Alterations in renal mitochondrial respiration in response to the reactive oxoaldehyde methylglyoxal

Mariana G. Rosca1, Vincent M. Monnier2, Luke I. Szweda3, and Miriam F. Weiss4

1 University of Medicine and Pharmacy of Iasi, Iasi, Romania 6600; and 2 Institute of Pathology and Department of Biochemistry, 3 Department of Physiology, and 4 Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chronic hyperglycemia has been linked to alterations in mitochondrial function, suggesting an important role in the pathophysiology of the complications of diabetes mellitus. In the diabetic kidney, ultrastructural changes in mitochondria are associated with impaired tubular function. The goal of this study was to determine if methylglyoxal (MGO), a dicarbonyl compound reaching high levels in hyperglycemic conditions, has direct toxicity for renal mitochondria. Intact mitochondria isolated from the renal cortex of rats were incubated with MGO to determine 1) its effect on mitochondrial respiration, 2) the conditions under which MGO exerts these effects, and 3) the potential mitochondrial targets of MGO influence. This study demonstrates that MGO has an inhibitory effect on both the tricarboxylic acid cycle and the electron respiratory chain. The modifications appear to be specific to certain mitochondrial proteins. Alterations of these proteins lead to disturbances in mitochondria that may play an important role in renal cellular toxicity and in the development of diabetic nephropathy.

dicarbonyl; diabetic nephropathy; mitochondria


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DIABETIC NEPHROPATHY is commonly characterized by progressive extracellular matrix accumulation, glomerular hypertrophy, and evidence of tubular cell loss (44). These structural modifications can be explained as the effect of chronic hyperglycemia upon the cell cycle (44), leading to proliferation of both interstitial fibroblasts and mesangial cells (45) and to apoptosis of tubular cells (18). How chronic hyperglycemia causes such cellular alterations remains incompletely defined. Mitochondria seem to be an important gear in the mechanistic pathways leading to cellular damage in diabetes. Significant relations between the disturbances of mitochondrial metabolism and each of the main molecular mechanisms involved in chronic diabetic complications were recently described (27). Short-term hyperglycemic conditions, as produced in cell culture conditions, have been associated with increased mitochondrial potential and superoxide formation (46). Conversely, in chronic diabetes mellitus, a diminution of mitochondrial respiration has been demonstrated, particularly in those tissues that are highly dependent on aerobic metabolism, such as heart (10, 11, 39) and brain (21). Complexes I, III, and IV of the electron respiratory chain (ERC) seem to be the main mitochondrial targets of hyperglycemia-induced injury. In addition, diabetes decreases the capacity of the tricarboxylic acid cycle to generate reducing equivalents in the mitochondria (1, 3, 8, 14, 32, 37). The intensity of mitochondrial failure has been directly correlated with the duration of diabetes. Moreover, in early stages of diabetic nephropathy, ultrastructural changes in renal proximal tubular mitochondria have been found to correlate with disturbances in the main functions of renal tubular cells (19). Although it has been suggested that chronic hyperglycemia may alter the proper function of mitochondria by causing alterations to mitochondrial proteins through glycation pathways (20), the specific compound(s) responsible for this effect have not been identified.

Methylglyoxal (MGO), a physiological but chemically highly reactive alpha -dicarbonyl metabolite of glucose degradation pathways, is the major source of intracellular and plasma advanced glycation end products (AGEs) in diabetes. In chronic diabetes mellitus, increased MGO is derived from the triose phosphate pool of glycolytic metabolites, the increased formation and metabolism of acetone, and the catabolism of threonine in body muscles (41). It is highly chemically reactive with arginine, lysine, and sulfhydryl groups on proteins (24) and nucleic acids (29), inducing the formation of a variety of structurally identified AGEs. As a result of such interactions, MGO can influence multiple aspects of cellular biology (6, 7, 22, 24, 28, 40, 41). In mitochondria isolated from malignant cells, it rapidly and irreversibly inhibits the respiratory rate (33) and aerobic glycolysis (15), resulting in a drastic decrease of cellular ATP. MGO has been shown to have a direct suppressive effect on mitochondrial complex I (34), without inhibition of succinate- and alpha -ketoglutarate-dependent respiration (2).

In this study, the MGO-induced inactivation of mitochondrial respiratory rates was investigated as a contributing factor to the development of diabetic complications. Because the intracellular concentration of both free and bound MGO that is induced by diabetes is still unknown (4, 5), the experiments below were designed to elucidate the potential role of MGO as a renal mitochondrial toxin, independent of the intracellular glucose level. Intact mitochondria isolated from rat renal cortex were incubated with MGO to determine the effects of MGO on mitochondrial respiratory rates, the conditions under which MGO exerts these effects, and the specific mitochondrial sites of MGO-induced dysfunction. This study demonstrates that MGO has an inhibitory effect on both the tricarboxylic acid cycle and the ERC. It decreases NADH-linked respiration and has no effect on succinate-linked respiration. MGO-induced modifications are specific to certain mitochondrial proteins. We identify intracellular dicarbonyl stress as a potential mechanism by which chronic hyperglycemia may give rise to mitochondrial dysfunction. This study provides the basis for future investigation of the etiological role of mitochondrial damage in renal cellular toxicity leading to diabetic nephropathy.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

MGO of the highest analytical grade was obtained from Fluka Chemicals (Milwaukee, WI) and then was further purified by vacuum distillation (30, 38). Pyruvic acid, acetol, D-lactic acid, L-lactic acid, and, except as noted, all other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Measurement of Mitochondrial Respiration

Renal mitochondrial isolation. Mitochondria were isolated from the cortical nephrons of young adult Sprague-Dawley rats (150-200 g) according to a previously published standard technique (26) with modifications of constituents of the isolation buffer. Briefly, before processing, the resected kidneys were decapsulated and halved sagitally, and the medulla was dissected out with iridectomy scissors, leaving only the cortex. Isolation buffer consisted of 0.25 M sucrose, 2 mM EDTA, and 25 mM Tris · HCl, pH 7.4. With the use of this technique, mitochondria could be stored at 4°C for up to 5 h without a change in state 3 or state 4 respiratory rates. Only mitochondria that had a respiratory ratio of about six or higher at 0 min incubation in respiratory buffer were used in this study.

Mitochondrial respiration. Mitochondrial respiration was measured using a polarographic Clark electrode (model 1060; Instech Laboratories, Philadelphia, PA) kept at a constant 24°C temperature with a pumping water bath. Mitochondria at a concentration of 0.5 mg/ml were incubated for varying times in oxphos buffer (125 mM KCl and 5 mM KH2PO4 at pH 7.25) in the presence or absence of varying concentrations of MGO. Time-dependent O2 consumption was measured every 15 s. State 2 respiration was initiated by adding either NADH-linked substrates (10 mM glutamate and 5 mM malate) or FAD-linked substrates (10 mM succinate, in the presence of rotenone). After stabilization for 2 min in the chamber at 24°C, ADP (0.3 mM) was added to begin state 3 respiration. After ADP exhaustion, state 4 respiration was monitored. For measuring respiration under the influence of a classic uncoupler, 60 µM 2,4-dinitrophenol (DNP) was added instead of ADP. O2 consumption was expressed either as nanomoles O2 per minute per milligram mitochondrial protein, as a percentage from control mitochondria, or as a respiratory quotient. All measurements were made within the first 3 h after mitochondrial isolation.

Assay for Mitochondrial NADH Levels

A spectrofluorophotometer (model RF-5301PC; Shimadzu Biotech) was used to measure production and consumption of NADH in intact mitochondria (0.5 mg mitochondrial protein/ml) at room temperature with excitation and emission wavelengths of 360 and 430 nm, respectively. Known quantities of NADH were added to 0.5 mg/ml mitochondria for calibration.

Assay for ERC-NADH Oxidase

To determine the ability of mitochondria to utilize exogenously added NADH, we used a protocol developed by Humphries et al. (17) with some modifications. Mitochondria (0.5 mg/ml) were diluted to a protein concentration of 0.2 mg/ml with a hypotonic buffer (10 mM MOPS and 0.5 mM EDTA, pH 7.4) and then sonicated for 30 s to provide access of NADH and cytochrome c to the electron transport chain. O2 consumption was monitored via a Clark-style electrode, after the addition of 9 µM cytochrome c and 0.4 mM NADH.

Western Blot Detection of Specific Mitochondrial Proteins Modified by MGO

Isolated rat renal mitochondria were incubated at varying concentrations and for varying times, under conditions identical to those used in measuring respiration. A 1:200 dilution of protease inhibitor cocktail (Sigma) was added to each mixture to prevent proteolytic damage to proteins during the incubations. After incubation, any remaining MGO was scavenged by the addition of 10 mM aminoguanidine. Mitochondria were separated by centrifugation at 5,000 g for 5 min and resuspended in storage buffer (5 mM KH2PO4 and 0.5 mM EDTA, pH 7.25; see Ref. 25). Centrifugation did not result in a significant change in mitochondrial respiration.

The selective interaction of MGO with mitochondrial proteins and the molecular weight distribution of MGO-induced modifications were examined using a Western blotting technique. BSA, fraction V (essential fatty acid free; 50 mg/ml), incubated with 20 mM MGO in 0.1 M phosphate buffer (pH 7.3 at 37°C for 7 days) was used as a positive control. Mitochondrial proteins were loaded at 30 µg/lane, and control protein was loaded at 3.65 µg/lane on duplicate 14% Tris-glycine-polyacrylamide gels (Invitrogen Life Technologies, Carlsbad, CA). One gel of the duplicate was stained with Coomassie blue to assess the size distribution of mitochondrial proteins. The second gel was electroblotted on a nitrocellulose membrane and probed using a monoclonal antibody to MGO-derived imidazole AGE (1H7G5, gift of Dr. Michael Brownlee) at 1:10,000 dilution. Briefly, membranes were incubated overnight in the presence of blocking buffer with carbonyl-free Tween-based detergent and then in the presence of primary antibody for 3 h at room temperature. Binding of primary antibody was detected by incubating the membranes with a 1:1,000 dilution of anti-mouse IgG conjugated to alkaline phosphatase (Bio-Rad Laboratories, Hercules, CA) for 1 h at room temperature. After incubation with a chemiluminescent alkaline phosphatase substrate (Tropix; Promega, Madison, WI), primary antibody bound to MGO-modified proteins was observed by autoradiography.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mitochondrial Respiration

Incubation of renal cortical mitochondria with different concentrations of MGO for varying times results in a decline of the NADH-linked ADP-dependent respiration in a dose- and time-dependent manner. Control mitochondria had a state 3 respiratory rate of 96.58 ± 5.23 nmol O2 · mg mitochondrial protein-1 · min-1, and a state 4 respiratory rate of 16.09 ± 1.65 nmol O2 · mg-1 · min-1 with glutamate and malate as substrates, after incubation for 5 min in the respiratory buffer. The ADP-to-O2 ratio (ADP/O) was 3.1. In Fig. 1, a typical experiment demonstrates the effect of MGO on O2 consumption by renal mitochondria. Control mitochondria respond with a characteristic increase in the rate of O2 consumption after the addition of ADP (state 3) and a leveling off of consumption with ADP exhaustion (state 4). Mitochondria (0.5 mg/ml) exposed to MGO, 50 µM for 5 min at 24°C, exhibit an almost 50% diminution in the rate of O2 consumption in state 3 and an increase in the rate of O2 consumption in state 4. These values correspond to an ADP/O ratio of 2.72, indicating a slightly uncoupling effect.


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Fig. 1.   Effect of methylglyoxal (MGO) on renal mitochondrial respiration. Mitochondria at a concentration of 0.5 mg/ml were incubated for 5 min in oxphos buffer with NADH-linked substrates (10 mM glutamate, 5 mM malate) at room temperature in the absence (control, solid line) or presence (broken line) of 50 µM MGO. O2 consumption in state 2 was monitored beginning at 0 min. State 3 respiration was initiated with the addition of 0.3 mM ADP at 2 min (arrow). State 4 respiration is the rate of O2 consumption after depletion of ADP. The abscissa indicates the dynamic measurement of O2 consumption.

Exposure of mitochondria to increasing concentrations of MGO (10-200 µM) for 5 min at 24°C, before testing respiration, results in progressive inactivation of state 3 respiration in a saturable fashion (Fig. 2A). The concentration of MGO required for half-maximal inhibition (IC50) is ~50 µM. State 4 respiration, occurring after ADP is consumed, showed a progressive increase to a maximal level (average of 160% above control) at 75 µM MGO (Fig. 2B). At concentrations of MGO >75 µM, we measured a diminution of state 4, suggesting a generalized inhibition of both ERC and the tricarboxylic acid cycle. The respiratory quotient, defined as the ratio of state 3 to state 4 respiration, provides a measure of the leakiness of the inner mitochondrial membrane. The decline in respiratory quotient reaches a saturable level, and the ratio of the two states falls to 1.0 (Fig. 2C).


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Fig. 2.   Mitochondrial respiration as a function of MGO concentration. A: change in state 3 respiration is expressed as a percentage of baseline. B: change in state 4 respiration is expressed as a percentage of baseline. C: respiratory quotient (the ratio of state 3 to state 4 respiration) as a function of MGO concentration. Data are means ± SD of 3 experiments.

As shown in Fig. 3, the rate of state 3 respiration declined in a time-dependent manner in the presence of MGO. State 3 respiration, expressed as a percentage, fell to a steady-state level of <50% when mitochondria were preincubated with 50 µM MGO for >5 min (Fig. 3A). Similarly, state 4 respiration increased an average of 130% above control after 5 min of incubation with 50 µM MGO. At incubation times >5 min, there was a fall in state 4 respiration because of MGO's inhibitory effect (Fig. 3B). The decline in respiratory quotient reaches a saturable level, and the state 3-to-state 4 ratio falls to one if renal mitochondria are incubated with 50 µM MGO for >5 min (Fig. 3C).


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Fig. 3.   Effect of incubation time on mitochondrial respiration in the presence or absence of MGO. A: change in state 3 respiration is expressed as a percentage of baseline, for each of the conditions. B: change in state 4 respiration is expressed as a percentage of baseline. Solid line, control (no MGO); broken line, 50 µM MGO. C: respiratory quotient (the ratio of state 3 to state 4 respiration) as a function of incubation time. Data are means ± SD of 3 experiments.

We also observed that the inhibitory effect of MGO on O2 consumption by renal mitochondria was irreversible. After incubation with MGO for 5 min, centrifugation at 5,000 g for 10 min to remove the excess MGO, resuspension in isolation buffer, and monitoring of O2 consumption, the renal mitochondria did not regain their respiratory ability.

To test the specificity of the effect of MGO on mitochondrial respiration, structurally similar compounds and metabolites of MGO were incubated with normal rat renal cortical mitochondria for periods up to 15 min. Figure 4 shows that there was no change in state 3 respiration in the presence of acetol (500 µM), L-lactate (500 µM), or D-lactic acid (500 µM). Pyruvic acid (500 µM), a structurally similar compound and a potential substrate for mitochondrial respiration, induced a small 10% increase in state 3 respiration at 5 min compared with baseline. In comparison, 50 µM MGO induced a suppression of respiration in a time-dependent manner. Similarly, there was no change in state 4 respiration induced by acetol, L-lactate, D-lactate, and pyruvic acid, in contrast to a nearly 30% increase in state 4 respiration induced by 50 µM MGO at 5 min.


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Fig. 4.   Lack of change in state 3 (A) and state 4 (B) mitochondrial respiration in response to incubation with 500 µM D-lactate, L-lactate, acetol, and pyruvate contrasts with 50 µM MGO. Pyruvate is a normal substrate, which increases state 3 respiration ~10%.

Uncoupled mitochondrial respiration. MGO could inhibit ADP-dependent respiration by acting in one of two ways: through an effect on adenine nucleotide translocase and/or ATPase, or by suppressing the tricarboxylic acid cycle and ERC. To differentiate between these two mechanisms, the effect of MGO on respiration was examined in mitochondria treated with the classical uncoupling agent DNP. DNP is known to increase O2 consumption independently of ADP transport or ATP synthesis but dependent on the supply of reducing equivalents (tricarboxylic acid cycle) and ERC components. MGO also inhibits uncoupled respiration in a concentration- and time-dependent manner (data not shown). IC50 was similar to that observed for coupled respiration. This suggests that MGO acts on the tricarboxylic acid cycle or ERC components.

Mitochondrial NADH synthesis and consumption. Steady-state levels of NADH in mitochondria represent a balance between production and consumption. To determine the effect of MGO on this balance, NADH levels were monitored by spectrofluorophotometry during the respiration of intact mitochondria. MGO-mediated declines in mitochondrial respiration appeared because of both diminished levels of reducing equivalents available to the ERC and because of the decreased speed of NADH consumption by the ERC. Intact and MGO-incubated mitochondria showed an initial decline in mitochondrial NADH level upon addition of 0.3 mM ADP, reflecting the increased requirement for NADH to support maximal rates of respiration. As shown in Fig. 5A, preincubation of mitochondria (0.5 mg/ml) with 50 µM MGO for 5 min resulted in both a reduction in the level of NADH consumed upon addition of ADP and a slow recovery of NADH to a normal level. When NADH was allowed to reach steady-state levels with glutamate and malate as substrates, addition of 50 µM MGO resulted in a decrease in NADH concentration (Fig. 5B). This decline in NADH levels during state 2 respiration is likely the result of decreased rates of NADH production relative to NADH consumption.


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Fig. 5.   Representative experiment demonstrating mitochondrial NADH synthesis and consumption. A: solid line, control mitochondria; broken line, mitochondria exposed to 50 µM MGO for 5 min before testing. The ability of mitochondria to consume NADH was measured upon addition of 0.3 mM ADP at 2 min (arrow). In the presence of MGO, there is a delay in recovery and a decrease of NADH levels. B: mitochondria (0.5 mg/ml) were allowed to respire in respiratory buffer in the presence of NADH-linked substrates (state 2) followed by the addition of 0 µM (solid line) and 50 µM (broken line) MGO at 2 min.

NADH and succinate-linked ADP-dependent mitochondrial respiration. NADH and FADH are electron donors with parallel effects in the ERC. Both donate electrons to ubiquinone, which is reduced to ubiquinol. Glutamate and malate are NADH-linked substrates for electron flow through complexes I, III, and IV. Succinate is an FADH-linked substrate for electron flow through complexes II, III, and IV. To distinguish sites of MGO-induced inhibition of respiration, we tested the effect of MGO on respiration in the presence of these two groups of substrates. MGO inhibited both ADP-dependent and uncoupled respiration in the presence of glutamate and malate (NADH-linked substrates; Figs. 1-4). In contrast, there were no significant effects of MGO on succinate-linked respiration at concentrations ranging from 1.0 to 200 µM (data not shown). This suggests that the inhibitory effect is mainly the result of diminution of the electron flux through complexes II, III, and IV.

Effect of MGO on ERC. The NADH oxidase assay was employed to determine the ability of the ERC to utilize exogenously added NADH. Exposure of mitochondria to 50 µM MGO for 5 min before testing respiration resulted in a 27% inhibition in ERC activity. If renal mitochondria were allowed to respire in the presence of cytochrome c and NADH, the addition of 50 µM MGO resulted in ~30% inhibition of the activity (Fig. 6), indicating an extremely rapid effect of MGO on the ERC.


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Fig. 6.   NADH oxidase assay. Mitochondria (0.5 mg/ml) were diluted to a protein concentration of 0.2 mg/ml with a hypotonic buffer and sonicated so that NADH and cytochrome c were in contact with the electron respiratory chain. O2 consumption was monitored after the addition of cytochrome c followed by NADH at 1 min. MGO (50 µM) was added at 4 min (dashed line).

Mitochondrial Protein Modification by MGO

To determine whether MGO modifies mitochondrial proteins in a selective manner, the molecular weight distribution of mitochondrial proteins incubated with MGO was probed by Western blot analysis. Figure 7, left, shows the Coomassie blue stain for proteins. Figure 7, right, shows a second membrane loaded with the same quantities of proteins, probed with anti-imidazole AGE monoclonal antibody. Figure 7, lane A, left, demonstrates a single protein band for MGO-modified BSA (Coomassie stain), and lane A, right, demonstrates several bands of immunoreactivity for imidazole modifications of BSA (antibody 1H7G5). No MGO-derived imidazole modifications were detected in mitochondrial proteins from normal control mitochondria (lane B, right). With increasing time and dose of MGO, increasingly intense signals were detected in several specific protein bands (lanes C-F, right). We are currently working to identify the structure of the mitochondrial protein modified by MGO.


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Fig. 7.   Presence of MGO-derived imidazole-advanced glycation end products (AGE) in mitochondrial proteins. Control MGO-BSA, -3.65 µg/lane; mitochondrial proteins, -30 µg/lane. Left: Coomassie blue stain demonstrates a single protein band for MGO-modified BSA (lane A) and multiple bands for mitochondria (lanes B-F). Right: membrane was probed with an antibody against MGO-derived imidazole AGE (no. 1H7G5). Lane A, several bands of immunoreactivity for the imidazole modification of BSA, the presence of high-molecular-weight fractions suggesting the presence of AGE cross-linked products of albumin; lane B, intact renal mitochondria; lanes C and D, incubation of renal mitochondria for 5 min in the presence of 50 and 75 µM of MGO, respectively; lanes E and F, incubation for 15 min with 50 and 75 µM of MGO, respectively. With increasing time and dose of MGO, increasingly intense signals were detected in several specific protein bands.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Modification of mitochondrial metabolism is considered one of the main pathophysiological mechanisms of diabetic complications (27, 46). Protein components of the mitochondria may be the target of the glycation reactions (20) and, as a result of the ERC malfunction, the origin of the reactive oxygen species (ROS) that have been reported to be increased in diabetes (31, 43). Moreover, diabetes has been associated with decreased capacity for the generation of reducing equivalents in the tricarboxylic acid cycle (1, 8, 32, 37). Although it has been shown that chronic diabetes mellitus is associated with alterations in mitochondrial function (10, 11, 21, 39, 42), the specific targets and the mechanism of inactivation have not been deciphered. The aim of this study was to establish if MGO, one of the most reactive dicarbonyl compounds that accumulates in blood and organs in diabetic conditions, has a direct influence on intact renal cortical mitochondria. We provide evidence that MGO has an inhibitor effect on NADH-linked ADP-dependent respiration. MGO exerts an inhibition of both the tricarboxylic acid cycle and the ERC. Furthermore, we show that MGO attaches to specific mitochondrial proteins to form MGO-derived imidazole AGEs. These modifications were observed over a relatively short period of time and at concentrations of MGO that encompass the experimental range in cell culture and approach the pathological range in diabetes. Therefore, our study provides evidence that mitochondria are a subcellular target of dicarbonyl-induced modifications and that MGO-induced mitochondrial inhibition represents a potential mechanism by which chronic hyperglycemia mediates mitochondrial failure and cellular toxicity.

To understand the contribution of dicarbonyl-induced mitochondrial dysfunction in the pathogenesis of diabetic complications, it is first critical to define the mitochondrial sites that are more sensitive to MGO modifications. These in vitro data show that MGO decreases both the availability of NADH to complex I and the ERC.

There are a number of mechanisms by which MGO may affect the level of NADH. It is likely that MGO causes leakage of NAD+/NADH from the mitochondria, which is reflected by the increase in state 4 respiration, typical of permeabilized mitochondria. Alternatively, MGO exerts its effects on respiration by inhibiting the synthesis of NADH because of its high reactivity and possible damage of protein components of the tricarboxylic cycle enzymes.

The present study also shows that MGO inhibits the ERC. These results are concordant with observations from animal models of chronic diabetes in which particular dysfunction of the ERC takes place in highly O2-dependent tissues, i.e., the heart (10, 11, 39, 42) and the brain (21). The amount of ERC inhibition found in this study cannot be responsible for the diminution of whole mitochondrial respiration. At least 50% diminution in a given part of the ERC activity is necessary for metabolic expression (35, 36). MGO, in a concentration of 50 µM, slightly decreased the ADP/O ratio, a marker for mitochondrial ability to couple the consumption of O2 to the phosphorylation of ADP. The reduction in the ADP/O ratio occurred in mitochondria that did not have overt defects in the diabetic respiratory control ratio (23). These alterations were associated with increased formation of ROS. Therefore, we predict that, in chronic hyperglycemia, long-term exposure to low doses of MGO might result in greater and more generalized inhibition of mitochondrial function, extrapolating from the in vitro time courses demonstrated in this paper. Both the concentrations of MGO and the time required for inhibition of mitochondrial respiration during the conditions of our experiments suggest that MGO-mediated mitochondrial dysfunction will take place during degenerative events associated with chronic diabetes.

Modifications of mitochondrial proteins by MGO may lead to diminution of substrates to ERC, functional impairment of ERC complexes, and diminution of ATP synthesis, despite biochemical and physiological protection of renal mitochondria against injury (35). The first two effects seem to coexist under the conditions of this experiment. MGO exerts a rapid and irreversible effect on the tricarboxylic acid cycle activity, which appears in large part to be responsible for the inhibition of whole respiration. In the case of oxidative phosphorylation, the imbalance of the components of the ERC might be responsible for the formation of O2 free radicals. The redox centers upstream from the functional defect may become more reduced and hence donate electrons directly to molecular O2 to form superoxide (31, 43). Previous studies have shown that the cytotoxicity of MGO is associated with ROS production (22) and with the loss of the mitochondrial membrane potential (9). Chronic ROS exposure can inactivate the iron-sulfur centers of the ERC complexes I, II, and III and tricarboxylic acid cycle aconitase. Thus the imbalance between ERC components engages mitochondria in a vicious cycle in which it becomes both the source and the target of excess ROS (43). The mitochondrial permeability transition pore is particularly sensitive to ROS, and its opening results in uncoupling of oxidative phosphorylation, a decrease in ATP synthesis, mitochondrial depolarization, and release of proapoptotic molecules (12, 13, 16). Thus the inhibitory effects of MGO may be related to ROS production and may unleash signals leading to cell apoptotic death. These data lead to the hypothesis that apoptotic death of renal tubular cells in diabetes is a result of mitochondrial damage by intracellular MGO. In the case of ATP synthesis, the decrease of energy supply to tubular cells may be responsible for disturbances in tubular function found in early stages of diabetes.

Future research will be needed to identify 1) those subunits of the ERC and tricarboxylic acid cycle enzymes modified by MGO, 2) the biochemical mechanism(s) by which MGO modifies mitochondrial proteins, 3) the relation of the MGO effect on mitochondria and mitochondrial-induced oxidative stress, and 4) the relative contribution of these processes to the alteration of mitochondrial function associated with diabetic nephropathy.


    ACKNOWLEDGEMENTS

We thank Dr. Michael Brownlee for the generous gift of monoclonal anti-imidazole antibody.


    FOOTNOTES

This work was supported by a fellowship to M. G. Rosca from the Fulbright Foundation for International Scholars and by National Institutes of Health Grants DK-45619 (M. F. Weiss), DK-990020 (M. F. Weiss and V. M. Monnier), EY-07099 (V. M. Monnier), and AG-016339 (L. I. Szweda).

Address for reprint requests and other correspondence: M. F. Weiss, Dept. of Medicine, Univ. Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106 (E-mail: maf3{at}po.cwru.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 29, 2002;10.1152/ajprenal.00302.2001

Received 25 September 2001; accepted in final form 24 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Bessman, S, and Mohan C. Insulin as a probe of mitochondrial metabolism in situ. Mol Cell Biochem 174: 91-96, 1997[ISI][Medline].

2.   Biswas, S, Ray M, Misra S, Dutta D, and Ray S. Selective inhibition of mitochondrial respiration and glycolysis in human leucocytes by methylglyoxal. Biochem J 323: 343-348, 1997[ISI][Medline].

3.   Boquist, L, Ericsson I, Lorentzon R, and Nelson L. Alteration in mitochondrial aconitase activity and respiration, and in concentration of citrate in some organs of mice with experimental or genetic diabetes. FEBS Lett 183: 173-176, 1985[ISI][Medline].

4.   Chaplen, F, Fahl E, and Cameron C. Method for determination of free intracellular and extracellular methylglyoxal in animal cells grown in culture. Anal Biochem 238: 171-178, 1996[ISI][Medline].

5.   Chaplen, F, Fahl E, and Cameron C. Evidence of high levels of methylglyoxal in cultured Chinese hamster ovary cells. Proc Natl Acad Sci USA 95: 5533-5538, 1998[Abstract/Free Full Text].

6.   Che, S, Asahi M, Takahashi M, Kaneto H, Okado A, Higashiyama S, and Taniguchi N. Selective induction of heparin-binding epidermal growth factor-like activity by methylglyoxal and 3-deoxyglucosone in rat aortic smooth muscle cells. The involvement of reactive oxygen species formation and a possible implication for atherogenesis in diabetes. J Biol Chem 272: 18453-18459, 1997[Abstract/Free Full Text].

7.   Cook, L, Davies J, Yates A, Elliot A, Lovell J, Joule J, Pemberton P, Thornalley P, and Best L. Effects of methylglyoxal on rat pancreatic beta-cells. Biochem Pharmacol 55: 1361-1367, 1998[ISI][Medline].

8.   Craven, P, Melhem M, Phillis S, and DeRubertis F. Overexpression of Cu2+/Zn2+ superoxide dismutase protects against early diabetic glomerular injury in transgenic mice. Diabetes 50: 2114-2125, 2001[Abstract/Free Full Text].

9.   Du, J, Suzuki H, Nagase F, Akhand A, Yokoyama T, Miyata T, Kurokawa K, and Nagashima I. Methylglyoxal induces apoptosis in Jurkat leukemia T cells by activating c-Jun N- terminal kinase. J Cell Biochem 77: 333-344, 2000[ISI][Medline].

10.   Dzhavadov, S, Dzhokharidze T, Dzhaliashvili I, Gel'fgat E, Saks V, and Pogacha G. Energy metabolism and contractile function of the heart in diabetic cardiomyopathy: effect of ischemia and reperfusion. Biokhimiia 57: 1917-1929, 1992[Medline].

11.   Flarsheim, C, Grupp I, and Matlib M. Mitochondrial dysfunction accompanies diastolic dysfunction in diabetic rat heart. Am J Physiol Heart Circ Physiol 271: H192-H202, 1996[Abstract/Free Full Text].

12.   Gellerich, F, Trumbeckaire S, Opalka J, Seppet E, Rasmussen H, Neuhoff C, and Zierz S. Function of the mitochondrial outer membrane as a diffusion barrier in health and diseases. Biochem Soc Trans 28: 164-169, 2000[ISI][Medline].

13.   Green, D, and Reed J. Mitochondria and apoptosis. Science 281: 1309-1312, 1998[Abstract/Free Full Text].

14.   Grindley, PB, Omoruyi FO, Asemoto HN, and Morrison EY. Effect of yam (Dioscorea cayenensin) and dasheen (Colocassia esculenta) extracts on the kidney of streptozotocin-induced diabetic rats. Int J Food Sci Nutr 52: 429-433, 2001[ISI][Medline].

15.   Halder, J, Ray M, and Ray S. Inhibition of glycolysis and mitochondrial respiration of Erlich ascites carcinoma cells by methylglyoxal. Int J Cancer 54: 443-449, 1993[ISI][Medline].

16.   Halestrap, A, Doran E, Gillespie J, and O'Toole A. Mitochondria and cell death. Biochem Soc Trans 28: 170-177, 2000[ISI][Medline].

17.   Humphries, M, Yoo Y, and Szweda L. Inhibition of NADH- linked mitochondrial respiration by 4-hydroxy-2-nonenal. Biochemistry 37: 552-557, 1998[ISI][Medline].

18.   Ishii, N, Ogawa Z, Suzuki K, Numakami K, Saruta T, and Itoh H. Glucose loading induces DNA fragmentation in rat proximal tubular cells. Metabolism 45: 1348-1353, 1996[ISI][Medline].

19.   Kaneda, K, Iwao J, Sakata N, and Takebayashi S. Correlation between mitochondrial enlargement in renal proximal tubules and microalbuminuria in rats with early streptozotocin-induced diabetes. Acta Pathol Jpn 42: 855-860, 1992[Medline].

20.   Kang, Y, Edwards L, and Thornalley P. Effect of methylglyoxal on human leukemia 60 cell growth: modification of DNA, growth arrest and induction of apoptosis. Leuk Res 20: 397-405, 1996[ISI][Medline].

21.   Kaur, G, and Bhardwaj S. The impact of diabetes on CNS. Role of bioenergetic defects. Mol Chem Neuropathol 35: 119-131, 1998[ISI][Medline].

22.   Kikuchi, S, Shinpo K, Moriwaka F, Makita Z, Toshio M, and Tashiro K. Neurotoxicity of methylglyoxal and 3-deoxyglucosone on cultured cortical neurons: synergism between glycation and oxidative stress, possibly involved in neurodegenerative diseases. J Neurosci Res 57: 280-289, 1999[ISI][Medline].

23.   Kristal, B, Jackson C, Chung HY, Matsuda M, Nguyen H, and Yu B. Defects at center P underlie diabetes-associated mitochondrial dysfunction. Free Radic Biol Med 22: 823-833, 1997[ISI][Medline].

24.   Lo, T, Westwood M, McLellan A, Selwood T, and Thornalley P. Binding and modification of proteins by methylglyoxal under physiological conditions. A kinetic and mechanistic study with N alpha-acetylarginine, N alpha-acetylcysteine, N alpha-acetyl-lysine, and bovine serum albumin. J Biol Chem 269: 32299-32305, 1994[Abstract/Free Full Text].

25.   Lucas, T, and Szweda L. Cardiac reperfusion injury: aging, lipid peroxidation, and mitochondrial disfunction. Proc Natl Acad Sci USA 95: 510-514, 1998[Abstract/Free Full Text].

26.   Nedergaard, J, and Cannon B. Overview-preparation and properties of mitochondria from different sources. Methods Enzymol 55: 3-28, 1979[Medline].

27.   Nishikawa, T, Edelstein D, Xue LD, Yamagishi S, Matsumura T, Kaneda Y, Yorek A, Beebe D, Oates J, Hammes H, Giardino I, and Brownlee M. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycemic damage. Nature 404: 787-790, 2000[ISI][Medline].

28.   Okado, A, Kawasaki Y, Hasuide Y, Takahashi M, Teshima T, Fujii J, and Taniguchi N. Induction of apoptotic cell death by methylglyoxal and 3-deoxyglucosone in macrophage-derived cell lines. Biochem Biophys Res Commun 225: 219-224, 1996[ISI][Medline].

29.   Papoulis, A, Al-Abed Y, and Bucala R. Identification of N2- (1-carboxyethyl)guanine (CEG) as a guanine advanced glycation endproduct. Biochemistry 34: 648-652, 1995[ISI][Medline].

30.   Perrin, D, Armarego W, and Perrin D. Purification of Laboratory Chemicals. Oxford, UK: Pergamon, 1966.

31.   Pitkanen, S, and Robinson B. Mitochondrial complex I deficiency leads to increased production of superoxide radicals and induction of superoxide dismutase. J Clin Invest 98: 345-351, 1996[Abstract/Free Full Text].

32.   Punkt, K, Adams V, Linke A, and Welt K. The correlation of cytophotometrically and biochemically measured enzyme activities: changes in the myocardium of diabetic and hypoxid diabetic rats, with and without Gingko biloba extract treatment. Acta Histochem 99: 291-299, 1997[ISI][Medline].

33.   Ray, M, Halder J, Dutta K, and Ray S. Inhibition of respiration of tumor cells by methylglyoxal and protection by lactaldehyde. Int J Cancer 47: 603-609, 1991[ISI][Medline].

34.   Ray, S, Dutta S, Halder J, and Ray M. Inhibition of electron flow through complex I of the mitochondrial respiratory chain of Ehrlich ascites carcinoma cells by methylglyoxal. Biochem J 303: 69-72, 1994[ISI][Medline].

35.   Rossignol, R, Malgat M, Mazat J, and Letellier T. Threshold effect and tissue specificity. J Biol Chem 247: 33426-33432, 1999.

36.   Rossignol, R, Letellier T, Malgat M, Rocher C, and Mazat JP. Tissue variation in the control of oxidative phosphorylation: implication for mitochondrial diseases. Biochem J 347: 45-53, 2000[ISI][Medline].

37.   Seymour, A, and Chatham J. The effects of hypertrophy and diabetes on cardiac pyruvate dehydrogenase activity. J Mol Cell Cardiol 29: 2771-2778, 1997[ISI][Medline].

38.   Shipanova, I, Glomb M, and Nagaraj R. Protein modification by methylglyoxal: chemical nature and synthetic mechanism of a major fluorescent adduct. Arch Biochem Biophys 344: 29-36, 1997[ISI][Medline].

39.   Tanaka, Y, Konno N, and Kako K. Mitochondrial disfunction observed in situ in cardiomyocytes of rats in experimental diabetes. Cardiovasc Res 26: 409-414, 1992[ISI][Medline].

40.   Thornalley, P. Monosacharide autoxidation in health and disease. Environ Health Perspect 64: 297-307, 1985[ISI][Medline].

41.   Thornalley, P. Advanced glycation and the development of diabetic complications. Unifying the involvement of glucose, methylglyoxal and oxidative stress. Endocrinol Metab 3: 149-166, 1996.

42.   Tomita, M, Mukai S, Geshi E, Umetsu K, Nakatain M, and Katagiri T. Mitochondrial respiration impairment in STZ-induced diabetic rat heart. Jpn Circ J 60: 673-682, 1996[ISI][Medline].

43.   Wallace, D. Mitochondrial diseases in man and mouse. Science 283: 1482-1488, 1999[Abstract/Free Full Text].

44.   Wolf, G. Cell cycle regulation in diabetic nephropathy. Kidney Int 58: S59-S66, 2000[ISI].

45.   Wolf, G, and Ziyadeh F. Molecular mechanisms of diabetic renal hypertrophy. Kidney Int 56: 393-405, 1999[ISI][Medline].

46.   Yamagishi, S, Edelstein D, Du X, and Bownlee M. Hyperglycemia potentiates collagen-induced platelet activation through mitochondrial superoxide overproduction. Diabetes 50: 1491-1494, 2001[Abstract/Free Full Text].


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