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Retina; Metabolism; Age-related macular degeneration; Antioxidants; Oxidative stress

Introduction

Global prevalence of vision impairment and blindness due to retinal diseases such as age-related macular degeneration (AMD) is 200 million and is expected to increase to ~300 million by 2040. It is the most common cause of irreversible blindness in individuals >60 years of age in developed countries [1]. Additionally, about 103 million people are living with diabetic retinopathy (DR), another blinding disease; its prevalence is expected to rise 1.5 times by 2045 [2].

Both AMD and DR are multifactorial in their etiologies, advancing age and hyperglycemia, respectively, considered to be the primary reasons for the initiation of aberrations in a wide variety of biochemical, metabolic, and molecular-signaling pathways, and consequent damage to cell structure and function. One of the prominent and well-known pathogenetic factors in several retinal diseases, including AMD and DR, is the array of cellular molecules targeted for modifications by reactive oxygen species (ROS).

ROS viz. superoxide, hydrogen peroxide, hydroxyl radicals, and singlet oxygen, are generated in minor quantities during normal cellular respiration, and are known to have important roles in several physiological processes through redox signaling and second messenger mediated signaling pathways impacting inflammation, cell proliferation, apoptosis, etc. [3]. However, excess ROS can cause perturbation to many molecules in the cell. Lipid constituents of cell membranes are highly vulnerable to peroxidation by ROS and forming malondialdehyde (MDA) and 4-hydroxynonenal (HNE), which could form adducts with proteins and DNA, further affecting cell-structure and function [4,5]. Involvement of membrane lipids can adversely affect membrane permeability with consequent disruptions in intra & extra-cellular ionic balance. ROS are also known to induce DNA base modifications and nicks/breaks [6]. Oxidative modifications of structural and enzymatic proteins can lead to cell structure alterations and metabolic dysfunction/inhibition, respectively [7].

Mitochondria, which serve as a key source of physiological ROS, are also one of the main targets of these radicals. Proximity of mitochondrial DNA to the electron transport chain, a generator of superoxide, makes it highly susceptible to oxyradical damage. Mitochondrial dysfunction caused by high levels of ROS are known to initiate apoptosis [8]. ROS can also induce endoplasmic reticulum (ER) stress causing disruption in protein folding and buildup of unwanted misfolded proteins that may trigger the unfolded protein response (UPR) and cell death [9]. Moreover, peroxisomes, which play a significant role in removing ROS from cells are also impacted by oxidative stress. Despite containing enzymes like catalase that decompose hydrogen peroxide into water and oxygen, high levels of ROS can hinder their functioning and cause more stress [10].

Ocular tissues are especially more susceptible to oxidative stress due to constant exposure to light during photopic vision which initiates photochemical generation of ROS in the presence of tissue photosensitizers; this can be potentially incessant as long as light is reaching the retina and O2 and photosensitizers are available. The retina is particularly more susceptible to such damage because of its high oxygen consumption to fulfil ATP demands of neurons, and abundance of polyunsaturated fatty acids. In view of the high prevalence of progressive vision-impairing degenerative diseases of the retina and lack of adequate preventive and therapeutic measures to halt or slow down their progression, it is important to investigate the potential of novel compounds that are effective in preventing oxidative stress well-known to be a major etiologic factor [11-13].

Results from the Age-Related Eye Diseases Studies (AREDS) conducted by NEI, NIH, have shown that the risk of progressing to advanced stage AMD was ~25% lower with dietary supplementation of the AREDS-1 formulation containing vitamins C and E, copper, zinc, and Beta carotene. The risk was also similarly lowered when beta carotene was substituted with lutein + zeaxanthin, and docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA) in AREDS-2 formulation [14,15]. Although the above antioxidant formulations have shown partial effectiveness in some patients in slowing the disease progression and vision deterioration, they are not beneficial in all patients and in early stages of the disease. In addition to adversely affecting the performance of routine activities, progression of vision deterioration due to both AMD and DR, has a highly significant negative economic impact on the individual and the nation. The financial burden on the US economy due to late-stage AMD is ~ $49.4 billion/annum [16]. Although many laboratories worldwide are heavily invested in exploring newer pharmacological strategies and novel techniques to prevent and treat the disease effectively, outcomes have not been very promising so far, especially in progressive dry AMD, and vision deterioration continues to be a highly significant public health concern.

Pyruvic acid, an alpha-ketoacid and an end-product of glycolysis, can effectively scavenge H2O2; this was first demonstrated by Fenton in 1900 [17] and Holleman in 1904 [18], and in biological systems by Sevag and Maiweg in 1934 [19]. In fact, pyruvic acid was shown to react with all reactive oxygen species with high rate constants [20- [21]]. The effectiveness of pyruvate in preventing oxidative stress to the lens in vitro as well as in vivo has been amply demonstrated by us [22-24]. Its neuroprotective effect was also shown by us previously in in vitro studies wherein pyruvate prevented oxidative stress-induced biochemical aberrations, as indicated by the maintenance of glutathione (GSH) levels and prevention of lipid peroxidation in the neural retina [25]. In addition, neural retinas incubated in pyruvate-fortified media were found to maintain glycolytic activity even when exposed to ROS, reflected in the levels of glycolytic end products pyruvate and lactate.

The levels of these metabolites decreased in the retinas incubated in an ROS generating medium without pyruvate supplementation, indicating a potential oxidative inactivation of glycolytic enzymes [26]. In that regard, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) are both known to be susceptible to inactivation by oxyradicals. The active site of GAPDH has a cysteine -SH which has been shown to be the site of oxidation and consequent inactivation [27]. Pyruvate kinase, the M2 isoform in particular, is also known to be inactivated by ROS. Both M1 and M2 isoforms of the enzyme are expressed, although differentially, in the various retinal cells. The inner plexiform and ganglion cell layers express PKM1 predominantly, while rod inner segments and outer plexiform layers express PKM2 predominantly [28-30].

The main goal of the current study was to determine whether the previously observed decrease in glycolysis in the retina exposed to ROS was due to inactivation of glycolytic enzymes, and whether pyruvate is effective in preventing this inactivation. The correlation between the duration of ROS exposure and status of enzyme activities, and the effect of pyruvate supplementation therein were also determined. Additionally, the status of energy production was evaluated by measurement of ATP levels. These studies were conducted using bovine retinas incubated in a medium generating ROS in the absence and presence of pyruvate.

Materials and Methods

Freshly enucleated bovine eyes were acquired from a local abattoir. Eyes were transported on ice until the dissection was initiated, usually within 15 minutes. The neural retina was then gently removed and incubated in medium 199 (Thermo Fisher Scientific, Cat # 11043- 023) containing 0.5mM sodium xanthine (XA) (Sigma X3627, St. Louis, MO) in a humidified incubator set at 5% CO2. Medium 199 is a serum-free basal tissue/cell culture medium composed of amino acids, inorganic salts, vitamins, glucose, sodium bicarbonate, adenosine, hypoxanthine, etc. to ensure tissue viability for the duration of incubation. The complete formulation is available from Thermo Fisher. The retinas were divided into 3 groups, Control, Experimental, and Pyruvate. The Control group was incubated in 0.5 ml medium 199 containing 0.5mM sodium xanthine without any further additions. Experimental group was incubated in the medium above but with the addition of 0.07U xanthine oxidase (XO) (Sigma X2252, St, Louis, MO). XA-XO reaction served as the source of ROS. For the Pyruvate group, medium 199 containing 0.5mM XA was supplemented with sodium pyruvate (Sigma P2256, St. Louis, MO) to a concentration of 10mM, and ROS was generated by the addition of 0.07U XO. Total duration of incubation was 6 hours. Incubation of some samples from each group was terminated at 2 hours, while it was continued for the rest for additional 4 hours (total 6 hours). Retinas from each of the 3 groups were processed for protein, GAPDH, PK, and ATP assays as follows.

The retinas were homogenized in 0.05M sodium phosphate buffer, pH 7.4, for GAPDH and PK assays, and in dH2O for ATP assay, ensuring the culture tubes were placed on ice during this process. The homogenates were centrifuged at 4°C at 17,500rpm for 10 minutes and the supernatant was used for determining the water-soluble protein content, GAPDH and PK activity, and ATP levels.

Protein assay

Protein content in the supernatant was determined by Bradford’s assay using Coomassie blue dye-based reagent (Sigma B6916, St. Louis, MO). An aliquot of the supernatant was reacted with the reagent and absorbance of the protein-bound dye was read spectrophotometrically at 595nm using an Agilent Biotek multimode plate reader. Protein standards using Bovine serum albumin (BSA) were run simultaneously, and the protein content in the sample was then calculated using absorbance values of standards.

GAPDH assay

GAPDH activity in the sample was determined by using a coupled reaction wherein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes oxidation of glyceraldehyde-3-phosphate (GAP) to 1,3-bisphosphoglycerate (BPG) with simultaneous reduction of its cofactor, NAD+ to NADH. The formation of NADH is monitored by noting the increase in its absorbance at λ340 nm in a plate reader.

The reagent mixture/well consisting of (1) 133μl of 15mM sodium pyrophosphate buffer, pH 8.5, containing 30mM sodium arsenate (Catalog # A6756, Sigma Aldrich, St. Louis, MO), (2) 7μl of 7.5mM NAD+ (Catalog # N0362, Sigma, St. Louis, MO), and (3) 7μl 0.1M dithiothreitol (DTT) (Catalog # 43816, Sigma-Aldrich, St. Louis, MO), was dispensed in a 96-well plate. An aliquot of the supernatant was then added to the wells and the initial absorbance (OD1) was recorded on a plate reader. Seven microliters of 15mM DL-glyceraldehyde-3- phosphate (Catalog # 17865, Cayman Chemical Company, Ann Arbor, MI), the substrate for GAPDH, was added to start the reaction and absorbance was monitored over 10 minutes until no significant further increase in absorbance was observed. OD2 at 10 minutes was noted. Appropriate positive and negative controls were run simultaneously. The change in absorbance ΔOD340nm was calculated by subtracting OD1 from OD2. GAPDH activity was calculated and expressed as ΔOD/mg protein.

Pyruvate kinase assay

PK activity was determined by using a coupled reaction; in the first reaction pyruvate kinase transfers a phosphate group from phosphoenolpyruvic acid to ADP, forming pyruvate and ATP. The second reaction involves reduction of the pyruvate (generated in the first reaction) to lactate by lactate dehydrogenase (LDH) with simultaneous oxidation of NADH to NAD+. The amount of NAD+ formed is proportional to the amount of pyruvate generated in the first reaction. The formation of NAD+ is monitored by noting the decrease in the absorbance of NADH at λ340nm.

Briefly, the assay was carried out in a 96-well plate as follows: Each well contained a reaction mixture consisting of 173μl 50mM imidazole buffer (pH 7.6, containing 1.79mg KCl, and 1.482 mg MgSO4), 7μl of 4.7mM ADP (Catalog # L14029.03, Thermo Fisher Scientific), 7μl 3mM phosphoenolpyruvic acid (Catalog # 19192, Cayman Chemical Company, Ann Arbor, MI), 7μl 7.5mM NADH (Catalog # J61638, Alfa Aesar/Thermo Fisher Scientific) and 2 μl LDH (Catalog # 10127876001, Roche Diagnostics, Indianapolis, IN). Initial absorbance OD1 was noted in the plate reader, and then an aliquot of the supernatant was added to the reaction mixture. Absorbance was monitored over 5 minutes after which no further decrease was observed. The change in absorbance ΔOD was calculated by subtracting OD5min from OD1. PK activity was then calculated by dividing ΔOD by the amount of protein in the sample.

ATP assay

ATP level in the supernatant was determined by a luminescencebased method using luciferin and luciferase present in firefly lantern extracts. The assay is based on quantifying the luminescence (photons) generated when luciferase oxidizes luciferin to oxyluciferin using O2 and ATP. The amount of luminescence is proportional to the amount of ATP.

Lyophilized firefly lantern extract (FLE6303 Sigma, St. Louis, MO) was reconstituted with 5 ml dH2O. After reconstitution, the FLE contains 50mM potassium phosphate, pH 7.4 and 20mM MgSo4. Fifty microliters of the sample supernatants prepared in dH2O as described above was pipetted out in white 96-well plates. ATP standards with known ATP concentrations were also run simultaneously. Agilent- Biotek multimode plate reader with dual injector module was set to luminescence mode, and was programmed to dispense 50μl of reconstituted FLE in each well after the plate was inserted into the reader. Luminescence was read simultaneously. ATP concentration in the sample was calculated by comparing the luminescence values of the samples with the standards and expressed as nanomoles/mg protein.

Statistical analysis: Data was analyzed using one-tailed t-test to determine the statistical significance of the differences in the values between the controls, experimentals and pyruvate groups.

Results

The main purpose of the current experiments was to determine the effect of ROS, with and without pyruvate supplementation, on the activity of glycolytic enzymes in the retina as a function of duration of exposure to the ROS generated by the xanthine-xanthine oxidase reaction, and to evaluate the status of energy generation.

As shown in Figure 1, after incubation with XA+XO for 2 hours, the activity of GAPDH decreased to 73±5% of the controls incubated without XO. The activity was lower than the controls even in the retinas incubated in pyruvate-fortified medium. In the tissues incubated for 6 hours, the activity in the experimental group was 92±10% of the controls, while it remained lowered to ~76% of controls in the pyruvate group.

Figure 1: Effect of sodium pyruvate supplementation on GAPDH activity in retinas exposed to ROS. Results are expressed as % of controls incubated without xanthine oxidase. n≥15 in each group at both 2-hrs and 6-hrs. p <0.4 and <0.05 between experimental and pyruvate groups at 2 hours and 6 hours, respectively.

The activity of pyruvate kinase was observed to decrease significantly in the experimental group to 57±10% of controls after 2 hours of ROSexposure, as shown in Figure 2. However, pyruvate supplementation maintained PK activity to ~93% of the controls and was 1.6 times the experimental levels. At 6-hour post-incubation, PK activity in the experimental group remained low- 60±11% of the controls, while its activity in the pyruvate group was still maintained at 96% of the control values and was ~1.6 times the experimental values.

Figure 2: Pyruvate kinase activity in retinas incubated with xanthine & xanthine oxidase +/- sodium pyruvate. Results are expressed as % of controls. p<0.001 between control and experimental, and experimental and pyruvate groups at both 2 hr-and 6 hr time points. n≥15 in each group at both time intervals.

ATP level after 6 hours of incubation was significantly lowered in the experimental group to 55±7% of controls, while it was substantially higher in the pyruvate group wherein it was ~140% of the controls (Figure 3).

Figure 3: ATP levels in the retinas incubated with xanthine & xanthine oxidase +/- sodium pyruvate. Results are expressed as % of controls. n≥15; p<0.001 between control and experimental, experimental and pyruvate, and control and pyruvate groups.

Discussion

Effectiveness of pyruvate as a H2O2 scavenger was demonstrated more than a century ago by Fenton and Holleman [17,18]. Its ability to protect ocular tissues against oxidative stress was first demonstrated in the rat lens by Varma et al. in 1988 [22]. Pyruvate prevented the peroxide-induced inhibition of active transport of Na+ and K+ in lens organ culture experiments [31]. It was also found to prevent lens damage and cataract formation induced by exposure to UV-light, in rodent lenses [32,33]. Biochemical and physiological aberrations such as decrease in the levels of the endogenous antioxidant glutathione, decrease in active transport, and decrease in ATP production, all were prevented when pyruvate was added to the culture medium. In addition to preventing cataract formation in vitro, pyruvate and its ester ethyl pyruvate, both, were also effective in preventing lens damage and cataract formation in vivo in animal models of oxidative stress such as the streptozotocin-induced diabetes rodent models [34,35]. We further found it to be highly effective in preventing ROS-induced biochemical and metabolic dysfunction in the mouse neural retina, reflected in maintenance of glutathione level, decrease in lipid peroxidation, and preventing inhibition of glycolysis indicated by production of pyruvate and lactate [25,26].

Many studies have shown pyruvate to be an effective antioxidant in many non-ocular tissues and cells, such as the myocardium, liver, brain, neuronal cell cultures, neuroblastoma cells, etc., protecting the tissues against ROS directly as well as against mitochondrial toxins. Sodium pyruvate protected striatal neurons from undergoing cell death, increasing neuronal survival [36]. In addition to its effectiveness in decomposing extracellular H2O2 by nonenzymatic decarboxylation, pyruvate was also shown to exert its neuroprotective effect by directly acting on the mitochondria, indicated by decrease in mitochondrial ROS generation and stabilization of mitochondrial membrane potential when neuroblastoma cells were exposed to oxidative insult [37-39]. Pyruvate was also found to protect the myocardium against ischemia reperfusion injury due to its antioxidant properties, combined with anti-inflammatory, and metabolic effects [40]. Studies have also shown pyruvate treatment to be effective in decreasing liver damage resulting from hemorrhagic shock, indicated by improvement in the redox status (NADH/NAD and GSH/GSSG), decreased pro-apoptotic biomarkers, and decreased mitochondrial release of cytochrome c [41,42].

The current study was primarily conducted to investigate whether the previously demonstrated effect of pyruvate in inhibiting glycolysis is due to prevention of oxidative inactivation of glycolytic enzymes. Our results clearly show that pyruvate maintained flux of glucose through glycolysis primarily by preventing inactivation of pyruvate kinase (PK) by ROS generated by xanthine-xanthine oxidase reaction. Although pyruvate didn’t prevent GAPDH from ROS-induced inactivation, maintenance of PK activity was effective in driving glycolysis forward. This is indicated by the significantly higher levels of glycolytic endproducts lactate and pyruvate generated in the retina exposed to ROS with pyruvate supplementation as compared to the levels of these metabolites in the absence of such supplementation, as reported by us previously [26]. ATP results (fig. 3) clearly demonstrate that, as would be expected, tissues exposed to ROS have significantly lowered energy production. Addition of pyruvate to the medium stimulated retinal metabolism such that ATP level was not only higher than the experimental group but was also higher compared to the controls. The level of ATP in the pyruvate group, as shown in Figure. 3, is 2.5 times higher than the level in the experimental group, and 1.4 times higher than even the control group.

Preventing oxidative inactivation of PK, as shown here, is considered one of the ways in which pyruvate helped in maintaining glycolysis. Another mechanism by which it could do so is by its reduction to lactate by lactate dehydrogenase with simultaneous oxidation of the coenzyme NADH, thereby making NAD+ available for the GAPDH reaction. The significantly higher levels of ATP in the pyruvate group could be due to its utilization in the citric acid cycle after being transported through the mitochondrial monocarboxylate transporter and oxidized to acetyl-coenzyme. We plan to further investigate the effect of ROS on utilization of pyruvate through Krebs’ cycle, and glucose utilization through other metabolic pathways such as the pentose phosphate pathway, in detail in our future studies.

Conclusion

Overall, these results clearly demonstrate that pyruvate is effective in preventing metabolic inhibition in the neural retina exposed to oxygen radicals by preventing inactivation of pyruvate kinase, a mechanism demonstrated for the very first time in the neural retina. It also increases ATP levels. Being an efficient scavenger of all ROS, combined with its ability to provide metabolic support to the tissue, this alpha keto acid has the potential to be useful therapeutically to reduce tissue damage in oxidative-stress induced pathologies.

Acknowledgement

The authors would like to acknowledge the contribution of Medha Hegde, Year 3 undergraduate student at Robert H. Smith School of Business, University of Maryland College Park, for statistical analysis of the data.

Ethics Statement: Ethical approval is not required for this study in accordance with local or national guidelines.

Ethical Review Board: N/A

Conflict of Interest Statement: The authors have no conflicts of interest to declare.

Funding Source: The authors are thankful to the University System of Maryland for the Elkins Professorship research funds awarded to Dr. Kavita Hegde for the work described herein.

Data Availability Statement: All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Authors Contribution

Kavita Hegde: conceptualizing the project, planning and implementation of experiments; Vansh Kapoor and Christian Stevens: conducting all assays and data calculations.

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