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Lactate dehydrogenase deficiency increases methylglyoxal and induces cardiomyopathy

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저작자표시-비영리-변경금지 2.0 대한민국 이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게 l 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다. 다음과 같은 조건을 따라야 합니다: l 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건 을 명확하게 나타내어야 합니다. l 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다. 저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다. 이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다. Disclaimer 저작자표시. 귀하는 원저작자를 표시하여야 합니다. 비영리. 귀하는 이 저작물을 영리 목적으로 이용할 수 없습니다. 변경금지. 귀하는 이 저작물을 개작, 변형 또는 가공할 수 없습니다.

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Master

’s Thesis in the Department of

Biomedical Sciences

Lactate dehydrogenase deficiency

increases methylglyoxal and induces

cardiomyopathy

The Graduate School of Ajou University

Department of Biomedical Sciences

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Lactate dehydrogenase deficiency

increases methylglyoxal and induces

cardiomyopathy

Supervised by

Chan Bae Park, PhD

I submit this thesis as the Master’s thesis in the

Department of Biomedical Sciences.

August, 2020

The Graduate School of Ajou University

Department of Biomedical Sciences

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i - Abstract –

Lactate dehydrogenase deficiency increases methylglyoxal and induces

cardiomyopathy

Methylglyoxal (MGO) is a highly reactive compound that breaks protein function and forms advanced glycation end products (AGEs). Advanced glycation end products (AGEs) cause a variety of diseases because they disrupt cell function. Methylglyoxal, which causes AGEs, is produced during the glycolysis. Glycolysis converts glucose into pyruvate, and pyruvate enters the mitochondrial TCA cycle to produce ATP or it is converted to lactate by lactate dehydrogenase (LDH), an enzyme that converts pyruvate to lactate in the cytoplasm. Continuous influx of glucose enhances the conversion of dihydroxyacetone phosphate (DHAP) to methylglyoxal by nonenzymatic reactions. D-lactate is produced as a final product via the glyoxalase system, the detoxification mechanism of methylglyoxal. In this study, we found that L-lactate dehydrogenase (LDH) plays an important role in D-lactate metabolism and LDHB in LDH isoenzymes has high enzyme activity for D-lactate. In this paper, in vivo and in vitro LDHB removal assays were performed to demonstrate. Lactate dehydrogenase B deficiency has been shown to increase D-lactate levels, accumulate methylglyoxal and cause mitochondrial dysfunction. LDHB deficiency also induces cardiomyopathy in mice.

Keywords: lactate dehydrogenase, methylglyoxal, d-lactate, Advanced glycation end products,

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ii

TABLE OF CONTENTS

ABSTRACT

--- i

TABLE OF CONTENTS

---ⅱ

LIST OF FIGURES

--- iv

I. INTRODUCTION

---1

II. MATERIALS AND METHODS

---7

A. HL-1 cell culture--- 7

B. LDH Isoenzyme pattern assay--- 7

C. Cell viability assay --- 8

D. LDHB, LDHA siRNA transfection age --- 8

E. D-lactate measurement ---8

F. L-lactate measurement --- 9

G. Western blotting assay--- 10

H. Quantification of methylglyoxal by HPLC --- 10

I. Methylglyoxal Determination by ELISA --- 11

J. Mitochondria isolation --- 11

K. Complex activity Ⅰ enzyme activity assay --- 12

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iii

III. RESULTS

--- 13

A. LDHB plays a major role in D-lactate metabolism---13

B. LDH enzyme activity to D-lactate---15

C. Loss of LDHB in cardiomyocytes increased D-Lactate---17

D. Loss of LDHB in cardiomyocytes increased Methylglyoxal ---19

E. Loss of LDHB in cardiomyocytes increased AGEs ---21

F. Loss of LDHB in cardiomyocytes decreased complex activity---23

G. Generation of LDHB heart specific KO mouse---25

H. LDHB knockout mouse heart increased methylglyoxal---27

I. LDHB knockout mouse heart increased AGEs---29

J. LDHB deficiency cause mitochondrial dysfunction---31

K. Loss of LDHB induces cardiomyopathy---33

IV. DISCUSSION

--- 36

VI. REFERENCES

---38

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iv

LIST OF FIGURES

Figure 1. Methylglyoxal (MGO) is the major a precursor of advanced glycation end products

(AGEs)--- 4

Figure 2. Glyoxalase pathway---5

Figure 3. Lactate Dehydrogenase ---6

Figure 4. LDH enzyme activity in extract ---14

Figure 5. LDH enzyme activity in pure enzyme---16

Figure 6. LDHB knockdown HL-1cells increased D-Lactate level---18

Figure 7. LDHB Knockdown increased Methylglyoxal in HL-1 cell---20

Figure 8. LDHB Knockdown increased MG-H1(AGEs) in HL-1 cell---22

Figure 9. LDHB Knockdown reduced Complex Ⅰ activity in HL-1 cell---24

Figure 10. Generation of heart specific knockout mouse---26

Figure 11. Loss of LDHB increase methylglyoxal in knockout mouse---28

Figure 12. Loss of LDHB increase AGEs in knockout mouse---30

Figure 13. Loss of LDHB causes mitochondrial dysfunction---32

Figure 14. LDHB knockout mouse Survival rat---34

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I. INTRODUCTION

Energy metabolism is the hundreds of chemical reactions that occur in the process of breaking down, synthesizing and converting glucose, amino acids and fatty acids to provide the body with the energy it needs. Cellular energy metabolism involves many processes and ultimately results in the production of adenosine triphosphate (ATP) from nutrients for many essential functions such as signaling and protein synthesis.

Glycolysis is used for energy production in all cells of the body. Glycolysis, the cytoplasmic pathway, produces energy by breaking down six carbon glucoses into two three carbon compounds. Glycolysis takes place in 10 steps. ATP is generated by substrate level phosphorylation by high energy compounds, such as 1,3-bisphosphoglycerate and phosphoenolpyruvate. Pyruvate is the final product decomposed under aerobic conditions, lactate is the final product under anaerobic conditions. In the cytoplasm, pyruvate generated through glycolysis enters mitochondrial TCA cycle for additional energy production (Asha Kumari, in Sweet Biochemistry, 2018).

Normal cells produce pyruvate using glucose under aerobic conditions to produce NADH via the TCA cycle in the mitochondria. NADH produces oxidative phosphorylation by giving electrons, generating ATP. This process is called mitochondrial respiration because both the TCA cycle and oxidative phosphorylation occur in the mitochondria. Normal cells use glycolysis as an energy source only under oxygen-deficient conditions. Because the glycolysis produces 2 molecules of ATP, oxidative phosphorylation produces about 30 to 36 molecules of ATP, so fermentation or anaerobic metabolism is less efficient than aerobic metabolism (Alberts B, 2002; Sepideh Aminzadeh-Gohari,2019).

During the glycolysis process, fragmentation of glyceraldehyde-3-phosphate (GAP) and dihydroxy acetone phosphate (DHAP) results in the formation of methylglyoxal (MGO) and glyoxal (GO) (Wetzels et al., 2017). Methylglyoxal(MGO), a highly reactive dicarbonyl compound, is unavoidably formed by glycolysis products .Methylglyoxal (MGO) is spontaneously formed from triphosphates of all organisms with anaerobic glycolysis and from other non-enzymatic and enzymatic pathways of different meanings depending on the organism (Thornalley, 2008).

Methylglyoxal (MGO) has two carbonyl groups, an aldehyde and a ketone group and is highly reactive. Methylglyoxal(MGO) interacts with lysine or arginine residues to form Advanced glycation

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end products (AGEs) (Chen et al., 2015). The main precursor of advanced glycosylated end products (AGE) is Methylglyoxal (MGO) (Fig.1A) (Allaman et al., 2015).

Currently, insulin resistance and hyperglycemia are known as the root cause of type 2 diabetes. This probably leads to an increase in methylglyoxal levels due to increased flux through glycolysis. An increase in methylglyoxal can mediate much of the tissue damage caused by hyperglycemia. Recent studies show that elevated levels of methylglyoxal due to increased production of methylglyoxal or impaired detoxification of methylglyoxal may contribute to type 2 diabetes. Increased methylglyoxal in organisms due to detoxification disorders has been shown to lead to gradual development of obesity, hyperglycemia and insulin resistance. It can be placed methylglyoxal not only downstream, but also upstream, in terms of the causal chain of insulin resistance and hyperglycemia (Moraru et al., 2018). Advanced glycation end products (AGEs) cause cardiovascular disease, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, CNS disorders, and aging (Fig.1B). During glycosylation, the dicarbonyl compounds methyl glyoxal (MGO) and glyoxal (GO) react with the amino acids of the protein to form advanced glycation end products (AGEs) into stable final products (Wetzels et al., 2017). These compounds are also called a-oxoaldehydes and are produced by lipid and protein metabolism and glycolysis. Methylglyoxal is one of the most strong glycation-inducing agents found endogenously (Chang and Wu, 2006; Thornalley, 2008). In order to prevent disease caused by AGEs, there is a method to eliminate methylglyoxal (MGO), a main precursor of high-grade glycation products (AGEs).

To avoid the toxic effects of methylglyoxal (MGO), it has different decoding mechanisms, such as glyoxalase, aldose reductase, aldehyde dehydrogenase, and carbonyl reductase pathways (Allaman et al., 2015; Thornalley, 1993; Vander Jagt and Hunsaker, 2003). Cells are protected from toxicity under normal conditions by the glyoxalase system, which is the major detoxification pathway for methylglyoxal (MGO) and other reactive dicarbonyl compounds. Thus, the glyoxalase system performs an important role in cellular defense against glycation and oxidative stress (Allaman et al., 2015; Kalapos, 2008; Thornalley, 1993).

The glyoxalase pathway detoxifies methylglyoxal (MGO) through two sequential enzymatic reactions catalyzed by glyoxalase-1 (Glo-1) and glyoxalase-2 (Glo-2) and uses glutathione as a cofactor. The spontaneous reaction of methylglyoxal with reduced glutathione (GSH) to form hemithioacetal and be converted into S-D-lactoyl-glutathione by Glo-1. The compound is metabolized by Glo-2 to D-lactate, which recycles glutathione during the conversion process (Fig.2)

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(Allaman et al., 2015; Jain et al., 2018). Accumulation of D-lactate increases methylglyoxal (MGO) due to feedback inhibitions.

Lactate exists in nature in two stereoisomeric forms known as L-lactate and D-lactate. L-lactate rotates the light clockwise and D-lactate rotates the light counterclockwise. It is therefore sometimes used in the nomenclature of D (-) and L (+) lactates. The predominant form is L-lactate. L-lactate is produced and metabolized from pyruvate by the action of the enzyme lactate dehydrogenase (Chris Higgins, 2011). However, it is known that enzymes are isomer specific and therefore require different enzymes for production and metabolism. Most of L-lactate is metabolized by Lactate dehydrogenase (LDH) in cytosol.

Pyruvate and lactic acid are converted by lactate dehydrogenase (LDH). Lactate dehydrogenase (LDH) is a tetramer enzyme composed of one or more of two subunits, LDHA (subunit A) and LDHB (subunit B), which is forms five LDH isozymes. Five LDH isozymes (B4 or LDH1, B3A or LDH2, B2A2 or LDH3, BA3 or LDH4, and A4 or LDH5) are formed by the ration of LDHA to LDHB subunits (Fig.3A). Even though structurally they are highly similar, each LDH isoenzyme has different kinetic properties. Each LDHA subunit prefers converting pyruvate to lactate and NADH to NAD+, whereas each LDHB subunit prefers converting lactate to pyruvate and NAD+ to NADH (Fig.3B) (Valvona et al., 2016). In this way, lactate dehydrogenase (LDH) is a reversible reaction enzyme, the role of which is that convert bidirectionally between pyruvate and lactate. LDH isoenzymes are expressed in many organs and tissues. LDHB subunits are expressed in the heart and LDHA subunits are expressed in the liver and muscles. (Drent et al., 1996).

Higher glucose will increase dihydroxy acetone phosphate (DHAP) and increase methylglyoxal (MGO) produced by non-enzymatic dephosphorylation. Increased methylglyoxal will induce a variety of diseases caused by protein modification and AGEs. The metabolism of D-lactate, the product of methylglyoxal (MGO) detoxification, is not known. Reduction of D-lactate metabolism can be expected to increase methylglyoxal (MGO). So, I believe that lactate dehydrogenase (LDH) may play an important role in D-lactate metabolism. Experiments were performed to demonstrate the involvement of L-lactate dehydrogenase in D-lactate metabolism.

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Figure 1. Methylglyoxal (MGO) is the main a precursor of advanced glycation end products (AGEs)

Methylglyoxal (MGO) interacts with lysine or arginine residues to form Methylglyoxal-derived hydroimidazolone (MG-H1) and Arg pyrimidine (AP), Ne- (1-carboxyethyl) -lysine (CEL).

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Figure 2. Glyoxalase pathway

Glyoxalase I and II. Glyoxalase I enzyme converts methylglyoxal into S-D-lactoyl glutathione which is converted to D-lactate by Glyoxalase II. (Jain et al., 2018).

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Figure 3. Lactate dehydrogenase

(A) Lactate dehydrogenase (LDH) homo‐ and tetramer formation. LDH isoenzymes (LDH1, LDH2, LDH3, LDH4 and LDH5) are formed with different proportions of LDHA and LDHB subunits transcribed from LDHA and LDHB, respectively (B) Lactate dehydrogenase (LDH) catalyzes the reversible conversion of pyruvate and NADH to lactate and NAD +.

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II. MATERIALS AND METHODS A. HL-1 cell culture

HL-1 cardiomyocyte cell lines derived from AT-1 mouse atrial myocardial cell tumor strains were purchased and used. HL-1 cells were incubated in an atmosphere of 5% CO2 and 37℃. Claycomb medium supplemented with 10% fetal bovine serum, 0.2 mM L-glutamine, 0.1mM norepinephrine, 100 units/ml penicillin and 100 μg/ml streptomycin (all products purchased from Sigma-Aldrich, USA) is used. Cells were grown on T75 culture flasks precoated for at least 1 h to overnight with a coating solution diluted 1 ml of 1 mg/ml fibronectin in 199 ml of 0.02% gelatin solution. Medium replaced every 1-2 days. When HL-1 cells reach about 100% confluence, the cells culture was subcultured 1: 3 and assigned to passages (Claycomb et al., 1998).

B. LDH Isoenzyme pattern assay

Native polyacrylamide gel electrophoresis (Native-PAGE) analysis was used to confirm the isoenzyme pattern of lactate dehydrogenase (LDH). Native-PAGE analysis is non-denaturing gel electrophoresis and allows direct identification of LDH isoenzymes expressed in protein native states. Sample protein extracts containing 20 mM Tris (pH 7.4), 5 mM EDTA, 120 mM KCl, protease inhibitor cocktail was added. The samples were then sonicated with a bioruptor and centrifuged at 12,000 x g for 10 minutes. Supernatants were used to quantify total protein concentration using Bradford protein assay (DC ™ protein assay, BIO-RAD).

Make 8% polyacrylamide gel in 25 mM Tris-HCl and 190 mM glycine (pH 8.3) buffer and prepare loading protein sample into a with a 2× gel loading buffer consisting of 0.1% bromophenol blue, 40% sucrose, and 20 mM Tris-HCl (pH 7.4), 5mM EDTA, 12 mM KCl (Monica L. Acosta, 2005; Jaime M. Ross, 2010). Pre-running the gel at 80V current for 30 min before loading the protein sample. The protein sample is loaded and run for 5 h at 100V current. To visualize LDH isoenzyme, using solution containing β-nicotinamide adenine dinucleotide (NAD+; 0.3 mg/ml), nitro blue tetrazolium (NBT) (0.8 mg/ml), phenazine methosulfate (PMS) (0.167 mg/ml), and L-lactate (3.24 mg/ml), or D-lactate(12.96 mg/ml) dissolved in 10 mM Tris-HCl (pH 8.5) buffer (Nissen and Schousboe, 1979; Pathak and Vinayak, 2005) incubated at 37 ˚C for 20 min, or longer time until saw the expected bands, stopped the reaction by 5% acetic acid.

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C. Cell viability assay

Cell viability assay was performed by MTT assay. Once the cells reach confluence, cells were plated in 48-well plates at a density of 4 x 104 cells/well, treated in the control and experimental groups and incubated overnight. MTT solution (5 mg/ml) was then added to each well and incubated at 37° C for 4 h Viable cells metabolize MTT, producing a purple color. And MTT solvent consisting of 0.1% NP-40 in 4 mM HCl, isopropanol is added to each well of 200 μl to dissolve formazan crystals. Mix for 15 min at RT to ensure complete solubilization and the absorbance values of each well was measured at 570-595 nm using a microplate reader. Normalized to the absorbance of the untreated control, the results indicated by the viability of the control. All experiments were determined in triplicate.

D. LDHB, LDHA siRNA transfection

Bioneer negative control siRNA and Bioneer LDHB siRNA (sequence: 5’-3’GAAAUGUCAACGUGUUCAA), LDHA siRNA (sequence: 5’-3’ GAG CAU AAU GAA GAA CCU U). Use Neon electroporation system (Thermo Fisher Scientific). Whole HL-1 cells with 80-90% confluency is used for transfection. Remove HL-1 cell media and collect cell pellet 1500 x g 3 min after trypsin treatment. The pellet was resuspended with 100 μl of buffer R and 10 μl of 10 pmol siRNA. 3 ml of Buffer E was added to the NEON electroporation system instrument and transfection was performed using NEON TIP under 1300 V, 20 ms and two pulse conditions. After transfection, siRNA-transfected cells were incubated for 48h-96h and then processed for further study.

E. D-lactate measurement

Prepare 100 ul of 1M perchloric acid homogenized HL-1 cell pellet and culture soup for deproteinization. After centrifugation (13000 rpm, 15 min, 4 ℃), take supernatant carefully. Take 1 x aqueous phase. Add 0.34 x 2 M KOH, then placed in the ice for 10 min. Next, samples centrifuged at 13000 rpm for 15 min at 4 ℃ and collect supernatant. This supernatant is D-lactate sample for the next step.

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D-lactate quantification requires two enzymatic reactions. In a first reaction, D-lactate is catalyzed by D-lactate dehydrogenase (D-LDH) and oxidized to pyruvate in the presence of nicotinamide adenine dinucleotide (NAD+). However, further reactions are required to capture pyruvate products because the reaction equilibrium is advantageously present in D-lactate and NAD+. This is accomplished by converting pyruvate to D-alanine and 2-oxoglutarate using the D-glutamate-pyruvate transaminase (D-GPT) enzyme in the presence of excess D-glutamate. In the coupled reaction, the amount of NADH formed is stoichiometric with the amount of D-lactate. NADH is measured by the increase in absorbance at 340 nm. The D-lactate was quantified using an enzymatic kit (Megazyme K-DATE, Megazyme International Ireland Ltd.), according to the manufacturer's recommendations. Experiments were performed on 96 well microplates. 10ul of sample or standards were used. 150 μl of Distilled water 50 μl of buffer, 10 μl of NAD+, 2 μl of D-GPT are added to each well. Mix and read the absorbance at 340 nm after 3 min. Add 2 μl of D-LDH into each well. Mix and read absorbance at 340 nm after 5-20 min (at the end of the reaction).

F. L-lactate measurement

The L-lactate was quantified using an enzymatic kit (Lactate Assay Kit, MAK064, Sigma-Aldrich), according to the manufacturer's recommendations. In this assay, lactate concentration is measured by enzymatic analysis, which produces a colorimetric (570 nm) product in proportion to the lactate present Lactate standards for colorimetric detection dilute 10 μl of 100 nmole/μl lactate standard with 990 μl lactic acid assay buffer to generate 1 nmole/μl standard solution. And add 0, 2, 4, 6, 8, 10ul 1nmole / μl lactate standards to 96 well plates to create 0 (blank), 2, 4, 6, 8, 10nmole / well standards. Add Lactate Assay Buffer to each well to make a volume of 50 μl. The assays require 50 μl of sample for each reaction (well). Tissue or cells are homogenized with 4 volumes of the Lactate Assay Buffer. To remove insoluble material, samples centrifuged at 13,000 rpm for 10 min. And to remove lactate dehydrogenase, samples should be deproteinized with a 10 kDa MWCO spin filter Soluble fractions can be analyzed directly. Directly by adding in duplicate to 96 well plate. Bring samples to final volume of 50 μl /well with Lactate Assay Buffer.

Add 50 μl of master reaction mix (Lactate Assay Buffer 46 μl, Lactate Enzyme Mix 2 μl, Lactate Probe 2 μl) required for each reaction (well). Use a shaker or pipette and protect the plate from light and incubate the reaction for 30 min at RT. Measure absorbance at 570 nm.

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G. Western blotting assay

Protein extracts from cultured HL-1 cardiomyocytes. Preparation HL-1 cell pellet and lysed in RIPA buffer containing proteinase inhibitor cocktail. The cell lysates were further homogenized by short sonication, centrifuged at 13,000 rpm for 10 min at 4°C. and the resulting supernatant collected.

DC ™ Protein Assay (BIO-RAD) was performed for protein concentration quantification. Samples containing the same amount of protein were run under conditions of 80 V 20 min, 120 V 1 h using 10-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the protein was then Transferred to vinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% BSA for 1h and incubated with primary , Actin (Santa Cruz [sc1616] 1:2,000), anti-Tubulin (Santa Cruz [sc32293] 1:2,000), anti-Lamin A (Abcam [ab26300] 1:5,000), (anti-LDHA (Abcam [ab9002] 1:10,000), anti-LDHB (Abcam [ab85319] 1:5,000),anti VDAC (Abcam [ab140817] 1:5,000) and anti-MG-H1(AGE) (cell biolabs [STA-011] 1:1,000) for 2 h at RT or overnight at 4 ℃. After washing with TBS containing Tween-20 (TBST), the membranes were incubated with peroxidase-conjugated secondary mouse, rabbit and sheep antibodies (1:5,000) for 1 h. Protein bands incubated with enhanced chemiluminescence reagents (ECL) were visualized using Developer. To ensure equal protein loading, Anti-Tubulin, Actin and lamin A were used as a control for protein level quantification.

H. Quantification of methylglyoxal by HPLC

0.1 g of cell harvest or tissue homogenized with 1 ml of 10 mM ortho-phenylenediamine (o-PD) in 0.45 N perchloric acid (PCA). After Bead homogenization and sonication, incubated for 24 h in RT on orbital shaker in dark. PCA treatment allowed protein to precipitate proteins from the samples and to inhibit metabolic reactions. Samples were centrifuged at 12,000 rpm for 10 min at 4°C to remove precipitated protein. Methyl glyoxal (MGO) was derivatized with a final concentration of 10 mM o-PD to form stable 2-methyl quinoxaline (2-MQ). In 10 minutes of PCA treatment, after removing the precipitated protein, o-PD was added to the supernatant. On the other hand, for 3 h or 24 h of PCA treatment, o-PD was added to the sample with PCA for 3 h or 24 h and incubated for 3 h or 24 h at RT (Chaplen et al., 1996).

Before performing HPLC system, the sample was further centrifuged at 13000 rpm for 10 min at 4℃, supernatant is used as a sample. 20 μl of samples were quantified on Agilent 1200 HPLC system

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via ZORBAX eclipse plus c18 4.6 x 150mm, 5-micron column, by plotting the concentration of standard 2-methylquinoxaline (2-MQ) derivative as a function of peak area detected at 315 nm. Acetonitrile 20% was used for mobile phase. Each samples was run for 30 min with a flow rate of 1 ml/min. 5-Methylquinoxaline (5-MQ) was use as internal standard (Dhar et al., 2009).

I. Methylglyoxal Determination by ELISA

The methylglyoxal was quantified using Competitive ELISA (Cell biolabs, STA-811, OxiSelect™ Methylglyoxal (MG) Competitive ELISA Kit), according to the manufacturer's recommendations. The OxiSelect™ Methylglyoxal (MG) ELISA Kit is an enzyme immunoassay developed for rapid detect and quantify of MG-H1 (methylglyoxal-hydroimidazolone) protein adducts. The amount of MG adducts in a protein sample is calculated by comparing the absorbance with the absorbance of a known MG-BSA standard curve. Just before use, prepare MG-conjugate coated ELISA plate. 100 μl of 500 ng/ml of MG-conjugate to each well to tested and incubate overnight at 4 ℃. Remove the MG-conjugate coating solution and wash twice with 1 x PBS. Then, 200 μl of Assay diluent is added to each well and blocked for 1 hour at RT in an orbital shaker. Transfer the plate to 4 ℃ and remove the assay diluents immediately before use. Add 50μl of samples and standards to the wells of the MG-conjugate coated plate. Add 50μl of diluted anti-MG antibody to each well and incubate at RT for 1 h on an orbital shaker. After incubation, anti-MG antibody is added followed by HRP conjugated secondary antibody. Add 100 μl of substrate solution to each well. Incubate for 20 min at RT on an orbital shaker. To stop the reaction, 100 μl of stop solution is added to each well to stop the enzyme reaction. Results should be read immediately.

Read the absorbance of each well using 450 nm on a microplate reader. Protein adducts in protein samples are quantified by comparison with a predetermined MG-BSA standard curve.

J. Mitochondria isolation

Mitochondria isolation was performed using the Mitochondria Isolation Kit for Cultured Cells (cat.89874, Thermo Scientific). Add protease inhibitors to Reagent A and Reagent C immediately before use. Add 800 μl of mitochondrial isolation reagent A, vortex for 5 s at medium speed and incubate the tube for exactly 2 min on ice. Add 10 μl mitochondrial isolation reagent B and vortex at maximum speed for 5 s. Incubate the tube for 5 min on ice and vortex every minute at full speed. Add 800μL Mitochondrial isolation reagent C. Invert the tube several times to mix (do not vortex).

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Centrifuge tubes for 10 min at 700 x g at 4°C. Transfer the supernatant to a new 2.0 mL tube and centrifuge at 12,000 x g for 15 min at 4°C. The supernatant containing the cytosol fraction is transferred to a new tube. The isolated mitochondria are contained in pellets. Add 500 μl Mitochondrial separation reagent C to the pellet and centrifuge for 5 min at 12,000 x g. Remove the supernatant. Store mitochondrial pellets on ice before further experimental treatment.

K. Complex activity Ⅰ enzyme activity assay

Mitochondria OXPHOS complex Ⅰ (NADH dehydrogenase) enzyme activity assay was measured using Complex I Enzyme Activity Assay Kit (Abcam, ab109721). Antibodies captured on precoated microplates immobilize complex I, whose activity has been determined upon oxidation of NADH to NAD+ and simultaneous reduction of provided dyes to increase absorbance at OD 450 nm. The prepared samples are put in a microplate wells and incubated at RT for 3 h. Assay solution is prepared by mixing 1 x dilution buffer, 20 x NADH, 100 x Dye. Wash plate wells with wash buffer and add 200 μl of Assay solution to each well. Read absorbance of each well on a microplate reader using 450 nm in kinetic mode at RT for up to 30 min.

L. Statistical analysis

All data were calculated as the average of at least three independent samples. Results were reported as mean ± SEM. Data was analyzed using Student's t-test and one-way ANOVA. Statistical significance was determined if P value ≤ 0.05.

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III. RESULTS A. LDHB plays a major role in D-lactate metabolism

Five LDH isozymes are formed by combining LDHA (subunit A) and LDHB (subunit B) in different proportions. The LDHA subunit prefers to convert pyruvate to lactate and the LDHB subunit prefers to convert lactate to pyruvate. LDH1 has a large LDHB subunits ratio and LDH5 has a large LDHA subunits ratio. LDH activity was confirmed when L-lactate and D-lactate were added to isoenzyme pattern gel (Fig.4A). D-lactate was also confirmed to have L-LDH activity and compared to L-lactate, the activity of LDH1 was greater than that of LDH5 in D-lactate (Fig.4B). We thought that LDH1 plays an important role in L-lactate and D-lactate metabolism.

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Figure 4. LDH enzyme activity in extract

(A) Native-PAGE and LDH activity staining, (B) analyzed activity percentage bar graph. (*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)

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B. LDH enzyme activity to D-lactate

In order to confirm the D-lactate specific activity of LDH1, the enzyme activities of LDH1 and LDH5 were measured under D-lactate 20 mM and NAD 5 mM conditions. The activity of LDH5 was only 37.8% of LDH1 (LDH1-3.86 D-lactate (mM) / mg / min, LDH5- 1.46 D-lactate (mM) / mg / min) (Fig.5). LDH 1 was found to be important in the D-form lactate because it consists only of LDHB subunits.

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Figure 5. LDH enzyme activity in pure enzyme

Human LDH1 and LDH5 activity to D-lactate (*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)

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C. Loss of LDHB in cardiomyocytes increased D-lactate

I performed knockdown LDHB in cardiomyocyte cell line HL-1 with siRNA, and knockdown efficiency was verified by Western blotting (Fig. 6A) and LDH isoenzyme staining (Fig. 6B). In addition, D-lactate level was increased by 200% in the LDHB knockdown HL-1 cells (Fig. 6C).

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Figure 6. LDHB knockdown HL-1cells increased D-lactate level

(A) Western blot, LDHB and LDHA was normalized with lamin A, (B) LDH isoenzyme assay with L-lactate, (C) D-lactate measurement, (D) Mitochondria complex1 activity assay.

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D. Loss of LDHB in cardiomyocytes increased Methylglyoxal

The most generally used method for measurement of methylglyoxal involves the derivatization of methylglyoxal with 1,2-diaminobenzene derivatives, such as o-phenylenediamine (o-pd), subsequent quantify of the resulting quinoxaline with high performance liquid chromatography (HPLC). 2-MQ derivatized with methylglyoxal by o-phenylenediamine (o-pd). Methylglyoxal was determined by quantification of 2MQ via HPLC analysis (Agilent 1200 HPLC system via ZORBAX eclipse plus c18 4.6 x 150mm, 5-micron column). I analyzed MG-derivatives to identify peaks in chromatograms of samples at the same retention time (Fig.7A; retention time: 2MQ 8.1 min, 5MQ 15.5 min) to analyze the internal standard 5MQ and 2MQ. In the LDHB KD HL-1 cells, methylglyoxal level was increased by 35% (Fig.7B). D-lactate was not metabolized, and D-lactate was increased, confirmed that methylglyoxal was increase.

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Figure 7. LDHB Knockdown increased Methylglyoxal in HL-1 cell

(A) HPLC results (B) Methylglyoxal measurement analyzed percent bar graph by HPLC (*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)

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E. Loss of LDHB in cardiomyocytes increased AGEs

The interaction of methylglyoxal with arginine leads to the formation of the specific AGEs, methylglyoxal-derived hydroimidazolone (MG-H1) and tetra hydro pyrimidine (THP) (Vistoli et al., 2013). methylglyoxal-derived hydroimidazolone (MG-H1) is the major product of MG-specific glycation and about 90% of all adducts. Methylglyoxal levels were measured indirectly with MG-H1. In the LDHB KD HL-1 cells, AGEs (MG-H1: hydroimidazolone) level was increased by 150% (Fig.8A,8B). The methylglyoxal accumulation was again measured using MG-H1 ELISA kit (Fig.8C). In other words, AGEs accumulated in LDHB knockdown cells. The experiment was performed triple and calculated as the average value

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Figure 8. LDHB Knockdown increased MG-H1(AGEs) in HL-1 cell

(A) Western blot analysis for AGEs, (B) MG-H1 was normalized with Actin, (C) MG-H1 ELISA. (*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)

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F. Loss of LDHB in cardiomyocytes decreased complex activity

In addition, I found that mitochondrial complex activity was reduced in LDHB KD cells (Fig.9). It can be seen that LDHB deficiency induces mitochondrial defects.

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Figure 9. LDHB Knockdown reduced Complex Ⅰ activity in HL-1 cell

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G. Generation of LDHB heart specific KO mouse

Previously our lab generated LDHB gene knockout mouse by Cre/loxP system (Fig. 10A). Conditional knockout is referred to as tissue specific knockout. Conditional mouse has floxed alleles with loxP sites on either side of the target gene to be removed. To knock out tissue-specific knockdown, Cre recombinase is expressed using a promoter of a gene that expresses only in the desired tissue. When Cre recombinase is expressed, target genes between the two loxP sites are removed. LDHB genes with loxP sites on both sides can be recombinated and inactivated by Cre recombination enzyme that recognizes two loxP sites. Recombination occurs only in cells that express Cre recombinase. Thus, LDHB remains active in all cells and tissues that do not express Cre recombinase. I recombined Cre recombination enzymes only in heart specific tissue. Through Western blot (Fig.10C) and isoenzyme patterning analysis (Fig.10D), I confirmed successful generation of Heart specific LDHB knockout mouse (Saihali, 2014; Song Mi, 2017).

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Figure 10. Generation of heart specific knockout mouse

(A) LDHB Knockout mouse model by Cre/loxP system, (B) analysis of control and targeted ES cells, (C) protein level of LDHB expression in LDHB knock out mouse heart tissue, (D) LDH isozyme pattern in LDHB knock out mouse heart tissue

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H. LDHB knockout mouse heart increased methylglyoxal

Methylglyoxal increased in LDHB knockdown HL-1 cell experiments, and the same experiment was performed in LDHB knockout mouse. As expected, in the LDHB knockout heart, Methylglyoxal level was increased by 24.4% (Fig.11).

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Figure11. Loss of LDHB increase methylglyoxal in knockout mouse

Methylglyoxal level was determined with HPLC and normalized with tissue wet weight (*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)

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I. LDHB knockout mouse heart increased AGEs

In knockout of LDHB hearts, measurements were performed using the MG-H1 ELISA kit. As expected, Methylglyoxal level was increased by 45% (Fig.12A). Furthermore, AGEs accumulated in LDHB knockout heart (Fig.12B), and interestingly, Western blot data showed mitochondria was more sensitive to compare with whole lysate (Fig.12C). More AGEs were found in mitochondria than whole cells.

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Figure 12. Loss of LDHB increase AGEs in knockout mouse

(A) Monoclonal anti-AGEs (MG-H1) antibody was used for ELISA, (B),(C) Western blot for AGEs in isolated mitochondria and whole cells (*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)

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J. LDHB deficiency cause mitochondrial dysfunction

Mitochondrial function is not only important for energy metabolism, but also plays an important role in the aging process. Mitochondrial function depends on the oxford complex of the mitochondrial lining. In addition, the essential subunits of the complex are encoded by mitochondrial DNA, so mitochondrial DNA homeostasis is important for the aging process.

Methylglyoxal, on the other hand, can damage not only proteins, but also lipids and DNA. The increased methylglyoxal levels in the LDHB knockout heart were thought to damage mitochondria more than WT. Indeed, LDHB knockout heart decreased mitochondrial DNA levels (Fig.13A, 13B), mitochondrial mRNA levels (Fig.13C) and tRNA levels also decreased. (Fig.13D) Also, LDHB knockout heart reduced mitochondrial complex activity and Mitochondrial transcription factor A (TFAM) (Fig.13E). TEM imaging showed that most of the mitochondria morphology were unhealthy in the LDHB knockout heart (Fig.13F).

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Figure 13. Loss of LDHB causes mitochondrial dysfunction

(A) Southern blot, mitochondrial DNA was normalized with 18s rRNA, (B) % mt DNA analysis, (C) northern blot, mitochondrial mRNA levels, (D) and mitochondrial tRNA levels, (E), western blot, mitochondrial protein levels, (F) TEM image (*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)

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K. Loss of LDHB induces cardiomyopathy

Heart specific LDHB knockout cause cardiomyopathy. Survival was reduced in mouse at 30 weeks of age (Fig.14).

40-week-old knockout mouse significantly increased the heart / weight ratio indicating myocardial hypertrophy, but 20-weeks- knockout mouse slightly increased but were not significant (Fig.15A). In addition, 40-week-old KO mouse had myofibrosis (Fig.15B).

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Figure 15. Loss of LDHB induces cardiomyopathy

(A) Heart weight (g)/ body weight (g) ratio, (B) Trichrome staining (*p<0.05,**p<0.01,***p<0.001 by Student’s t-test)

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Ⅳ. DISCUSSION

The mechanism of D-lactate, the detoxification product of methylglyoxal at present, is not yet known. It is hypothesized that accumulation of D-lactate will increase methylglyoxal due to feedback inhibition. Increasing methylglyoxal will result in the accumulation of AGEs, disruption of cell function and various diseases. So, I think that the metabolism of D-lactate is important for methylglyoxal detoxification. I think LDH, which converts lactate to pyruvate, will play an important role in D-lactate metabolism.

Lactate dehydrogenase (LDH) activity was confirmed when L-lactate and D-lactate were added to the isoenzyme pattern gel (Fig. 4A). This result shows that L-lactate dehydrogenase also metabolizes D-lactate. In addition, the activity of LDHB was higher than that of LDHA in D-lactate compared to L-lactate (Fig. 4B). This means that isoenzyme with LDHB subunits play a greater role in D-lactate metabolism. I found that LDH plays a role in D-lactate metabolism and that LDHB plays an important role.

To demonstrate whether LDHB is involved in D-lactate metabolism, experiments were performed with LDHB removal from HL-1 cells. siRNA LDHB knockdown in HL-1 cells showed an increase in D-lactate compared to the control (Figure 6C). I demonstrate a decrease in D-lactate metabolism in the absence of LDHB. As hypothesized, experiments were conducted to confirm that an increase in D-lactate caused an increase in methylglyoxal. Quantitative results of Methylglyoxal in LDHB knockdown Cells, methylglyoxal level increased 20% compared to control (Fig. 7). As expected, AGE increases as methylglyoxal increases, confirming the accumulation of AGEs in LDHB knockdown cells (Fig.8). It was measured by whether AGEs that interfere with cell function will destroy the mitochondria of my interest. In the absence of LDHB, mitochondrial complex activity was reduced (Fig.9). Further experimentation is needed to measure the change in glyoxalase Ⅰ, Ⅱ levels in the LDHB deficiency of the glyoxalase pathway, the detoxification pathway of methylglyoxal.

Based on the previous results, experiments were performed with the mouse removed from LDHB to determine if the actual disease was induced. Methylglyoxal was quantified in LDHB knockout mouse cardiac extract as in cell experiments (Fig.11), and AGEs were also increased by 40% (Fig.12A). In addition, mitochondria were isolated from LDHB knockout mouse heart tissues and confirmed the

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accumulation of AGEs (Fig.12B). I thought that LDHB deficiency damaged mitochondria. So, I confirmed that it causes mitochondrial dysfunction in LDHB KO mouse.

The function of mitochondria depends on the electron transport complex of the inner membrane. Mitochondrial DNA levels were decreased in LDHB knockout mouse (Fig.13A, 13B) and RNA levels were also decreased (Fig.13C, 13D). Similarly, mitochondrial transcription factor A (TFAM) protein and mitochondrial complex subunit protein levels were also reduced in LDHB knockout mouse (Fig.13E). In addition, electron microscopy confirmed that the morphology of mitochondria was not normal in the LDHB knockout mouse heart (Fig.13F). LDHB knockout mouse had a reduced survival rate after 30 weeks (Fig.14), and 40-week-old LDHB knockout mouse had cardiac hypertrophy (Fig.15A) and myofibrosis (Fig.15B). This confirmed that LDHB deficiency caused cardiomyopathy. Since LDHB is present in many hearts, I used only cardiac specific mouse. Further experimentation with the whole body LDHB knockout mouse is considered necessary.

In summary, LDH uses D-lactate as a substrate as well as L-lactate. LDHB plays an important role in D-lactate metabolism. LDHB loss increases D-lactate and methylglyoxal. It also causes mitochondrial defects and induces cardiomyopathy.

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Ⅴ. REFERERNCES

1. Allaman, I., Belanger, M., and Magistretti, P.J. (2015). Methylglyoxal, the dark side of glycolysis. Front Neurosci 9, 23.

2. Chang, T., and Wu, L. (2006). Methylglyoxal, oxidative stress, and hypertension. Can J Physiol Pharmacol 84, 1229-1238.

3. Chaplen, F.W., Fahl, W.E., and Cameron, D.C. (1996). Method for determination of free intracellular and extracellular methylglyoxal in animal cells grown in culture. Anal Biochem 238, 171-178.

4. Chen, S.J., Aikawa, C., Yoshida, R., and Matsui, T. (2015). Methylglyoxal-derived hydroimidazolone residue of plasma protein can behave as a predictor of prediabetes in Spontaneously Diabetic Torii rats. Physiol Rep 3.

5. Claycomb, W.C., Lanson, N.A., Jr., Stallworth, B.S., Egeland, D.B., Delcarpio, J.B., Bahinski, A., and Izzo, N.J., Jr. (1998). HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci U S A 95, 2979-2984.

6. Dhar, A., Desai, K., Liu, J., and Wu, L. (2009). Methylglyoxal, protein binding and biological samples: are we getting the true measure? J Chromatogr B Analyt Technol Biomed Life Sci 877, 1093-1100.

7. Drent, M., Cobben, N.A., Henderson, R.F., Wouters, E.F., and van Dieijen-Visser, M. (1996). Usefulness of lactate dehydrogenase and its isoenzymes as indicators of lung damage or inflammation. Eur Respir J 9, 1736-1742.

8. Jain, M., Nagar, P., Sharma, A., Batth, R., Aggarwal, S., Kumari, S., and Mustafiz, A. (2018). GLYI and D-LDH play key role in methylglyoxal detoxification and abiotic stress tolerance. Sci Rep

8, 5451.

9. Kalapos, M.P. (2008). Methylglyoxal and glucose metabolism: a historical perspective and future avenues for research. Drug Metabol Drug Interact 23, 69-91.

10.Moraru, A., Wiederstein, J., Pfaff, D., Fleming, T., Miller, A.K., Nawroth, P., and Teleman, A.A. (2018). Elevated Levels of the Reactive Metabolite Methylglyoxal Recapitulate Progression of Type 2 Diabetes. Cell Metab 27, 926-934 e928.

11. Nissen, C., and Schousboe, A. (1979). Activity and isoenzyme pattern of lactate dehydrogenase in astroblasts cultured from brains of newborn mouse. J Neurochem 32, 1787-1792.

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12. Pathak, C., and Vinayak, M. (2005). Modulation of lactate dehydrogenase isozymes by modified base queuine. Mol Biol Rep 32, 191-196.

13. Saihali (2014). Early onset of age related hearing loss in LDHB knock out mouse (KR: 아주대학교).

14. Song Mi, H. (2017). LDHB 유전자 삭제에 의한 허혈성 신경세포 사멸 감소 (GG: 아주대학교).

15. Thornalley, P.J. (1993). The glyoxalase system in health and disease. Mol Aspects Med 14, 287-371.

16. Thornalley, P.J. (2008). Protein and nucleotide damage by glyoxal and methylglyoxal in physiological systems--role in ageing and disease. Drug Metabol Drug Interact 23, 125-150.

17. Valvona, C.J., Fillmore, H.L., Nunn, P.B., and Pilkington, G.J. (2016). The Regulation and Function of Lactate Dehydrogenase A: Therapeutic Potential in Brain Tumor. Brain Pathol 26, 3-17. 18. Vander Jagt, D.L., and Hunsaker, L.A. (2003). Methylglyoxal metabolism and diabetic complications: roles of aldose reductase, glyoxalase-I, betaine aldehyde dehydrogenase and 2-oxoaldehyde dehydrogenase. Chem Biol Interact 143-144, 341-351.

19. Wetzels, S., Wouters, K., Schalkwijk, C.G., Vanmierlo, T., and Hendriks, J.J. (2017). Methylglyoxal-Derived Advanced Glycation Endproducts in Multiple Sclerosis. Int J Mol Sci 18.

20. Jaime M. Ross., (2010). High brain lactate is a hallmark of aging and caused by a shift in the lactate dehydrogenase A/B ratio. PNAS 107 (46), 20087-20092;

21. Monica L. Acosta., (2005). Early markers of retinal degeneration in rd/rd mouse. Mol Vis. 11:717-28

22. Alberts B, Johnson A, Lewis J, et al. (2002) ‘The Mitochondrion’. Molecular Biology of the Cell. 4th edition. Garland Science

23. Sepideh Aminzadeh-Gohari, René Günther Feichtinger, Barbara Kofler., Chapter 7. Energy Metabolism and Metabolic Targeting of Neuroblastoma. (2019) Neuroblastoma. Molecular Mechanisms and Therapeutic Interventions. 2019, Pages 113-132.

24. Asha Kumari (2018) Glycolysis, in Sweet Biochemistry.

25. Chris Higgins.(2011) L-lactate and D-lactate - clinical significance of the difference. acutecaretesting.org

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- 국문요약 –

젖산

탈 수소효소 결핍은 메틸글리옥살을 증가시키고 심근병증을 유도한

메틸글리옥살 (Methylglyoxal)은 단백질 기능을 망가뜨리고 최종 당화 산물 (Advanced glycation end products, AGEs)를 형성하는 반응성이 높은 화합물이다. 최종 당화 산물 (AGEs)은 세포 기능을 망가뜨리기 때문에 다양한 질병을 유발한다. 최종 당화 산물 (AGEs)를 유발하는 메틸글리옥살은 해당 과정에서 생성된다. 해당과정은 포도당을 피 루브산으로 변환하고 피루브산은 미토콘드리아 TCA 주기로 들어가 ATP를 생성하거나 세포질에서 피루브산을 젖산으로 전환시키는 효소인 젖산 탈수소 효소 (lactate dehydrogenase, LDH)에 의해 젖산으로 전환된다. 포도당의 지속적인 유입은 비 효소 반 응에 의해 디하이드록시 아세톤 포스페이트 (DHAP)의 메틸글리옥살로의 전환을 향상 시킨다. D-젖산은 메틸글리옥살의 해독 메커니즘 인 글리옥살라제 시스템을 통해 최종 생성물로서 생성된다. 이 연구에서 L-젖산 탈 수소 효소 (LDH)가 D-젖산 대사에서 중 요한 역할을 하고, 젖산 탈 수소 효소 5개의 동종 효소 중의 LDHB 소단위체를가진 효 소가 D-젖산에 대한 높은 효소 활성을 갖는다는 것을 발견했다. 이 논문에서는, 생체 내 및 시험 관내 LDHB 제거 분석을 수행하여 입증한다. 젖산 탈 수소 효소 B 결핍은 D-젖산 수준을 증가시키고 메틸글리옥살을 축적하며 미토콘드리아 기능 장애를 유발하 는 것으로 나타났다. LDHB 결핍은 또한 마우스에서 심근 병증을 유도한다 Keywords: 젖산 탈 수소 효소, 메틸글리옥살, d-젖산, 최종 당화 산물, 심근병증

수치

Figure 1. Methylglyoxal (MGO) is the main a precursor of advanced glycation end products  (AGEs)
Figure 2. Glyoxalase pathway
Figure 3. Lactate dehydrogenase
Figure 4. LDH enzyme activity in extract
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