Reductive Metabolism of AGE Precursors: A Metabolic Route for Preventing AGE Accumulation in Cardiovascular Tissue
Article Figures & Tables
Figures
- FIG. 1.
High glucose–induced AGE formation. A: Western blot analysis of total cell lysates prepared from HUVECs cultured in the presence of normal (NG; 5.5 mmol/l) or high (HG; 30 mmol/l) glucose for 7 days and probed with anti-argpyrimidine antibodies. Western blots from two representative experiments are shown. Inset shows the major immunopositive spot resolved by two-dimensional gel analysis of the cell lysates. The spot, corresponding to a molecular weight of 27 kDa and a pI of 7.83, was identified to be HSP27 by MALDI/MS analysis. B: Fluorescence-activated cell sorter data obtained from HUVECs cultured in normal (i, iii) and high (ii, iv) glucose. Extracellular AGEs were detected by labeling the cells with the anti-argpyrimidine antibody and phycoerythrin-conjugated secondary antibody. To detect total (extracellular + intracellular) AGEs, the cells were permeabilized before antibody treatment. Relative mean fluorescence was calculated by subtracting the fluorescence obtained from the isotype-matched antibody control. Group data are presented as means ± SE. *P < 0.05 vs. normal glucose; n = 3–4.
- FIG. 2.
Methylglyoxal metabolism in endothelial cells. A: GC-MS analysis of acetol formation in HUVECs. For acetol quantification, 13C3-methylglyoxal was synthesized from 13C3-acetone and 13C3-acetol was prepared by incubating 13C3-methylglyoxal with AKR1B1 and 0.15 mmol/l NADPH. i: Natural and 13C3-methylglyoxal were derivatized using O-(2,3,4,5,6 pentaflourobenzyl)-hydroxylamine hydrochloride (PFBHA) extracted in hexane and separated by gas chromatography. The 1,2 dioxime methylglyoxal eluted with a retention time of 13.1 min (inset). On MS analysis, the fragmentation pattern of 12C-1,2, dioxime methylglyoxal showed a parent ion with m/z 462. Ions with m/z 432 and 265 represented the loss of nitric oxide (NO) [M-30] and OC7H3F5 [M-197] groups, respectively, from 1,2 dioxime methylglyoxal. Corresponding ions with m/z 465, 435, and 268 are because of the 13C3-methylglyoxal. ii: Acetol was derivatized using PFBHA and N,O-bis (trimethylsilyl) trifluoroacetamide with trimethylchlorosilane (BSTFA). Derivatized acetol eluted with a retention time of 7.5 min (inset). Ions with m/z 326 and 285 were assigned to TMS-2-oxime acetol suffering a loss of CH3 [M-15] or C3H4O [M-56], respectively. Corresponding ions with m/z 329 and 286 are because of 13C3-acetol. iii: Acetol formation in HUVECs, cultured in media containing 1 mmol/l methylglyoxal for 24 h in the absence and presence of the AKR1B inhibitors sorbinil (50 μmol/l) or tolrestat (25 μmol/l). After treatment, 13C3-acetol was added to cell lysates and lysates were derivatized and analyzed by GC-MS. *P < 0.05 vs. control. B: Western analysis of lysates prepared from HUVECs cultured in normal (NG) or high (HG) glucose, with or without sorbinil (50 μmol/l), probed with anti-argpyrimidine and anti-HSP27 antibodies. Intensity of immunopositive band (argpyrimidine) was normalized to HSP27. Data are presented as means ± SE. *P < 0.05 vs. normal glucose (n = 4) and #P < 0.05 vs. high glucose without sorbinil (n = 4).
- FIG. 3.
AKR-catalyzed reduction of methylglyoxal in mouse heart. A: Acetol generated in effluents of isolated wild-type (C57) or aldose reductase–transgenic hearts perfused with 20 μmol/l methylglyoxal. Acetol concentration was measured by GC-MS after derivatization with PFBHA and BSTFA. 13C3 Acetol was used as an internal standard. Data are presented as means ± SE. *P < 0.01 vs. wild type (n = 4). B: Western blot analysis of cardiac myocytes isolated from adult male C57 mice probed with antibodies raised against AKR1B1 (aldose reductase), AKR1B8 (FR-1), and AKR1A4 (ALDR). Figure shows bands from three different mice. C: Rate of methylglyoxal reduction in homogenates prepared from hearts of wild-type mice (n = 6) or mice with cardiac myocyte–specific transgene expressing AKR1B4 (rat aldose reductase; n = 6) or AKR1B8 (FR-1; n = 6). The enzyme activity was determined with 1 mmol/l methylglyoxal and 0.15 mmol/l NADPH, with or without 1 μmol/l sorbinil. Inset shows Western blots from wild-type and transgenic hearts developed with anti–aldose reductase and anti–FR-1 antibodies. *P < 0.01 vs. wild type (control), #P < 0.01 vs. aldose reductase–transgenic (control), and §P < 0.01 vs. FR1–transgenic (control). AR-TG, aldose reductase–transgenic; WT, wild type.
- FIG. 4.
Genetic ablation of AKR1B3 (aldose reductase) diminishes the reduction of AGE precursors. A: Rate of reduction of glyceraldehyde (i), methylglyoxal (ii), 3-deoxyglucosone (iii), and glyoxal (iv) in cardiac homogenates prepared from wild-type and akr1b3-null mice. The enzyme activity was determined with glyceraldehyde (10 mmol/l), glyoxal (1 mmol/l), methylglyoxal (1 mmol/l), or deoxyglucosone (1 mmol/l) and 0.15 mmol/l NADPH, with or without 1 μmol/l sorbinil. Values are presented as means ± SE. *P < 0.05 vs. wild type (n = 6). Inset shows the expression of the proteins in wild-type and aldose reductase–knockout mice. B: Rate of formation of S-d-lactoylglutathione (i) and S-glycolylglutathione (ii) in homogenates prepared from wild-type and akr1b3-null hearts. Glyoxalase I activity was measured with methylglyoxal (1 mmol/l) or glyoxal (1 mmol/l) and GSH (1 mmol/l) in the absence or presence of glyoxalase I inhibitor BBGC (0.2 mmol/l). Inset to panel i shows Western blots developed from wild-type and akr1b3-null (knockout) hearts using the anti–glyoxalase-I antibody. Data are means ± SE (n = 6). *P < 0.01 vs. wild-type (methylglyoxal or glyoxal) and #P < 0.01 vs. aldose reductase–null mice (methylglyoxal or glyoxal). Inset shows the expression of glyoxalase I in wild-type and aldose reductase–knockout mice. C: Computer simulations for the relative contributions of aldose reductase and glyoxalase I in the metabolism of glyoxal (i) and methylglyoxal (ii). Relative contribution of the enzymes was calculated on the basis of measurement of aldose reductase and glyoxalase I enzyme activities assuming that the concentration of AGE precursors is in steady state achieved between the processes of formation and those of elimination. AR, aldose reductase; WT, wild type.
- FIG. 5.
Increased accumulation of plasma AGEs in the aldose reductase–null mice. Western blots of plasma from nondiabetic and diabetic wild- type and akr1b3-null (aldose reductase–null) mice, probed with anti-argpyrimidine (A) and anti-CML (B) antibodies. Inset shows positive recognition of glyoxlyic acid–treated BSA. Bar graphs show the intensity of indicated anti-argpyrimidine- or anti-CML–positive bands normalized to Amido-Black–stained blots. Data are presented as means ± SE. *P < 0.01 vs. wild type (control), #P < 0.01 vs. aldose reductase–null (control), and §P < 0.01 vs. wild-type diabetic plasma. AR, aldose reductase; WT, wild type.
- FIG. 6.
Increased AGE accumulation in the hearts of aldose reductase–null mice. A: Western blots of heart homogenates from diabetic and nondiabetic wild-type and aldose reductase–null mice were probed with anti-argpyrimidine (A) and anti-CML (B) antibodies. Nondiabetic wild-type and aldose reductase–null hearts served as respective controls. The expression of aldose reductase, FR-1, and ALDR in the hearts of these mice was examined by Western blots developed using anti–aldose reductase, FR-1, and ALDR antibodies. Recombinant proteins were used as positive controls. Bar graphs show the intensity of the indicated anti-argpyrimidine- or anti-CML–positive bands normalized to GAPDH. Data are presented as means ± SE. *P < 0.01 vs. wild type (control), #P < 0.01 vs. aldose reductase null (control), and §P < 0.01 vs. wild-type diabetic. C: Immunohistochemical analyses of AGE accumulation in hearts of diabetic wild-type and aldose reductase–null mice. Sections were stained with anti-argpyrimidine antibody, and staining was quantified by image analysis. Group data shows the extent of staining quantified using the MetaMorph imaging software. Data are presented as means ± SE. *P < 0.01 vs. wild type (diabetic). AR, aldose reductase; RP, recombinant proteins; WT, wild type. (A high-quality color digital representation of this figure is available in the online issue.)
- FIG. 7.
Genetic ablation of aldose reductase exacerbates diabetic lesion formation and AGE accumulation. A: Photomicrographs of cross sections of innominate arteries of 20-week-old nondiabetic (control) and diabetic apoE-null and akr1b3-apoE–null mice. Sections were stained with hematoxylin and eosin, and the lesion area was quantified by image analysis. Data are presented as means ± SE. *P < 0.01 vs. apoE-null (control), #P < 0.01 vs. aldose reductase/apoE–null (control) and §P < 0.01 vs. apoE-null (diabetic). Arterial sections of diabetic apoE-null and akr1b3-apoE–null mice stained with anti-argpyrimidine (B), anti-CML (C), and anti–3-deoxyglucosone imidazolone (D) antibodies. The extent of staining was quantified by image analysis. Data are presented as means ± SE. *P < 0.05 vs. apoE-null (diabetic). AR, aldose reductase. (A high-quality color digital representation of this figure is available in the online issue.)
Tables
- TABLE 1
Steady-state kinetic parameters for the reduction of AGE precursors by AKRs
Substrates Genbank accession number Proteins Km (μM) kcat (min−1) kcat/Km (min−1/μM−1) Glyoxal NM_021473 AKR1A4 3,229 ± 904 45.8 ± 4.2 0.014 NM_001628 AKR1B1 350 ± 20 29.5 ± 2.02 0.082 NM_009658 AKR1B3 333 ± 62 12.1 ± 0.36 0.036 NM_009731 AKR1B7 7,450 ± 1,500 5.6 ± 0.35 0.001 NM_008012 AKR1B8 635 ± 81 20 ± 0.52 0.031 NM_020299 AKR1B10 7,800 ± 359 8.87 ± 0.63 0.002 NM_030611 AKR1C3 NDA — — NM_013778 AKR1C6 12,073 ± 1,031 12.8 ± 0.67 0.001 NM_134066 AKR1C18 NDA — — NM_025337 AKR7A5 5,931 ± 1,393 12.87 ± 0.27 0.002 Methylglyoxal AKR1A4 874 ± 91 483 ± 0.27 0.55 AKR1B1 22 ± 2 37.2 ± 2.21 1.698 AKR1B3 35 ± 4 19.5 ± 2.3 0.56 AKR1B7 1,996 ± 274 8.9 ± 0.31 0.004 AKR1B8 184 ± 15 18.2 ± 1.21 0.098 AKR1B10 1,250 ± 89 26.82 ± 5 0.389 AKR1C3 NDA — — AKR1C6 139 ± 35 7.65 ± 0.7 0.054 AKR1C18 NDA — — AKR7A5 9,075 ± 1,250 22 ± 0.7 0.002 Furfural AKR1A4 5,918 ± 1,379 17.4 ± 0.78 0.002 AKR1B1 316 ± 34 30.86 ± 1.91 0.097 AKR1B3 69 ± 10 13.36 ± 0.93 1.91 AKR1B7 NDA — — AKR1B8 622 ± 100 2.6 ± 0.27 0.001 AKR1B10 1,617 ± 287 178 ± 15 0.11 AKR1C3 156 ± 35 3.09 ± 0.33 0.019 Hydroxymethyl Furfural AKR1A4 6,428 ± 1,479 24.05 ± 4 0.003 AKR1B1 482 ± 92 14.55 ± 2 0.03 AKR1B3 98 ± 15 4.15 ± 0.3 0.042 AKR1B7 NDA — — AKR1B8 1,197 ± 117 7.82 ± 0.05 0.016 AKR1B10 1,117 ± 135 100 ± 15 0.085 AKR1C3 1,939 ± 425 167 ± 0.02 0.008 Deoxyglucosone AKR1A4 2,067 ± 178 777 ± 25 0.376 AKR1B1 112 ± 5 38.9 ± 2 0.345 AKR1B3 89 ± 5 11.6 ± 1 0.13 AKR1B10 3,293 ± 118 14.36 ± 1.9 0.004 AKR1B10 3,293 ± 118 14.36 ± 1.9 0.004 Enzyme activity was measured in 0.1 mol/l potassium phosphate (pH 7.0) using the indicated substrates and 0.15 mmol/l NADPH at room temperature. Proteins were reduced with dithiothreitol before assay. NDA, no detectable activity.
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