LM significantly increased NRF1 only at 40 μmol/L (by 74%). It increased the NRF2 α-subunit expression at 5, 10, and 20 μmol/L, with the most effective concentration of 20 μmol/L causing a 3.2 fold increase. LM increased Tfam at 40 μmol/L (by 75%) and at 80 μmol/L (Fig 2B).
LM was more potent than LA in inducing PPARGC1a expression and increasing mtDNA copy number Since LA has been proved to be a mitochondrial biogenesis inducer [37], we compared the abilities of LM and LA in inducing mitochondrial biogenesis. Consistent with previous results, the key transcriptional coactivator PPARGC1a was significantly induced by LM treatment (40 μmol/L for 48 h), but not by LA at the same concentration ( Fig 2C). For mitochondrial DNA, we found both LM and LA could significantly increase mitochondrial DNA copy number, but the effect of LM treatment was more significant than that of LA ( Fig 2D).

LM stimulated oxygen consumption, increased MMP and inhibited ROS production
AS the above results strongly indicate the fact that LM stimulates mitochondrial biogenesis, we further evaluated mitochondrial function by measuring oxygen consumption and mitochondrial membrane potential (MMP) in ARPE-19 cells. LM treatment (40 μmol/L for 48 h) caused a 34% increase in oxygen consumption (Fig 3A), and a significant increase (40%) in MMP ( Fig  3B) compared with untreated control. We were also concerned whether the higher oxygen consumption and MMP might lead to higher ROS production. However, LM treatment did not cause an increase-on the contrary, it showed a small but still statistically significant inhibition of ROS (Fig 3C). At the same time, we surprisingly found the cellular ATP level was decreased by 10% by LM treatment (Fig 3D), which seemed to be conflict with higher oxygen consumption. This unexpected phenomenon was addressed in the latter part of this article.

LM promoted Nrf2 expression in both nuclear and cytosolic protein fractions
We have previously shown that LM pretreatment protects ARPE-19 cells from the acrolein-induced oxidative stress. Then we asked whether LM could directly react with free radicals. However, in the DPPH assay, we found neither LA nor LM directly scavenge DPPH free radical when compared with ascorbic acid (S1 Fig). These results implied that LA and LM may boost cellular antioxidant ability acting as indirect antioxidant. Nrf2 (nuclear factor erythroid 2-related factor 2) is the key transcription factor for regulating antioxidant response element-(ARE)based gene expression of phase II detoxifying enzymes. We demonstrated that LM treatment at 20, 40 and 80 μmol/L promoted Nrf2 expression (Fig 4A). LM treated at 40μmol/L was more potent than LA at the same concentration in inducing Nrf2 nuclear translocation ( Fig 4B). As we concerned that the cell density may affect the effect of LM and LA in activating Nrf2, we also used fully confluent cells to verify our data. The results (S3 Fig) showed that nuclear Nrf2 protein levels in fully confluent cells were also increased by LM and LA treatment.

LM increased expression and/or enzymatic activities of phase II antioxidant enzyme (NQO-1, GST, Cu/ZnSOD and G6PD)
NQO-1 is one of the phase II antioxidant enzyme that is regulated by Nrf2. We examined the expression and activity of NQO-1. Consistent with findings on Nrf2, the protein expression of NQO-1 was increased by LM treatment at 20, 40 and 80 μmol/L (Fig 4C). We also compared the effects of LM and LA at 40 μmol/L on NQO-1 expression and activity, and found that both LM and LA could significantly stimulate NQO-1 expression and activity (Fig 4D and 4E); LM showed a more potent effect than LA, but the difference was not statistically significant. GST is another important phase II enzyme. The activity of total GST was increased by 19% and 27% by treatment with 40 μmol/L LM and LA, respectively (Fig 5B). However, protein expression of total GST was not increased by either LM or LA treatment (Fig 5A and 5B).
Catalase and Cu/ZnSOD are important endogenous antioxidant enzymes, and both were regulated by Nrf2. We tested the effect of LM and compared its effect with that of LA. LM treatment (40 μmol/L, 48 h) caused a significant increase in both expression and activity of catalase, independent experiments. Optical densities of four independent images were analyzed with Quantity One software; results are expressed as ratios of PPARGC1a to β-actin. (D) Effects on mitochondrial DNA copy number. Real-time PCR was employed for assaying the D-LOOP region of mitochondrial DNA. The results are expressed as ratios of D-LOOP to 18S rDNA. Values are means ± SEM from four independent experiments. Results are in arbitrary units normalized by setting the ratio of control cells to 100. In both C and D, C stands for control, LM stands for 40 μmol/L LM treatment and LA stands for 40 μmol/L LA treatment. For both A and B, statistical significance was established by student's t test. * p<0.05, and **p<0.01 vs. untreated control (0 μmol/L). For both C and D, statistical significance was established by one way ANOVA followed by the LSD test. * p<0.05, ** p<0.01 vs. untreated control (C, 0 μmol/L), and # p<0.05, vs. LA.   (Fig 5C and 5D). We also found both LA and LM could significantly increase the Cu/ZnSOD mRNA level, but the effect of LM was not potent (Fig 5E). Besides, glucose-6-phosphate dehydrogenase (G6PD), the key enzyme in NADPH generation, is also an Nrf2-regulated enzyme, and we found its activity exibited a more than two-fold increase after LM treatment, while only 50% increase after LA treatment (Fig 5F).
LM increased GCL expression, enzymatic activity, GSH levels and GSH/ GSSG ratio GCL, which ligates L-glutamate and L-cysteine, is the key enzyme in GSH de novo synthesis. We examined the expression of GCLc, the catalytic subunit of GCL, and the activity of GCL. With 40 μmol/L LM treatment, GCLc expression was significantly increased (Fig 6A and 6B). Similarly, GCL activity was also increased significantly (by 45%) by LM treatment (Fig 6B). LA treatment showed no significant effect on either the expression of GCLc or the activity of GCL (Fig 6A and 6B).
GSH is the final product of GCL catalysis and is considered to be the most important low weight antioxidant molecule, as GSH levels usually determines the redox status of the cell. LM treatment induced a 1.7 fold increase in the GSH level compared with control; LA treatment also induced a significant but much smaller increase (20%) compared with control, and the effect of LM was significantly greater than LA (Fig 6C). GSH/GSSG ratio, the indicator of cellular redox status, increased by 70% with LM treatment; while LA treatment doesn't significantly increase the ratio (Fig 6D).

Enhanced mitochondrial function is to sustain high cellular GSH levels with LM treatment
To find out whether the increase of GSH level was due to GCL activation, the GCL inhibitor BSO was used to block de novo GSH generation; and we found that the increased GSH level caused by LM treatment was very sensitive to BSO inhibition (Fig 6E). It's reported that the depletion of GSH by diethyl maleate cannot be replenished if GCL is inhibited [38]. We found that the GSH level recovered faster in the presence of LM compared to control after we depleted cellular GSH with diethyl maleate treatment (Fig 6F). These results demonstrated that LM treatment increased de novo GSH synthesis through GCL activation.
As we mentioned above, LM treatment increased oxygen consumption by about 40% compared to control (Figs 3A and 6G). However, intracellular ATP levels did not increase but declined 10% (Fig 3D). We speculated that the LM-induced energy expenditure may have been used for de novo GSH synthesis. We then treated the cells with BSO to inhibit GCL activity. Under these conditions, in both LM-treated and LM-untreated cells, GSH levels decreased to the same level, about 60% of control. Oxygen consumption also decreased to the same level showing similar trends. (D) LM treatment decreased cellular ATP level. Values are means ± SEM from 3 independent experiments. (E) Expression of MnSOD,Trx2,Prx3,and Prx5. ARPE-19 cells were treated with 40 μmol/L LM or LA for 48 h; then RNA was isolated and reverse-transcribed to cDNA. Real time PCR was employed to measure expression levels of the indicated genes. The results (from 5 independent experiments) are expression ratios of the target genes to 18SrRNA, and are normalized to control (control = 100). C stands for control, LM stands for 40 μmol/L LM treatment and LA stands for 40 μmol/L LA treatment. Statistical significance was established by one way ANOVA followed by the Tukey test (A, B, C, D) or LSD test (E). * p<0.05, ** p<0.01 vs. untreated control (0 μmol/L); # p<0.05, ## p<0.01 vs. LA. doi:10.1371/journal.pone.0128502.g003 ( Fig 6G). The mitochondrial membrane potential and ROS level had no significant change with BSO treatment (S2 Fig), which indicated the decrease of oxygen consumption after BSO treatment was not due to GSH decline caused oxidative stress. These results indicate that the additional oxygen consumption was used to generate de novo GSH.

Discussion
Accumulated evidences suggest oxidative damage and mitochondrial dysfunction are the major causes of aging and age-related degenerative diseases and that improving mitochondrial function and inhibiting oxidative damage are effective strategies for delaying aging and preventing age-related diseases [7,39]. LA has been proven to be a mitochondrial antioxidant   [20]. More recently, LA has been shown to promote mitochondrial biogenesis and function in 3T3-L1 adipocytes [11] and in SK-N-MC neuroblastoma cells [40]. LM is the amide derivative of LA, and was found to be more potent than LA in protecting ARPE-19 cells from acrolein-induced oxidative damage and mitochondrial dysfunction [22]. In this study, we demonstrated that LM stimulated mitochondrial biogenesis by increasing PPARGC1a expression and induced Nrf2-regulated phase II antioxidant enzymes in ARPE-19 cells; meanwhile, we showed LM was more potent than LA in the above-mentioned capabilities.
LM acts as a stimulator of mitochondrial biogenesis and simultaneously as an inducer of phase II antioxidant enzyme. It suggests that mitochondrial biogenesis and phase II antioxidant systems are closely related or coupled. The relation between mitochondrial biogenesis and ROS production has been well debated [41]. Mitochondrial biogenesis can lead to enhancement of both intracellular and mitochondrial antioxidant system. PPARGC1a, the most important transcriptional coactivator in inducing mitochondrial biogenesis, also affects the cellular antioxidant system. Liang et al. [3] found that overexpression of PPARGC1a promoted the antioxidant ability of 3T3 fibroblasts; Valle et al. [31] found the expression of PPARGC1a promoted the expression of mitochondrial antioxidant enzymes. St-Pierre et al. [42] proved PPARGC1a is required for the expression of antioxidant enzymes, and PPARGC1a expression enhances antioxidant ability and reduces ROS in neurons. In our previous study, we found LM pretreatment protected ARPE-19 cells from acrolein-induced oxidant stress and mitochondrial dysfunction. In this study, we further showed that LM-induced increase in PPARGC1a expression led to an increase in mitochondrial biogenesis and function, and also enhanced gene expression of mitochondrial antioxidant enzymes, including SOD, Trx2, Prx3 and Prx5 (Fig 3E). Moreover, we found that increased mitochondrial mass was accompanied by an increase in MMP (Fig 3B), mitochondrial complex expression (Fig 1), oxygen consumption and a decrease in ROS production (Fig 3A and 3C).
However, the small but significant decline (10%) in ATP was unexpected ( Fig 3D). We propose that the ATP decrease may be related to the increased antioxidant levels, especially to the increased generation of GSH. There may be an energy link between the mitochondrial biogenesis stimulation and phase II enzyme induction. The de novo generation of the most important phase II enzyme product-GSH, is highly related to energy supply, because generation of a new GSH molecule requires consumption of two ATP molecules [43,44]. With a concentration in the micromolar scale, GSH is abundant in the cell. LM increased GSH levels by 50-70% in ARPE-19 cells (Fig 6C). This must require a large amount of energy, which may be compensated for by the increased mitochondrial mass and improved mitochondrial function. Pantothenic acid is reported to increase intracellular GSH levels by promoting ATP production [45] and mitochondrial uncouplers or ATP synthase inhibitors can lower intracellular GSH levels [46]. We showed that LM-treated cells tended to maintain higher GSH level increased GSH generation was related to increased oxygen consumption, and blocking GSH synthesis recovered the oxygen consumption to near normal level (Fig 6G). These results imply that improvement of mitochondrial biogenesis or mitochondria function is necessary for generating sufficient ATP to maintain high GSH level. established by one way ANOVA followed by the Tukey test. In A, B, C and D* p<0.05, **p<0.01 vs. untreated controls (0 μmol/L), # p<0.05 vs. LA, and ## p<0.01 vs. LA. In E and G, **p<0.01 vs. untreated controls, and ## p<0.01 vs. LM treatment doi:10.1371/journal.pone.0128502.g006 NADPH-consuming antioxidant enzymes (NQO-1, and enzymes involved in GSH recycling, etc.) may also be affected by energy supply. Glucose-6-phosphate dehydrogenase (G6PD), the key enzyme in NADPH generation, and the first rate-limiting enzyme in pentosephosphate pathway, is also an Nrf2-regulated enzyme, and we found its activity had a more than two-fold increase after LM treatment (Fig 5F). The increased requirement for NADPH and greater G6PD activation should shunt more glucose into the pentose-phosphate pathway, which may inhibit glycolysis and make the cell more dependent on mitochondrial function. That provides a possible explanation linking induction of phase II antioxidant enzymes and stimulation of mitochondrial biogenesis and function. However, whether induction of other phase II antioxidant enzymes correlate with mitochondrial biogenesis and function for the sake of ATP supplementation, warrants further investigation.
In conclusion, we found LM treatment stimulated mitochondrial biogenesis and function and induced phase II enzyme expression in ARPE-19 cells. Enhanced mitochondrial biogenesis, improved mitochondrial function and strengthened antioxidant defense might account for the protecting mechanism of LM against acrolein-induced oxidant damage and mitochondrial dysfunction.
Supporting Information S1 Fig. LA or LM doesn't directly scavenge DPPH free radical. (A) Doses of 40 μM ascorbic acid, lipoic acid or lipoic amide were added to 60 μM DPPH free radical. Absorbance was measured after incubation for 90 minutes at 37°C following addition (One-way ANOVA followed by Tukey's test, n = 8 per group); (B) Different doses of lipoic acid, lipoamide or ascorbic acid were added to 50 μM DPPH, and absorbance was measured after incubation for 30 minutes at 37°C following addition (n = 4 per group).