Mitochondrial complex I impairment in leukocytes from type 2 diabetic patients

Free Radical Biology & Medicine 50 (2011) 1215–1221

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Mitochondrial complex I impairment in leukocytes from type 2 diabetic patients Antonio Hernandez-Mijares a,b,1, Milagros Rocha a,c,d,1, Nadezda Apostolova d,e, Consuelo Borras f, Ana Jover a,c, Celia Bañuls a,c, Eva Sola a, Victor M. Victor a,c,d,e,f,⁎ a

Endocrinology Service, University Hospital Dr. Peset, Valencia, Spain Department of Medicine, University of Valencia, Valencia, Spain University Hospital Dr. Peset Foundation, 46017 Valencia, Spain d CIBERehd, Valencia, Spain e Department of Pharmacology, University of Valencia, Valencia, Spain f Department of Physiology, University of Valencia, Valencia, Spain b c

a r t i c l e

i n f o

Article history: Received 19 August 2010 Revised 13 January 2011 Accepted 13 January 2011 Available online 22 January 2011 Keywords: Antioxidant Complex I Diabetes Mitochondria Oxidative stress Reactive oxygen species Free radicals

a b s t r a c t Diabetes is associated with oxidative stress. This study evaluated the rates of oxidative stress and mitochondrial impairment in type 2 diabetes patients. The study population consisted of 182 diabetic patients and 50 bodycomposition- and age-matched controls. We assessed anthropometric and metabolic parameters and mitochondrial function by evaluating mitochondrial oxygen (O2) consumption, reactive oxygen species (ROS) production, glutathione (GSH) levels, GSH/GSSG ratio, mitochondrial membrane potential, and mitochondrial complex I activity in polymorphonuclear cells from diabetes type 2 patients. We found an increase in waist circumference and augmented serum levels of triglycerides, proinflammatory cytokines (IL-6 and TNF-α), homocysteine, glycated hemoglobin, ultrasensitive C-reactive protein, glucose, insulin, and homeostasis model assessment of insulin resistance score in diabetic patients versus controls. There was an impairment of mitochondrial function in diabetic patients, evidenced by a decrease in mitochondrial O2 consumption, an increase in ROS production, decreased GSH/GSSG ratio, a drop in GSH levels, and an undermining of the mitochondrial membrane potential. Furthermore, an impairment of mitochondrial complex I was detected. This study supports the hypothesis of an association of type 2 diabetes and the rate of impaired mitochondrial function. We also propose that one of the targets of oxidative stress responsible for diabetes is mitochondrial complex I. © 2011 Elsevier Inc. All rights reserved.

Diabetes is one of the medical conditions that represent the greatest risk of developing cardiovascular disease [1]. Glucose overload may damage the cells through oxidative stress, suggesting that hyperglycemiainduced oxidative stress may account for the pathogenesis of most diabetic complications [2]. It has been speculated that multiple metabolic abnormalities associated with diabetes, such as hyperglycemia, hyperlipidemia, and insulin resistance, contribute to adverse outcomes in diabetes [3,4] and

DOI of original article: 10.1016/j.freeradbiomed.2011.02.015. Abbreviations: BMI, body mass index; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; DBP, diastolic blood pressure; FA, fatty acid; GSH, glutathione; GSSG, oxidized glutathione; HbA1c, glycated hemoglobin; HBSS, Hanks’ balanced salt solution; HDL, highdensity lipoprotein; HOMA-IR, homeostasis model assessment of insulin resistance; IL-6, interleukin-6; Lp(a), lipoprotein a; LDL, low-density lipoprotein; VO2max, maximal O2 consumption; MCB, monochlorobimane; PMN, polymorphonuclear leukocyte; ROS, reactive oxygen species; SBP, systolic blood pressure; TMRM, tetramethylrhodamine methyl ester; TG, triglycerides; TNF-α, tumor necrosis factor α; US-CRP, ultrasensitive C-reactive protein. ⁎ Corresponding author at: University Hospital Dr. Peset Foundation, 46017 Valencia, Spain. Fax: + 34 961622492. E-mail addresses: [email protected], [email protected] (V.M. Victor). 1 These authors contributed equally to this work. 0891-5849/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2011.01.019

severely affect mitochondrial metabolism, leading to the development of impaired glucose-induced insulin secretion in type 2 diabetes [5]. Oxidative stress has been implicated in the etiology of insulin resistance associated with type 2 diabetes [6–8], and in experimental models, insulin resistance is commonly detected in tissues [9,10]. The pathogenesis of mitochondrial dysfunction in obesity or diabetes-related disease is multifactorial and includes mitochondrial uncoupling and oxidative damage [11,12]. Furthermore, it has been demonstrated that mitochondria undergo biogenesis in response to hyperglycemia, but the increased biogenesis is insufficient to accommodate the metabolic load [13]. In addition to providing energy for the cell, mitochondria are recognized as an important site for the generation of reactive oxygen species (ROS) at complex I and III. In this sense, hydrogen peroxide (H2O2) is emitted from mitochondria and is considered an important barometer of mitochondrial function and a modulator of the overall cellular redox environment [14]. Furthermore, there are several studies that support the idea that the rate of mitochondrial H2O2 emission is significantly greater when basal respiration is supported by fatty acids [15,16], raising the question of whether mitochondrial H2O2 emission is a primary factor in the etiology of insulin resistance.

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There is considerable evidence that a chronic, low-grade inflammatory response is ongoing in and actually precedes type 2 diabetes and related syndromes [17,18], and this inflammation may be due, in part, to the effects of hyperglycemia or other metabolic abnormalities on white blood cells [19]. These cells, especially polymorphonuclear leukocytes (PMNs), constitute a critical host defense against most organisms that cause the infections associated with diabetes. Furthermore, PMNs may be contributors to the oxidative stress and inflammation associated with diabetes. In this sense, it has been demonstrated that oxidative stress occurs in the PMNs of polycystic ovary syndrome patients and that it is related to an impairment of mitochondrial function [20]. This study sought to throw light on the relationship between the rate of the impairment of mitochondrial function and type 2 diabetes. Several mitochondrial alterations were detected, such as a decrease in the mitochondrial oxygen (O2) consumption, an increase in ROS production, a drop in GSH levels paralleled by a decrease in GSH/GSSG ratio, as well as changes in TNF-α expression and undermining of the mitochondrial membrane potential. Moreover, we have identified mitochondrial complex I as one of the targets at which the oxidative stress responsible for diabetes takes place. Materials and methods Methods One hundred eighty-two patients (age 56.3 ± 0.7) that consecutively attended the Outpatient Department of Endocrinology of the University Hospital Dr. Peset, Valencia, Spain, were diagnosed with type 2 diabetes according to the criteria established by the American Diabetes Association. Patients were defined as type 2 diabetic if one or more of the following criteria were fulfilled: (1) exhibiting at least twice a fasting serum glucose ≥7.0 mmol/L (126 mg/dl) or a random serum glucose ≥11.1 mmol/L (200 mg/dl), (2) having been informed by their physician that they have diabetes, or (3) taking antidiabetic medication. Patients were not taking allopurinol or any oral antioxidant supplements. The patients were enrolled in the study along with 50 control subjects (age 53.8 ± 1.2). In accordance with the Declaration of Helsinki, all participants were informed of the purpose, risks, procedures, and possible benefits of the study and gave their express consent. The study was approved by the local ethics committee. Study procedure During their first visit, subjects provided their case history and were subjected to a physical examination that included the measurement of body mass index (BMI) and waist circumference. Subjects were given an appointment for the following week and were instructed to attend after fasting for at least 10 h. This appointment took place between 8:00 and 10:00 AM at the Endocrinology Department's functional testing unit for collection of blood for biochemical determinations. At the same time, the subjects were fitted with a Holter to monitor blood pressure over 24 h. Biochemical determinations Blood samples were collected in SST tubes, which were immediately centrifuged at 4 °C for 10 min at 3000 rpm. Aliquots of serum were obtained to perform the following determinations on the day of extraction: glucose, total cholesterol, and triglycerides (TG) using an enzymatic method and high-density lipoproteins (HDL) by precipitation with dextran sulfate in a Beckman LX20 automated analyzer (Beckman Corp., Brea, CA, USA). The intraseries coefficient of variation (CV) was b3.5% for all these determinations. Low-density lipoproteins (LDL) content was calculated using Friedewald's formula for values of triglycerides below 3.39 mmol/L. Lipoprotein a (Lp(a)) levels were

measured by the enzyme-linked immunosorbent assay (ELISA) using a Biopool kit (Trinity Biotech, County Wicklow, Ireland). Insulin was determined by immunochemiluminescence in a DPC Immulite analyzer (Block Scientific Corp., CA, USA) (intratest CV b4%). Insulin resistance was calculated using the homeostasis model assessment of insulin resistance (HOMA-IR) in patients not taking insulin. Ultrasensitive C-reactive protein (US-CRP) levels were quantified by a latex-enhanced immunonephelometric assay (Behring Nephelometer II; Dade Behring, Newark, DE, USA) with an intra-assay CV of 8.7% and sensitivity of 0.01 mg/L. Plasma homocysteine was measured using the Abbott IMX homocysteine assay (Abbott Laboratories, Abbott Park, IL, USA; CV 4%). The proinflammatory cytokines TNF-α and interleukin-6 (IL-6) were measured by ELISA (R&D Systems, Minneapolis, MN, USA). Cells PMNs were isolated from citrated blood samples and incubated with dextran (3%) for 45 min. The supernatant was released over Ficoll– Hypaque and centrifuged at 250 g for 25 min. The pellet was resuspended in lysis buffer, and after centrifugation at room temperature (100 g, 5 min), PMNs were counted in a Neubauer chamber, washed in HBSS medium, and resuspended in a complete RPMI medium. Measurement of O2 consumption PMNs were resuspended (5× 106 cells/ml) in HBSS medium and placed in a gas-tight chamber. O2 consumption was then measured with a Clark-type O2 electrode (Rank Brothers, Bottisham, UK) [20]. Sodium cyanide (10− 3 mol/L) was employed to confirm that O2 consumption was mainly mitochondrial (95 to 99%) [20]. Measurements were collected using the data-acquisition device Duo.18 (WPI, Stevenage, UK) and following a published method [21]. The maximal rate of O2 consumption (VO2max, with endogenous substrates) was calculated using the GraphPad program. A trypan blue exclusion test revealed no changes in cell viability. Measurement of ROS production Two methods were employed to evaluate ROS. Total ROS production was assessed by fluorimetry using a Fluoroskan plate reader or fluorescence microscopy after incubation (30 min) with the fluorescent probe (5 × 10− 6 mol/L) 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), as described elsewhere [22]. Quantitative assessment of H2O2 was performed with the Amplex Red H2O2/Peroxidase Assay kit [22]. Positive control was obtained by treatment of cells with 0.5×10− 4 mol/L H2O2. Determination of glutathione content GSH content was assessed after incubation (30 min) with the fluorescent probe monochlorobimane (MCB; 40 × 10− 6 mol/L) [20]. To summarize, cells seeded on 96-well plates were washed once with phosphate-buffered saline and then incubated with MCB diluted in phosphate-buffered saline. Fluorescence intensities were measured after being maintained at 37 °C for 15 min, using excitation and emission wavelengths of 380 and 485 nm, respectively. The intracellular GSH level was expressed as arbitrary units of fluorescence. Total glutathione and its oxidized form (GSSG) were spectrophotometrically evaluated by monitoring variations in absorbance at 412 nm in the presence of 0.6 mM 5,5′-dithiobis-(2-nitrobenzoic acid), 0.21×10− 3 mol/L NADPH, and 0.5 units of glutathione reductase per milliliter of assay mixture in 50 mM phosphate buffer, pH 7.4 [23]. To calculate the GSH/GSSG ratio, GSH (reduced form) was obtained by subtracting the GSSG values from the total glutathione values, and the values obtained were corrected for spontaneous reactions in the absence

A. Hernandez-Mijares et al. / Free Radical Biology & Medicine 50 (2011) 1215–1221 Table 1 Baseline anthropometric characteristics of diabetes type 2 patients and control subjects.

Number of participants Age (years) Duration of diabetes (years)a Mean SBP (mm Hg) Mean DBP (mm Hg) BMI (kg/m2) Waist circumference (cm) Number of active smokers

Control subjects

Diabetic patients

50 53.8 ± 1.2 — 126.7 ± 2.9 73.6 ± 5.9 27.4 ± 1.6 97.8 ± 1.4 14

182 56.3 ± 0.7 5.2 (2.0–13.0) 121.3 ± 2.6 74.5 ± 3.4 30.1 ± 1.4 107.9 ± 2.1* 19

Data are expressed as mean ± SD. Statistical analysis consisted of Student's t test for unpaired samples (GraphPad Software) and for comparison of means of the normally distributed variables and the Mann–Whitney U test for variables without a normal distribution. a Variables not normally distributed in which the results are expressed as median (quartiles). *P b 0.05 vs control.

of the sample. Proteins were determined using the BCA protein assay kit (Pierce, Rockford, IL, USA). In addition, we also evaluated GSH and GSSG in whole blood collected in heparinized tubes using an HPLC method. For GSSG determination blood samples (0.5 ml) were treated, at 4 °C, with 0.5 ml ice-cold perchloric acid (12%) containing 40× 10− 3 mol/L N-ethylmaleimide (Sigma Chemical Co., St. Louis, MO, USA), to prevent GSH oxidation, and 2 mM BPDS (bathophenanthroline disulfonic acid; Sigma Chemical Co.), as described by Asensi et al. [24]. To measure total glutathione, blood (0.5 ml) was treated at 4 °C with 0.5 mL trichloroacetic acid (30%). Samples were then centrifuged at 15,000 g for 5 min, at 4 °C, and the acidic supernatants were used for GSH, GSSG, and total glutathione measurements. Determination of GSSG by HPLC GSSG was measured by HPLC as previously described [24]. Namely, 500 μl of the acidic supernatants (see above) was derivatized by adding 50 μl of 1 mM γ-glutamylglutamate (Sigma Chemical Co.) prepared in 0.3% perchloric acid. Afterward, the pH was adjusted to pH 8.0 with KOH (2 mol/L)/morpholinopropane sulfonic acid (0.3 mol/L), the samples were centrifuged, and then an aliquot of 25 μl of the supernatant was mixed with 50 μl of 1% 1-fluoro-2,4-dinitrobenzene (Sigma Chemical Co.). After this, derivatization was completed in 45 min and desiccated Table 2 Clinical and metabolic characteristics of diabetes type 2 patients and control subjects.

Number of participants Cholesterol (mmol/L)a TG (mmol/L)a HDL (mmol/L) LDL (mmol/L) Lp(a) (μmol/L)a IL-6 (pg/mL)a TNF-α (pg/mL) HbA1c (%) Homocysteine (μmol/L)a US-CRP (mg/L)a Glucose (mmol/L)a Insulin (pmol/L) HOMA-IR

Control subjects

Diabetic patients

50 5.38 ± 0.1 1.02 (0.88–1.6) 1.21 ± 0.07 3.54 ± 0.11 0.21 (0.10–0.60) 1.9 (1.0–3.2) 3.7 ± 0.4 4.2 ± 0.6 9.7 (8.1–12.3 1.9 (0.8–2.2) 5.38 (4.1–8) 60.3 (42–75) 3.13 (2.1–5.3)

182 5.02 ± 0.2 2.1 (1.6–2.7)* 1.07 ± 0.04* 3.01 ± 0.13* 0.18 (0.11–0.68) 3.7 (2–8.3)* 7.9 ± 0.5* 7.2 ± 1.6* 10.9 (8.2–13.1)* 3.8 (1.5–4.9)* 9.5 (7.2–13.8)* 100.4 (62–138)* 10.9 (6.8–13.5)*

Data are expressed as mean ± SD. Values of serum triglyceride concentrations were normalized using a log transformation. Statistical analysis consisted of Student's t test for unpaired samples (GraphPad Software) and for comparison of means of the normally distributed variables and the Mann–Whitney U test for variables without a normal distribution. The χ2 test was employed for comparison of percentages. HOMAIR = fasting insulin (mU/mL) × fasting glucose (mmol/L)/22.5. a Variables not normally distributed in which the results are expressed as median (quartiles). *P b 0.05 vs control.

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Table 3 Treatment. % of total patients Metformin Glitazone Sulfonylurea Statin Fibrate

60.8 15.2 28.1 49.2 18.7

samples remained stable at −20 °C for several weeks until injection. This procedure reduces GSH oxidation in blood to about 1%. Determination of mitochondrial membrane potential For evaluating mitochondrial membrane potential, cells were incubated (30 min) with the fluorescent probe tetramethylrhodamine methyl ester (TMRM; 1 × 10− 7 mol/L), and fluorescence was detected by fluorimetry using a Fluoroskan plate reader and fluorescence microscopy [20]. The fluorochrome Hoechst 33342 was used for nuclei staining. Analysis of mitochondrial complex I activity The activity of mitochondrial complex I was assessed by calculating the NADH oxidation rate employing a spectrophotometric method [22]. In brief, a cellular homogenate (20 μl, 0.3 mg) was added to 1 ml of potassium phosphate buffer (10− 2 mol/L) containing NADH (10− 4 mol/L) at a temperature of 37 °C. Basal absorbance (340 nm) was recorded for 1 min. Subsequently, 5 μl of ubiquinone (10− 2 mol/L) was added and the rate of NADH oxidation (defined as complex I activity) was measured over a 5-min period. The NADH oxidation rate was calculated from the time-dependent decrease in the slope of absorbance using an extinction coefficient for NADH of 6.81 × 10− 3 mol/L per centimeter at 340 nm. Isolated complex Idependent respiration was evaluated in digitonin-permeabilized cells in the presence of the complex I substrates malate (0.4 × 10− 3 mol/L) and glutamate (3 × 10− 2 mol/L), the complex II substrate succinate (10− 2 mol/L), or the complex I inhibitor rotenone (6 × 10− 6 mol/L) [20]. Drugs and solutions Sodium cyanide, NADH, ubiquinone, glucose, 5′-dithiobis-2nitrobenzoic acid, trypan blue, succinate, rotenone, glutamate, malate, digitonin, arginine, glutathione reductase, γ-glutamylglutamate, BPDS, rotenone and H2O2 were obtained from Sigma–Aldrich (St. Louis, MO, USA). Dextran was purchased from Fluka and Ficoll–Hypaque from Healthcare (Pittsburgh, PA, USA). HBSS was supplied by Cambrex (Verviers, Belgium) and RPMI by Invitrogen (Carlsbad, CA, USA). DCFHDA and Hoechst 33342 were provided by Calbiochem (San Diego, CA, USA). MCB, and TMRM were supplied by Molecular Probes (Eugene, OR, USA). Data analysis Quantitative variables are expressed as means and standard deviation if normally distributed; if not, they are expressed as medians and quartiles. Qualitative data are expressed as total number and percentage. Statistical analysis consisted of Student's t test for unpaired samples (GraphPad Software) and for comparison of means of normally distributed variables and the Mann–Whitney U test for variables without a normal distribution. The χ2 test was employed for comparison of percentages. Unless stated otherwise, all values are means±SEM. Significance was defined as Pb 0.05 and Pb 0.01.

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B O2 consumption

100

Control Diabetic patients 50

0 0

10

20

30

Oxygen consumption (nmol O2 /min/million cells)

Oxygen concentration (µM)

A

2.5

O2 consumption

2.0 1.5

**

1.0 0.5 0.0 Control

Diabetic patients

Time (min) Fig. 1. Effects of type 2 diabetes on mitochondrial O2 consumption. (A) Representative traces showing the rate of O2 consumption in PMNs from control subjects and diabetes type 2 patients in a closed respiration chamber. (B) Summary of oxygen consumption rate analysis. Data are represented as VO2 (nmol O2/min/million cells) and were statistically analyzed by Student's t test. **P b 0.01 vs control.

Results Clinical and metabolic characteristics The anthropometric characteristics of the diabetes type 2 patients and control subjects are presented in Table 1, which shows an increase

(P b 0.05) in waist circumference measurement in type 2 diabetes patients. Fasting levels of TG, IL-6, TNF-α, homocysteine, US-CRP, Hba1c, glucose, insulin, and HOMA-IR were higher in type 2 diabetes patients (P b 0.05; Table 2), whereas the levels of HDL and LDL were lower (P b 0.05). Table 3 shows the treatments being received by the diabetic patients in this study. Mitochondrial O2 consumption First we monitored the rate of O2 consumption in PMNs from the blood of both controls and diabetes type 2 patients when placed in an O2-tight chamber, as previously described [21,22]. The O2 requirement of the tissue was mainly mitochondrial, because addition of sodium cyanide resulted in almost a complete (95–99%) inhibition of O2 consumption (not shown). As can be seen by the change in the curve slopes (representative traces, Fig. 1A), the rate of O2 consumption was lower in diabetes type 2 patients. Fig. 1B presents the reduction in the O2 consumption in type 2 diabetic patients (P b 0.01), expressed as nmol O2/min/million cells. ROS production DCFH-DA fluorescence was significantly higher in the diabetes type 2 patients (Fig. 2A, P b 0.01). A similar increase was observed when the effects were evaluated by confocal microscopy (Fig. 2B). Diabetic patients presented higher levels of H2O2 (Fig. 2 C, P b 0.01). In these experiments we included the exogenous addition of H2O2 as a positive control. Namely, after the addition of 0.5 × 10− 4 mol/L H2O2, an increase of 500% in the fluorescence of DCFH-DA was obtained (data not shown). The addition of the rotenone at 1 × 10− 6 mol/L significantly increased ROS in controls but not in diabetic patients (data not shown). GSH levels Oxidative stress is related to both an increase in ROS production and a decrease in the antioxidant content. As shown in Figs. 3A and C and using two different detection methods, the levels of GSH were significantly lower (P b 0.01) in the diabetes type 2 group. Figs. 3B and D show that the GSH/GSSG ratio was lower among diabetes type 2 patients (P b 0.01), which is of great relevance with respect to oxidative stress.

Fig. 2. Effects of type 2 diabetes on total intracellular ROS content. (A, B) PMNs from control subjects and diabetes type 2 patients were stained with DCFH-DA and changes in the total cellular fluorescence were recorded by (A) fluorimetry and (B) fluorescence microscopy (representative images). (C) H2O2 production in PMNs of control subjects and diabetes type 2 patients detected by spectrophotometry. Data were statistically analyzed by Student's t test. **P b 0.01 vs control.

Mitochondrial membrane potential (Δψm) A diminution in TMRM fluorescence among diabetes type 2 patients (P b 0.01), which was indicative of a reduction in Δψm, was

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Type 2 diabetes impairs mitochondrial complex I Fig. 5A shows the inhibition of mitochondrial complex I activity in type 2 diabetic patients (Pb 0.01), calculated as the rate of NADH oxidation in PMNs, suggesting that complex I was one of the main targets of the mitochondrial dysfunction in PMNs of these subjects. The specificity of the action of type 2 diabetes was further characterized using an alternative method by which cells were permeabilized with digitonin, and isolated complex I-dependent respiration was then measured. Fig. 5B shows that O2 consumption in control PMNs by the complex I substrates malate (0.4×10− 3 mol/L) and glutamate (30×10− 3 mol/L) was inhibited almost 90% with the complex I inhibitor rotenone (6×10− 6 mol/L). The cells from type 2 diabetics respired very poorly with malate and glutamate (Pb 0.05), whereas rotenone-sensitive respiration did not differ from that observed in the absence of the inhibitor. When succinate (10×10− 3 mol/L), a complex II electron donor, was added to bypass complex I-dependent respiration, PMNs from type 2 diabetic patients exhibited O2 consumption rates similar to those of controls, further suggesting that complex I was the main target of mitochondrial dysfunction. Discussion The results of this study demonstrate, as expected, an increase in waist circumference and fasting levels of TG, IL-6, TNF-α, homocysteine, USCRP, HbA1c, glucose, insulin, and HOMA-IR in type 2 diabetic patients. Furthermore, we have demonstrated the grade of mitochondrial function impairment in PMNs that takes place during type 2 diabetes. This effect was evident in the decrease in mitochondrial O2 consumption, the increase in ROS production, the enhanced TNF-α levels and the decrease in GSH/GSSG ratio related to oxidative stress, the drop in GSH levels, and the diminished mitochondrial membrane potential that we observed in

MCB fluorescence (arbitrary units)

30

20

**

10

B

100

GSH/GSSG ratio

75

50

25

** 0

C 2000

GSH concentration (µM)

Total GSH content

Control

Diabetic patients

GSH concentration

** 1000

0 Control

0

Diabetic patients

D 150

[GSH]/[GSSG]

A

these patients. We propose that complex I of the electron transport chain is one of the targets of the ROS generated during type 2 diabetes and produces a decrease in the mitochondrial consumption of O2. Leukocytes from patients with oxidative stress have been reported to be in a proinflammatory state that is expressed by a heightened sensitivity to physiologic hyperglycemia and elevated plasma US-CRP [25]. Recently, it has been reported that insulin signaling per se also regulates mitochondrial O2 consumption and ATP synthesis rates [26,27]. The studies in question demonstrated a coordinate reduction in tricarboxylic acid and fatty acid (FA) enzymes that appeared to impair the delivery of reducing equivalents to the electron transport chain. These data highlight the important role of the insulin signaling pathway in modulating mitochondria bioenergetics and integrity. In addition to mitochondrial dysfunction, insulin resistance in patients with type 2 diabetes is characterized by a reduced VO2max [28]. This parameter, probably the best predictor of functional capacity [29], is positively correlated with insulin sensitivity [30] and is considered to be a strong determinant of insulin sensitivity index in both men and women [31]. Impairment of lipid oxidation has been observed when FA oxidation is measured in the whole body and skeletal muscle from obese or extremely obese (BMI ≥ 40 kg/m2) subjects and in patients with type 2 diabetes. In this sense, mitochondrial dysfunction seems to be caused by both a lower number and a decreased functional capacity of mitochondria [28,31]. Major mechanisms of mitochondrial dysfunction are oxidative stress and ROS-mediated mitochondrial damage. In this work, we have demonstrated an increase in the production of ROS by leukocytes of diabetes type 2 patients under basal conditions, suggesting an impairment of mitochondrial function. In this sense, our study also supports previous observations of mitochondrial dysfunction as a complication of hyperglycemia- and hyperlipidemia-induced ROS production in skeletal muscle from mice [8]. Furthermore, our data show an increase in the release of TNF-α and IL-6 from leukocytes. Given that these cytokines are proinflammatory, these findings provide further evidence of the role of inflammation in

[GSH]/[GSSG]

revealed by both the measurements performed using a Fluoroskan plate reader (Fig. 4A) and confocal microscopy (Fig. 4B).

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Control

Diabetic patients

GSH/GSSG ratio

100

**

50

0

Control

Diabetic patients

Fig. 3. Effects of type 2 diabetes on GSH levels and GSH/GSSG ratio. Total intracellular GSH levels were detected in PMNs from control subjects and diabetic patients, by (A) fluorimetry and (C) spectrophotometry, and (B) the GSH/GSSG ratio was determined spectrophotometrically. (D) GSH/GSSG ratio was also studied by HPLC using whole-cell blood. Data were statistically analyzed by Student's t test. **P b 0.01 vs control.

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Fig. 4. Effects of type 2 diabetes on mitochondrial membrane potential. PMNs from control subjects and diabetic patients were stained with TMRM and total intracellular fluorescence was measured by (A) fluorimetry or (B) confocal microscopy (representative images). Data were statistically analyzed by Student's t test. **P b 0.01 vs control.

the development of insulin resistance in type 2 diabetes. An augmented release of TNF-α from leukocytes after activation by ROS-induced oxidative stress may inhibit insulin signaling and impair glucose uptake [32]. Leukocytes are highly sensitive to the oxidative damage mediated by the ROS that are released from the endothelium and inflammatory cells [33]. Furthermore, leukocytes are especially linked to ROS generation. However, excessive amounts of ROS are harmful to cells, as they can initiate lipid peroxidation and apoptosis [34]. Other, nonmitochondrial sources of ROS have also been associated with type 1 diabetes. In this sense, it has been demonstrated that xanthine oxidase activity is increased in type 1 diabetic animals and that it is a significant cause of oxidative stress and responsible for the activation of the NF-κB signaling pathway [35]. These effects of ROS can be neutralized by the complex antioxidant system developed by the organisms. Thus, antioxidants in general and GSH in particular play a vital role in keeping cells in a reduced environment and protecting them from oxidative stress [36]. In this way, the increase in the production of ROS, decrease in GSH levels and GSH/GSSG ratio, and reduction in the mitochondrial membrane potential—all characteristics of diabetes— point toward a dysfunction within the respiratory chain that compromises the functioning of the mitochondrion as a source of energy. Oxidative stress plays a pivotal role in the development of diabetes complications [37], and atherosclerosis and cardiomyopathy in type 2

diabetes are caused in part by pathway-selective insulin resistance, which increases mitochondrial ROS production from free fatty acids. In this sense, our findings also provide evidence of an impaired mitochondrial function, particularly in mitochondrial complex I. Mitochondria are recognized as an important site for the generation of ROS. In fact, their emission of H2O2 can alter the redox state by either reacting directly with thiol residues within redox-sensitive proteins or altering the GSH/GSSG ratio, the main redox buffer of the cell. ROS are highly toxic to various sites of the mitochondrial respiratory chain, and inhibition of complex I would seem to be the most likely consequence of this toxicity [38]. Furthermore, a reduction in complex I enzyme activity leads to an accumulation of electrons in the initial part of the transport chain (complex I and coenzyme Q), which facilitates the direct transfer of electrons to molecular O2. This ultimately results in the generation of ROS. In addition to being a major site of production, the mitochondrion also represents a target for ROS action. In this study, we have demonstrated that type 2 diabetes is accompanied by a marked reduction in NADH oxidation that is indicative of an undermined complex I activity. Further experiments analyzing isolated complex I-dependent respiration in permeabilized PMNs have demonstrated that, in the presence of succinate, a complex II electron donor added to bypass complex I-dependent respiration, the cells of type 2 diabetes patients exhibit O2 consumption rates similar to those of controls, thereby confirming that this condition mainly affects complex I.

Complex I activity NADH oxidation

Oxygen consumption

A

B 2.0

10

* 5

0

Control

Diabetic patients

Oxygen consumption (nmol O2/min/million cells)

NADH oxidation rate (nmol NADH/min/mg protein)

15

basal

+malate/glutamate

+rotenone

+succinate

1.5

1.0

* 0.5

0.0 Control

Diabetic patients

Fig. 5. Effect of type 2 diabetes on the activity of complex I of the electron transport chain. (A) PMNs from control subjects and diabetic patients were used to determine complex I-dependent NADH oxidation rate. Data were statistically analyzed by Student's t test. *Pb 0.05 vs control. (B) Control and diabetic PMNs were used to determine isolated complex I-dependent respiration in digitonin-permeabilized cells in the presence of the complex I substrates malate (0.4×10− 3 mol/L) and glutamate (3×10− 2 mol/L), the complex II substrate succinate (10− 2 mol/L), or the complex I inhibitor rotenone (6×10−6 mol/L). Data were statistically analyzed by Student's t test. *Pb 0.05 vs corresponding values in the control subjects.

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In conclusion, this study demonstrates the contribution of PMNs to oxidative stress and inflammation in diabetes type 2 patients in relationship to an impairment of mitochondrial function. In addition, we demonstrate that hyperglycemia diminishes the activity of mitochondrial complex I by the insulin resistance that is associated with type 2 diabetes. These data support the hypothesis that mitochondrial activity, and concretely mitochondrial complex I, may represent an important new pharmacological target for the prevention and treatment of type 2 diabetes. Acknowledgments We thank Brian Normanly for his editorial assistance and Isabel Soria for her work in the extraction of the biological samples. This study was financed by Grants PI10/1195, PI09/01025, ACOMP 2010/169, CIBERehd, and PROMETEO 2010/060. V.M.V. and M.R. are recipients of Fondo de Investigacion Sanitaria and Generalitat Valenciana contracts (CES10/030 and CP10/0360, respectively). References [1] Ceriello, A.; Testa, R. Antioxidant anti-inflammatory treatment in type 2 diabetes. Diab. Care 32:232–236; 2009. [2] Brownlee, M. Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820; 2001. [3] Capes, S. E.; Hunt, D.; Malmberg, K.; Gerstein, H. C. Stress hyperglycaemia and increased risk of death after myocardial infarction in patients with and without diabetes: a systematic overview. Lancet 355:773–778; 2000. [4] Cao, J. J.; Hudson, M.; Jankowski, M.; Whitehouse, F.; Weaver, W. D. Relation of chronic and acute glycemic control on mortality in acute myocardial infarction with diabetes mellitus. Am. J. Cardiol. 96:183–186; 2005. [5] Jitrapakdee, S.; Wutthisathapornchai, A.; Wallace, J. C.; MacDonald, M. J. Regulation of insulin secretion: role of mitochondrial signalling. Diabetologia 53:1019–1032; 2010. [6] Evans, J. L.; Goldfine, I. D.; Maddux, B. A.; Grodsky, G. M. Oxidative stress and a stress-activated signalling pathways: a unifying hypothesis of type 2 diabetes. Endocr. Rev. 23:599–622; 2002. [7] Maddux, B. A.; See, W.; Lawrence, J. C.; Goldfine, A. L.; Goldfine, I. D.; Evans, J. L. Protection against oxidative stress-induced insulin resistance in rat L6 muscle cells by micromolar concentrations of alpha-lipoic acid. Diabetes 50:404–410; 2001. [8] Bonnard, C.; Durand, A.; Peyrol, S.; Chanseaume, E.; Chauvin, M. A.; Morio, B.; Vidal, H.; Rieusset, J. Mitochondrial dysfunction results from oxidative stress in the skeletal muscle of diet-induced insulin-resistant mice. J. Clin. Invest. 118: 789–800; 2008. [9] Mazumder, P. K.; O'Neill, B. T.; Roberts, M. W.; Buchanan, J.; Yun, U. J.; Cooksey, R. C.; Boudina, S.; Abel, E. D. Impaired cardiac efficiency and increased fatty acid oxidation in insulin-resistant ob/ob mouse hearts. Diabetes 53:2366–2374; 2004. [10] Park, S. Y.; Cho, Y. R.; Kim, H. J.; Higashimori, T.; Danton, C.; Lee, M. K.; Dey, A.; Rothermel, B.; Kim, Y. B.; Kalinowski, A.; Russell, K. S.; Kim, J. K. Unravelling the temporal pattern of diet-induced insulin resistance in individual organs and cardiac function in C57BL/6 mice. Diabetes 54:3530–3540; 2005. [11] Boudina, S.; Sena, S.; O'Neill, B. T.; Tathireddy, P.; Young, M. E.; Abel, E. D. Reduced mitochondrial oxidative capacity and increased mitochondrial uncoupling impair myocardial energetics in obesity. Circulation 112:2686–2695; 2005. [12] Shen, X.; Zheng, S.; Metreveli, N. S.; Epstein, P. N. Protection of cardiac mitochondria by overexpression of MnSOD reduces diabetic cardiomyopathy. Diabetes 55:798–805; 2006. [13] Edwards, J. L.; Quattrini, A.; Lentz, S. I.; Figueroa-Romero, C.; Cerri, F.; Backus, C.; Hong, Y.; Feldman, E. L. Diabetes regulates mitochondrial biogenesis and fission in mouse neurons. Diabetologia 53:160–169; 2010. [14] Schafer, F. Q.; Buettner, G. E. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic. Biol. Med. 30:1191–1212; 2001. [15] St Pierre, J.; Buckingham, J. A.; Roebuck, S. J.; Brand, M. D. Topology of superoxide production from different sites in the mitochondrial electron transport chain. J. Biol. Chem. 277:44784–44790; 2002.

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