- Email: [email protected]
High molecular weight adiponectin correlates positively with myeloperoxidase in patients with type 2 diabetes mellitus
diabetes research and clinical practice 82 (2008) 179–184
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/diabres
High molecular weight adiponectin correlates positively with myeloperoxidase in patients with type 2 diabetes mellitus P. Bobbert a,*, U. Rauch a,1, B. Stratmann b, P. Goldin-Lang a, S. Antoniak a, T. Bobbert c, H.P. Schultheiss a, D. Tschoepe b a
Department of Cardiology and Pneumology, Medical Clinic II, Charite´ – Universita¨tsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12203 Berlin, Germany b Heart and Diabetes Center NRW, Diabetes Center, Ruhr-University Bochum, 32545 Bad Oeynhausen, Germany c Department of Endocrinology, Diabetes and Nutrition, Charite´ – Universita¨tsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin, Germany
article info
abstract
Article history:
Adiponectin (APN) is present in human plasma as a low molecular weight (LMW), a middle
Received 18 April 2008
molecular weight (MMW) and a high molecular weight form (HMW). As a support to
Received in revised form
determine properties such as anti-atherogenic or atherogenic effects, recent clinical studies
16 July 2008
suppose to determine the ratio of each APN multimer to total APN but not the absolute
Accepted 22 July 2008
plasma concentration of APN. In the present study, the correlation of APN and its multimers
Published on line 7 September 2008
with myeloperoxidase (MPO), an enzyme with pro-inflammatory properties, was examined in patients with type 2 diabetes mellitus.
Keywords: Adiponectin
MPO and APN serum levels were assessed in 49 patients with type 2 diabetes mellitus at the beginning and at the end of an anti-diabetic treatment. After treatment a significant
Diabetes
increase in the ratio of HMW to total APN (from 0.43 0.16 to 0.59 0.14, p < 0.05) was found.
Atherosclerosis
Before treatment, HMW-APN was correlated positively with MPO (r = 0.314, p < 0.05). Moreover, a positive correlation was observed between the increased HMW ratio and MPO during treatment (r = 0.304, p < 0.05). HMW-APN correlates positively with MPO in patients with type 2 diabetes. Therefore, HMW-APN may exert possible pro-inflammatory effects in type 2 diabetes. # 2008 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
Atherosclerosis is a chronic inflammation of the arterial wall, initiated by endothelial cells in response to injury. The inflammatory response is maintained by reactions of lipoproteins, inflammatory cells such as macrophages or lymphocytes, different wall constituents and smooth muscle cells. Patients with type 2 diabetes mellitus are subject to an increased risk of the development of atherosclerosis. The link
between type 2 diabetes mellitus and vascular disease can be shown by the increased expression of vascular cell adhesion molecules or by procoagulant proteins such as plasminogen activator inhibitor 1 in patients with insulin resistance [1]. Furthermore, chronically elevated plasma glucose levels may induce advanced glycation of proteins, which trigger adverse vascular events [2]. A complete explanation underlying the link between diabetes and atherosclerosis still remains unclear. Recent studies point to adipose tissue playing an
* Corresponding author. Tel.: +49 30 8445 2362; fax: +49 30 8445 4648. E-mail address: [email protected] (P. Bobbert). 1 Contributing equally to the first author. 0168-8227/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2008.07.018
180
diabetes research and clinical practice 82 (2008) 179–184
microtitre plate having wells coated with an anti-human APN monoclonal antibody was used to assess the quantity of APN and its multimers directly or indirectly by final incubation with substrate. Colour intensities were determined using an ELISA plate reader at 37 8C (Molecular Devices, US).
important role in the pathogenesis of vascular complications in patients with insulin resistance [3]. Adipose tissue seems to act as an endocrine organ, which produces different cytokines such as adiponectin (APN), leptin, resistin or visfatin [4]. APN, a 30-kDa adipocyte derived hormone, is present in human plasma as a trimer known to exist as a low molecular weight (LMW), a hexamer as a middle molecular weight (MMW) and also as a high molecular weight form (HMW). It has been shown that the level of total APN and its isoforms correlates negatively with the body mass index (BMI) [5]. Moreover, circulating levels of APN are lower in patients with obesity and type 2 diabetes mellitus when compared with those of healthy controls [6,7]. Recent clinical studies support APN as having anti-diabetic properties due to insulin-mimetic and insulinsensitizing effects [8,9]. Myeloperoxidase (MPO) is a neutrophil and monocyte enzyme, which uses chloride as a cosubstrate with hydrogen peroxide in order to generate chlorinating oxidants such as hypochlorous acid (HOCl) [10,11]. Recent studies have demonstrated that MPO is able to contribute to cardiovascular disease [12,13] and that it may play an important role in the development of atherosclerosis, leading to endothelial dysfunction [14] and subsequently to atherosclerotic plaque vulnerability [12,15]. Moreover, high plasma MPO level is known to predict the future risk of cardiovascular diseases in apparently healthy individuals [16]. For that reason, this study was undertaken as an attempt to investigate a possible link between APN multimers and the pro-atherogenic MPO in patients with diabetes type 2 so as to achieve a deeper understanding of the pathogenesis of atherosclerosis in individuals with insulin resistance.
For statistical analyses, SPSS statistical software Version 11.0.1 was used. All data were expressed as mean S.E.M. A value of p < 0.05 was regarded as statistically significant. A ttest for parametrically distributed data and a Wilcoxon matched-pair signed rank test for non-parametrically distributed data were utilised to compare parameters at the beginning and at the end of the study. Quantitative variables with normal distribution were analyzed with a t-test, and variables without normal distribution with a two-tailed Mann– Whitney U-test. Correlation analysis was performed with Pearson’s and Spearman’s test.
2.
Methods
3.
Results
2.1.
Subjects and procedure
3.1.
Background data of the study
The study protocol was approved by the local ethics committee. It was performed in accordance to the ethics principles in the Declaration of Helsinki. Written informed consent was given by each patient before participation in the study. Forty nine patients with untreated or insufficiently treated diabetes mellitus type 2 were included in this study. Peripheral blood at the beginning and at the end of the study was obtained by venipuncture into heparinized tubes. Patients were treated with insulin, metformin, acarbose, repaglinide, glimepiride, glibenclamide and nateglinide for different time periods so that they could obtain an improved basal glucose plasma level. During this period, patients were treated at least with one of these medications.
2.2.
Measurement of APN
To assess total APN, HMW APN, middle weight APN and low weight APN in human heparinized plasma, a specific assay purchased from Alpco Diagnostics (Salem, NH, USA) was used. This system was capable of quantifying total APN and its different multimers in a single ELISA. In brief, proteases that selectively digested LMW-APN, MMW-APN or HMW-APN were used for determination of the various multimers. A special
2.3.
Measurement of MPO
MPO serum levels were measured by ELISA according to procedures recommended by the manufacturer (Immunology Consultants Laboratory, Inc., US). In short, polystyrene microtitre wells covered with anti-MPO antibodies were used to detect MPO in human plasma. After addition of plasma, secondary antibodies conjugated with horseradish peroxidase and a chromogenic substrate were added, and the quantity of enzyme bound was detected using an ELISA plate reader at 37 8C (Molecular Devices, US).
2.4.
Statistical analysis
This study examined 49 patients (37 men and 12 women) with untreated or insufficiently treated diabetes mellitus type 2 (Table 1). Before treatment, mean basal plasma glucose was 146.1 7.05 mg/dL with a mean HbA1c of 8.4 1.84%. No significant differences were measured between male or female subjects (males: 148.57 49.13, females: 138.08 55.39 mg/dL, p > 0.05). The mean period of treatment was 7.02 1.6 days. Anti-diabetic treatment led to significant decreases of weight and BMI (Table 2; weights: from 98.24 22.3 down to 97.15 21.05 kg, p < 0.05; BMIs: from 32.62 7.72 down to 32.25 7.28 kg/m2, p < 0.05). The success of treatment was determined by the occurrence of a significant decrease in the concentration of plasma basal glucose (133 5.1 mg/dL, p < 0.05). Plasma C-reactive protein (CRP) and MPO levels were not found to change significantly.
3.2. Change of APN plasma levels during anti-diabetic treatment Plasma APN did not differ significantly in regard to the total APN before and after treatment (from 4.05 2.34 to 3.94 1.95 mg/mL, p > 0.05). On the other hand, the ratio of HMW to APN and MMW to APN changed significantly after
diabetes research and clinical practice 82 (2008) 179–184
181
Table 1 – Anthropometrical and biochemical characteristics of study patients (male and female) at the beginning of treatment (n.s.: not significant) Characteristics Sex Age (years) Period of treatment (days) Weight (kg) BMI (kg/m2) Glucose (mg/dL) HbA1c (%) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglyceride (mg/dL) Lipoprotein a (mg/dL) CRP (mg/dL) APN (mg/dL) HMW (mg/dL) HMW-APN MMW (mg/dL) MMW-APN LMW (mg/dL) LMW-APN MPO (ng/dL) Leukocytes (N/nL)
Male
Female
37 62.93 10.29 6.78 1.4 99.37 20.88 31.95 6.75 148.57 49.13 8.46 1.8 183.53 46.86 39.15 8.35 109.85 39.05 257.95 251.23 20.43 38.79 0.93 2.19 4.15 2.55 1.84 1.42 0.41 0.15 1.09 0.64 0.28 0.1 1.22 0.93 0.31 0.12 12.75 10.89 7.77 2.02
12 65.67 9.79 7.75 2.01 94.76 26.86 34.67 10.26 138.08 55.39 8.23 2.02 202.67 53.16 44.58 10.66 127.33 41.05 203.5 103.68 12.5 41.04 0.41 0.23 3.72 1.56 1.86 1.4 0.46 0.2 0.88 0.59 0.24 0.12 0.98 0.37 0.29 0.12 11.29 8.16 8.16 1.36
treatment (Table 2) (HMW-APN: from 0.43 0.16 to 0.59 0.14, p < 0.05; MMW-APN: from 0.27 0.11 to 0.22 0.11, p < 0.05). LMW-APN remained apparently unaltered during the treatment period.
3.3.
Correlation of HMW and LMW with MPO
HMW-APN measured before treatment correlated positively with MPO (Fig. 1) (r = 0.314, p < 0.05) after corrections were made for BMI, CRP and leukocytes. Moreover, a negative correlation between LMW-APN and MPO was indicated at the beginning of the study (r = 0.309, p < 0.05). To examine the correlation of HMW-APN and LMW-APN to MPO during the anti-diabetic treatment, patients were separated into quartiles according to their HMW-APN increase (Table 3: quartile 1: highest HMW-APN increase, quartile 4: lowest HMW-APN increase). These quartiles were compared with the MPO differences before and after anti-diabetic treatment (Fig. 2). We found a positive correlation between the HMW-APN increase and MPO (r = 0.304, p < 0.05)
p n.s. n.s. n.s n.s n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
after correction for CRP, BMI and leukocyte count. Comparing that quartile which comprises the highest increase of HMWAPN with that of the lowest HMW-APN increase, a significant difference was seen in regard to mean MPO (quartile 1: 2.043 1.004 ng/dL; quartile 2: 0.7800 2.394 ng/dL; quartile 3: 0.6658 0.9101 ng/dL; quartile 4: 5.231 2.14 ng/dL; for quartile 1 versus quartile 4: p < 0.05). Characterisation of these quartiles revealed no significant dependencies on difference in age, BMI, HbA1c, period of treatment, CRP or glucose (Table 3). In contrast, LMW-APN was identified as tending to result in a negative correlation with MPO but without any significance (Fig. 3). A tendency but no significant difference in MPO was seen as well after subdividing the patients into those with highest LMW-APN reduction after treatment (quartile 4) and those with lowest LMW-APN reduction (quartile 1); (r = 0.156, p > 0.05; quartile 1: 4.048 1.784 ng/dL; quartile 2: 0.3915 1.237 ng/dL; quartile 3: 0.7700 1.897 ng/dL; quartile 4: 2.392 2.023 ng/dL; again, for quartile 1 versus quartile 4: p > 0.05).
Table 2 – Characteristics of all patients at the beginning and at the end of anti-diabetic treatment (n.s.: not significant) Characteristics
Start of treatment
End of treatment
APN (mg/mL) HMW (mg/mL) MMW (mg/mL) LMW (mg/mL) HMW-APN MMW-APN LMW-APN CRP (mg/dL) Weight (kg) BMI (kg/m2) MPO (ng/dL) Leukocytes (N/nL)
4.05 2.34 1.84 1.39 1.04 0.63 1.17 0.83 0.43 0.16 0.27 0.11 0.3 0.12 0.79 1.91 98.24 22.3 32.62 7.72 12.39 10.23 7.87 1.87
3.94 1.95 2.03 1.34 0.83 0.57 1.08 0.91 0.59 0.14 0.22 0.11 0.29 0.17 0.8 2.21 97.15 21.05 32.25 7.28 11.63 7.74 7.14 1.85
p n.s. n.s. <0.05 n.s. <0.05 <0.05 n.s. n.s. <0.05 <0.05 n.s. <0.05
182
diabetes research and clinical practice 82 (2008) 179–184
Fig. 1 – Correlation of HMW-APN (quotient of concentration of HMW and APN) and LMW-APN (quotient of concentration of LMW and APN) with MPO in the entire study group.
4.
Discussion
This study has shown HMW-APN to be positively correlated with the pro-atherogenic MPO in patients having insufficiently treated diabetes mellitus type 2. After treatment with different anti-diabetic drugs, which led to a significant improvement of basal glucose plasma level, a positive correlation of increased HMW-APN and MPO was detected, indicating that HMW-APN reflects the pro-inflammatory state in type 2 diabetes mellitus. Cardiovascular disease is the main cause of the increased mortality and morbidity in patients with insulin resistance. Hyperglycemia triggers different pathophysiological mechanisms, leading to atherosclerosis. Various studies point towards adipose tissue as the origin of cytokines such as APN, or towards tumor necrosis factor alpha, both of which play a potential role in the development for vascular complications [17,18]. The role of APN in the development of vascular disease under pro-inflammatory circumstances such as untreated diabetes still remains unclear. APN is mainly considered to be an anti-diabetic, anti-inflammatory and anti-atherogenic protein [19]. This idea was supported by studies in vivo and in vitro, which described a negative
correlation of APN and insulin resistance [6,20]. Moreover, plasma APN concentrations were observed to be decreased in patients with high BMI, hypertension and coronary heart disease [21–23]. Because of decreased HMW-APN in subjects with increased BMI or multiple cardiovascular risk factors, HMW-APN was mainly described as a possible protective multimer [24]. Interestingly, recent studies mentioned the importance of the ratios of the different APN multimers to total APN concerning the determination of pro-inflammatory or antiinflammatory properties [25]. Some of these studies have stressed that HMW-APN can indeed act as a possible proinflammatory or pro-atherogenic protein as well [26,27]. For instance, HMW-APN could be shown to enhance interleukin-6 release of human monocytes, whereas LMW-APN reduced the release of this pro-inflammatory cytokine. Moreover, HMWAPN was found to be positively correlated with the severity of retinopathy and nephropathy in patients with diabetes mellitus. In fact, the results of our study showed that HMWAPN increase was associated with altered MPO. MPO acts as a potent pro-atherogenic enzyme which is increased significantly in patients with cardiovascular disease and type 2
Table 3 – Characteristics of quartiles according to their HMW-APN increase Characteristics Number of patients Age (years) Period of treatment (d) CRP (mg/dL) BMI (kg/m2) HbA1c (%) Glucose (mg/dL) Therapy Insulin Metformin Acarbose Repaglinide Glimepiride Glibenclamide Nateglinide
1
2
3
13 59.9 2.1 7.51 0.6 0.51 0.2 34.1 1.9 7.99 0.6 151.5 17.2
12 66.3 3.1 7.14 0.2 0.56 0.1 34.1 2.5 8.74 0.5 151 11.4
12 62 3.4 6.33 0.5 0.31 0.13 28.5 1.7 8.97 0.4 139 9.7
8 6 3 2 1 1 1
9 7 2 1 0 1 0
9 6 3 2 1 0 0
4 12 66.7 2.7 7.12 0.3 1.8 1.1 33.2 2.6 8.03 0.6 140.9 17 10 4 4 1 1 0 1
diabetes research and clinical practice 82 (2008) 179–184
183
Fig. 2 – Correlation of HMW-APN with MPO during anti-diabetic treatment. The study group was divided into quartiles (quartile 1: highest increase of HMW-APN after treatment; quartile 4: lowest increase of HMW-APN after treatment; MPO difference: MPOStart S MPOStop; HMW-APN difference: HMW-APNStart S HMW-APNStop).
Fig. 3 – Correlation of LMW-APN with MPO during anti-diabetic treatment. The study group was divided into quartiles (quartile 1: lowest decrease of LMW-APN after treatment; quartile 4: highest decrease of LMW-APN after treatment; MPO difference: MPOStart S MPOStop; LMW-APN difference: LMW-APNStart S LMW-APNStop).
diabetes mellitus [15,28]. This positive correlation, that did not have any dependencies in age, BMI, HbA1c, period of treatment or glucose, was still significant even after correction for BMI, CRP and leukocytes. This may indicate the importance of HMW-APN in diabetic situations. In contrast to HMW-APN, LMW-APN was correlated negatively with MPO before antidiabetic treatment. However, after treatment only a trend to a negative correlation of LMW-APN with MPO could be demonstrated, indicating that larger cohorts are necessary to verify these correlations. In conclusion, this study demonstrated that HMW-APN might be a possible pro-atherogenic protein in patients with diabetes mellitus type 2. A positive correlation of HMW with MPO was found in a hypergylcemic situation of insufficiently treated patients with insulin resistance. Anti-diabetic treatment was associated with a significant increase of HMW-APN, which was positively correlated with an increase of MPO. Thus, HMW-APN may exert possible pro-inflammatory effects in type 2 diabetes mellitus.
Acknowledgments The study was supported by grants from Ra 799/3-1 and the DFG-Sonderforschungsbereich SFB-TR19. We thank Franziska Bleis for the excellent technical assistance.
Conflict of interest The authors declare that they have no conflict of interest.
references
[1] P.J. Boyle, Diabetes mellitus and macrovascular disease: mechanisms and mediators, Am. J. Med. 120 (2007) S12–S17. [2] J. Plutzky, The vascular biology of atherosclerosis, Am. J. Med. 115 (Suppl. 8A) (2003) 55S–61S.
184
diabetes research and clinical practice 82 (2008) 179–184
[3] I. Kowalska, Role of adipose tissue in the development of vascular complications in type 2 diabetes mellitus, Diabetes Res. Clin. Pract. (2007). [4] R.S. Ahima, Adipose tissue as an endocrine organ, Obesity (Silver. Spring) 14 (Suppl. 5) (2006) 242S–249S. [5] T. Bobbert, H. Rochlitz, U. Wegewitz, S. Akpulat, K. Mai, M.O. Weickert, et al., Changes of adiponectin oligomer composition by moderate weight reduction, Diabetes 54 (2005) 2712–2719. [6] K. Hotta, T. Funahashi, N.L. Bodkin, H.K. Ortmeyer, Y. Arita, B.C. Hansen, et al., Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys, Diabetes 50 (2001) 1126–1133. [7] C. Weyer, T. Funahashi, S. Tanaka, K. Hotta, Y. Matzuzawa, R.E. Pratley, et al., Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia, J. Clin. Endocrinol. Metab. 86 (2001) 1930–1935. [8] X. Fang, G. Sweeney, Mechanisms regulating energy metabolism by adiponectin in obesity and diabetes, Biochem. Soc. Trans. 34 (2006) 798–801. [9] T. Kadowaki, T. Yamauchi, N. Kubota, K. Hara, K. Ueki, K. Tobe, Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome, J. Clin. Invest. 116 (2006) 1784–1792. [10] J.K. Hurst, W.C. Barrette, Leukocytic oxygen activation and microbicidal oxidative toxins, Crit. Rev. Biochem. Mol. Biol. 24 (1989) 271–328. [11] S.J. Klebanoff, Oxygen metabolism and the toxic properties of phagocytes, Ann. Intern. Med. 93 (1980) 480–489. [12] S. Baldus, C. Heeschen, T. Meinertz, A.M. Zeiher, J.P. Eiserich, T. Munzel, et al., Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes, Circulation 108 (2003) 1440–1445. [13] J.W. Heinecke, Oxidative stress: new approaches to diagnosis and prognosis in atherosclerosis, Am. J. Cardiol. 91 (2003) 12A–16A. [14] J.P. Eiserich, S. Baldus, M.L. Brennan, W. Ma, C. Zhang, A. Tousson, et al., Myeloperoxidase, a leukocyte-derived vascular NO oxidase, Science 296 (2002) 2391–2394. [15] M.L. Brennan, M.S. Penn, L.F. Van, V. Nambi, M.H. Shishehbor, R.J. Aviles, et al., Prognostic value of myeloperoxidase in patients with chest pain, N. Engl. J. Med. 349 (2003) 1595–1604. [16] M.C. Meuwese, E.S. Stroes, S.L. Hazen, J.N. van Miert, J.A. Kuivenhoven, R.G. Schaub, et al., Serum myeloperoxidase levels are associated with the future risk of coronary artery
[17]
[18] [19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
disease in apparently healthy individuals: the EPIC-Norfolk Prospective Population Study, J. Am. Coll. Cardiol. 50 (2007) 159–165. H. Tilg, A.R. Moschen, Adipocytokines: mediators linking adipose tissue, inflammation and immunity, Nat. Rev. Immunol. 6 (2006) 772–783. W. Aldhahi, O. Hamdy, Adipokines, inflammation, and the endothelium in diabetes, Curr. Diab. Rep. 3 (2003) 293–298. Y. Matsuzawa, T. Funahashi, S. Kihara, I. Shimomura, Adiponectin and metabolic syndrome, Arterioscler. Thromb. Vasc. Biol. 24 (2004) 29–33. K. Hotta, T. Funahashi, Y. Arita, M. Takahashi, M. Matsuda, Y. Okamoto, et al., Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients, Arterioscler. Thromb. Vasc. Biol. 20 (2000) 1595– 1599. M. Adamczak, A. Wiecek, T. Funahashi, J. Chudek, F. Kokot, Y. Matsuzawa, Decreased plasma adiponectin concentration in patients with essential hypertension, Am. J. Hypertens. 16 (2003) 72–75. T. Hara, H. Fujiwara, T. Shoji, T. Mimura, H. Nakao, S. Fujimoto, Decreased plasma adiponectin levels in young obese males, J. Atheroscler. Thromb. 10 (2003) 234–238. M. Kumada, S. Kihara, S. Sumitsuji, T. Kawamoto, S. Matsumoto, N. Ouchi, et al., Association of hypoadiponectinemia with coronary artery disease in men, Arterioscler. Thromb. Vasc. Biol. 23 (2003) 85–89. T. Inoue, N. Kotooka, T. Morooka, H. Komoda, T. Uchida, Y. Aso, et al., High molecular weight adiponectin as a predictor of long-term clinical outcome in patients with coronary artery disease, Am. J. Cardiol. 100 (2007) 569–574. K. Hara, M. Horikoshi, T. Yamauchi, H. Yago, O. Miyazaki, H. Ebinuma, et al., Measurement of the high-molecular weight form of adiponectin in plasma is useful for the prediction of insulin resistance and metabolic syndrome, Diabetes Care 29 (2006) 1357–1362. K. Kato, H. Osawa, M. Ochi, Y. Kusunoki, O. Ebisui, K. Ohno, et al., Serum total and high molecular weight adiponectin levels are correlated with the severity of diabetic retinopathy and nephropathy, Clin. Endocrinol. (Oxf.). (2007). F. Schober, M. Neumeier, J. Weigert, S. Wurm, J. Wanninger, A. Schaffler, et al., Low molecular weight adiponectin negatively correlates with the waist circumference and monocytic IL-6 release, Biochem. Biophys. Res. Commun. 361 (2007) 968–973. S.L. Hazen, Myeloperoxidase and plaque vulnerability, Arterioscler. Thromb. Vasc. Biol. 24 (2007) 1143–1146.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/diabres
High molecular weight adiponectin correlates positively with myeloperoxidase in patients with type 2 diabetes mellitus P. Bobbert a,*, U. Rauch a,1, B. Stratmann b, P. Goldin-Lang a, S. Antoniak a, T. Bobbert c, H.P. Schultheiss a, D. Tschoepe b a
Department of Cardiology and Pneumology, Medical Clinic II, Charite´ – Universita¨tsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12203 Berlin, Germany b Heart and Diabetes Center NRW, Diabetes Center, Ruhr-University Bochum, 32545 Bad Oeynhausen, Germany c Department of Endocrinology, Diabetes and Nutrition, Charite´ – Universita¨tsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12203 Berlin, Germany
article info
abstract
Article history:
Adiponectin (APN) is present in human plasma as a low molecular weight (LMW), a middle
Received 18 April 2008
molecular weight (MMW) and a high molecular weight form (HMW). As a support to
Received in revised form
determine properties such as anti-atherogenic or atherogenic effects, recent clinical studies
16 July 2008
suppose to determine the ratio of each APN multimer to total APN but not the absolute
Accepted 22 July 2008
plasma concentration of APN. In the present study, the correlation of APN and its multimers
Published on line 7 September 2008
with myeloperoxidase (MPO), an enzyme with pro-inflammatory properties, was examined in patients with type 2 diabetes mellitus.
Keywords: Adiponectin
MPO and APN serum levels were assessed in 49 patients with type 2 diabetes mellitus at the beginning and at the end of an anti-diabetic treatment. After treatment a significant
Diabetes
increase in the ratio of HMW to total APN (from 0.43 0.16 to 0.59 0.14, p < 0.05) was found.
Atherosclerosis
Before treatment, HMW-APN was correlated positively with MPO (r = 0.314, p < 0.05). Moreover, a positive correlation was observed between the increased HMW ratio and MPO during treatment (r = 0.304, p < 0.05). HMW-APN correlates positively with MPO in patients with type 2 diabetes. Therefore, HMW-APN may exert possible pro-inflammatory effects in type 2 diabetes. # 2008 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
Atherosclerosis is a chronic inflammation of the arterial wall, initiated by endothelial cells in response to injury. The inflammatory response is maintained by reactions of lipoproteins, inflammatory cells such as macrophages or lymphocytes, different wall constituents and smooth muscle cells. Patients with type 2 diabetes mellitus are subject to an increased risk of the development of atherosclerosis. The link
between type 2 diabetes mellitus and vascular disease can be shown by the increased expression of vascular cell adhesion molecules or by procoagulant proteins such as plasminogen activator inhibitor 1 in patients with insulin resistance [1]. Furthermore, chronically elevated plasma glucose levels may induce advanced glycation of proteins, which trigger adverse vascular events [2]. A complete explanation underlying the link between diabetes and atherosclerosis still remains unclear. Recent studies point to adipose tissue playing an
* Corresponding author. Tel.: +49 30 8445 2362; fax: +49 30 8445 4648. E-mail address: [email protected] (P. Bobbert). 1 Contributing equally to the first author. 0168-8227/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2008.07.018
180
diabetes research and clinical practice 82 (2008) 179–184
microtitre plate having wells coated with an anti-human APN monoclonal antibody was used to assess the quantity of APN and its multimers directly or indirectly by final incubation with substrate. Colour intensities were determined using an ELISA plate reader at 37 8C (Molecular Devices, US).
important role in the pathogenesis of vascular complications in patients with insulin resistance [3]. Adipose tissue seems to act as an endocrine organ, which produces different cytokines such as adiponectin (APN), leptin, resistin or visfatin [4]. APN, a 30-kDa adipocyte derived hormone, is present in human plasma as a trimer known to exist as a low molecular weight (LMW), a hexamer as a middle molecular weight (MMW) and also as a high molecular weight form (HMW). It has been shown that the level of total APN and its isoforms correlates negatively with the body mass index (BMI) [5]. Moreover, circulating levels of APN are lower in patients with obesity and type 2 diabetes mellitus when compared with those of healthy controls [6,7]. Recent clinical studies support APN as having anti-diabetic properties due to insulin-mimetic and insulinsensitizing effects [8,9]. Myeloperoxidase (MPO) is a neutrophil and monocyte enzyme, which uses chloride as a cosubstrate with hydrogen peroxide in order to generate chlorinating oxidants such as hypochlorous acid (HOCl) [10,11]. Recent studies have demonstrated that MPO is able to contribute to cardiovascular disease [12,13] and that it may play an important role in the development of atherosclerosis, leading to endothelial dysfunction [14] and subsequently to atherosclerotic plaque vulnerability [12,15]. Moreover, high plasma MPO level is known to predict the future risk of cardiovascular diseases in apparently healthy individuals [16]. For that reason, this study was undertaken as an attempt to investigate a possible link between APN multimers and the pro-atherogenic MPO in patients with diabetes type 2 so as to achieve a deeper understanding of the pathogenesis of atherosclerosis in individuals with insulin resistance.
For statistical analyses, SPSS statistical software Version 11.0.1 was used. All data were expressed as mean S.E.M. A value of p < 0.05 was regarded as statistically significant. A ttest for parametrically distributed data and a Wilcoxon matched-pair signed rank test for non-parametrically distributed data were utilised to compare parameters at the beginning and at the end of the study. Quantitative variables with normal distribution were analyzed with a t-test, and variables without normal distribution with a two-tailed Mann– Whitney U-test. Correlation analysis was performed with Pearson’s and Spearman’s test.
2.
Methods
3.
Results
2.1.
Subjects and procedure
3.1.
Background data of the study
The study protocol was approved by the local ethics committee. It was performed in accordance to the ethics principles in the Declaration of Helsinki. Written informed consent was given by each patient before participation in the study. Forty nine patients with untreated or insufficiently treated diabetes mellitus type 2 were included in this study. Peripheral blood at the beginning and at the end of the study was obtained by venipuncture into heparinized tubes. Patients were treated with insulin, metformin, acarbose, repaglinide, glimepiride, glibenclamide and nateglinide for different time periods so that they could obtain an improved basal glucose plasma level. During this period, patients were treated at least with one of these medications.
2.2.
Measurement of APN
To assess total APN, HMW APN, middle weight APN and low weight APN in human heparinized plasma, a specific assay purchased from Alpco Diagnostics (Salem, NH, USA) was used. This system was capable of quantifying total APN and its different multimers in a single ELISA. In brief, proteases that selectively digested LMW-APN, MMW-APN or HMW-APN were used for determination of the various multimers. A special
2.3.
Measurement of MPO
MPO serum levels were measured by ELISA according to procedures recommended by the manufacturer (Immunology Consultants Laboratory, Inc., US). In short, polystyrene microtitre wells covered with anti-MPO antibodies were used to detect MPO in human plasma. After addition of plasma, secondary antibodies conjugated with horseradish peroxidase and a chromogenic substrate were added, and the quantity of enzyme bound was detected using an ELISA plate reader at 37 8C (Molecular Devices, US).
2.4.
Statistical analysis
This study examined 49 patients (37 men and 12 women) with untreated or insufficiently treated diabetes mellitus type 2 (Table 1). Before treatment, mean basal plasma glucose was 146.1 7.05 mg/dL with a mean HbA1c of 8.4 1.84%. No significant differences were measured between male or female subjects (males: 148.57 49.13, females: 138.08 55.39 mg/dL, p > 0.05). The mean period of treatment was 7.02 1.6 days. Anti-diabetic treatment led to significant decreases of weight and BMI (Table 2; weights: from 98.24 22.3 down to 97.15 21.05 kg, p < 0.05; BMIs: from 32.62 7.72 down to 32.25 7.28 kg/m2, p < 0.05). The success of treatment was determined by the occurrence of a significant decrease in the concentration of plasma basal glucose (133 5.1 mg/dL, p < 0.05). Plasma C-reactive protein (CRP) and MPO levels were not found to change significantly.
3.2. Change of APN plasma levels during anti-diabetic treatment Plasma APN did not differ significantly in regard to the total APN before and after treatment (from 4.05 2.34 to 3.94 1.95 mg/mL, p > 0.05). On the other hand, the ratio of HMW to APN and MMW to APN changed significantly after
diabetes research and clinical practice 82 (2008) 179–184
181
Table 1 – Anthropometrical and biochemical characteristics of study patients (male and female) at the beginning of treatment (n.s.: not significant) Characteristics Sex Age (years) Period of treatment (days) Weight (kg) BMI (kg/m2) Glucose (mg/dL) HbA1c (%) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglyceride (mg/dL) Lipoprotein a (mg/dL) CRP (mg/dL) APN (mg/dL) HMW (mg/dL) HMW-APN MMW (mg/dL) MMW-APN LMW (mg/dL) LMW-APN MPO (ng/dL) Leukocytes (N/nL)
Male
Female
37 62.93 10.29 6.78 1.4 99.37 20.88 31.95 6.75 148.57 49.13 8.46 1.8 183.53 46.86 39.15 8.35 109.85 39.05 257.95 251.23 20.43 38.79 0.93 2.19 4.15 2.55 1.84 1.42 0.41 0.15 1.09 0.64 0.28 0.1 1.22 0.93 0.31 0.12 12.75 10.89 7.77 2.02
12 65.67 9.79 7.75 2.01 94.76 26.86 34.67 10.26 138.08 55.39 8.23 2.02 202.67 53.16 44.58 10.66 127.33 41.05 203.5 103.68 12.5 41.04 0.41 0.23 3.72 1.56 1.86 1.4 0.46 0.2 0.88 0.59 0.24 0.12 0.98 0.37 0.29 0.12 11.29 8.16 8.16 1.36
treatment (Table 2) (HMW-APN: from 0.43 0.16 to 0.59 0.14, p < 0.05; MMW-APN: from 0.27 0.11 to 0.22 0.11, p < 0.05). LMW-APN remained apparently unaltered during the treatment period.
3.3.
Correlation of HMW and LMW with MPO
HMW-APN measured before treatment correlated positively with MPO (Fig. 1) (r = 0.314, p < 0.05) after corrections were made for BMI, CRP and leukocytes. Moreover, a negative correlation between LMW-APN and MPO was indicated at the beginning of the study (r = 0.309, p < 0.05). To examine the correlation of HMW-APN and LMW-APN to MPO during the anti-diabetic treatment, patients were separated into quartiles according to their HMW-APN increase (Table 3: quartile 1: highest HMW-APN increase, quartile 4: lowest HMW-APN increase). These quartiles were compared with the MPO differences before and after anti-diabetic treatment (Fig. 2). We found a positive correlation between the HMW-APN increase and MPO (r = 0.304, p < 0.05)
p n.s. n.s. n.s n.s n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
after correction for CRP, BMI and leukocyte count. Comparing that quartile which comprises the highest increase of HMWAPN with that of the lowest HMW-APN increase, a significant difference was seen in regard to mean MPO (quartile 1: 2.043 1.004 ng/dL; quartile 2: 0.7800 2.394 ng/dL; quartile 3: 0.6658 0.9101 ng/dL; quartile 4: 5.231 2.14 ng/dL; for quartile 1 versus quartile 4: p < 0.05). Characterisation of these quartiles revealed no significant dependencies on difference in age, BMI, HbA1c, period of treatment, CRP or glucose (Table 3). In contrast, LMW-APN was identified as tending to result in a negative correlation with MPO but without any significance (Fig. 3). A tendency but no significant difference in MPO was seen as well after subdividing the patients into those with highest LMW-APN reduction after treatment (quartile 4) and those with lowest LMW-APN reduction (quartile 1); (r = 0.156, p > 0.05; quartile 1: 4.048 1.784 ng/dL; quartile 2: 0.3915 1.237 ng/dL; quartile 3: 0.7700 1.897 ng/dL; quartile 4: 2.392 2.023 ng/dL; again, for quartile 1 versus quartile 4: p > 0.05).
Table 2 – Characteristics of all patients at the beginning and at the end of anti-diabetic treatment (n.s.: not significant) Characteristics
Start of treatment
End of treatment
APN (mg/mL) HMW (mg/mL) MMW (mg/mL) LMW (mg/mL) HMW-APN MMW-APN LMW-APN CRP (mg/dL) Weight (kg) BMI (kg/m2) MPO (ng/dL) Leukocytes (N/nL)
4.05 2.34 1.84 1.39 1.04 0.63 1.17 0.83 0.43 0.16 0.27 0.11 0.3 0.12 0.79 1.91 98.24 22.3 32.62 7.72 12.39 10.23 7.87 1.87
3.94 1.95 2.03 1.34 0.83 0.57 1.08 0.91 0.59 0.14 0.22 0.11 0.29 0.17 0.8 2.21 97.15 21.05 32.25 7.28 11.63 7.74 7.14 1.85
p n.s. n.s. <0.05 n.s. <0.05 <0.05 n.s. n.s. <0.05 <0.05 n.s. <0.05
182
diabetes research and clinical practice 82 (2008) 179–184
Fig. 1 – Correlation of HMW-APN (quotient of concentration of HMW and APN) and LMW-APN (quotient of concentration of LMW and APN) with MPO in the entire study group.
4.
Discussion
This study has shown HMW-APN to be positively correlated with the pro-atherogenic MPO in patients having insufficiently treated diabetes mellitus type 2. After treatment with different anti-diabetic drugs, which led to a significant improvement of basal glucose plasma level, a positive correlation of increased HMW-APN and MPO was detected, indicating that HMW-APN reflects the pro-inflammatory state in type 2 diabetes mellitus. Cardiovascular disease is the main cause of the increased mortality and morbidity in patients with insulin resistance. Hyperglycemia triggers different pathophysiological mechanisms, leading to atherosclerosis. Various studies point towards adipose tissue as the origin of cytokines such as APN, or towards tumor necrosis factor alpha, both of which play a potential role in the development for vascular complications [17,18]. The role of APN in the development of vascular disease under pro-inflammatory circumstances such as untreated diabetes still remains unclear. APN is mainly considered to be an anti-diabetic, anti-inflammatory and anti-atherogenic protein [19]. This idea was supported by studies in vivo and in vitro, which described a negative
correlation of APN and insulin resistance [6,20]. Moreover, plasma APN concentrations were observed to be decreased in patients with high BMI, hypertension and coronary heart disease [21–23]. Because of decreased HMW-APN in subjects with increased BMI or multiple cardiovascular risk factors, HMW-APN was mainly described as a possible protective multimer [24]. Interestingly, recent studies mentioned the importance of the ratios of the different APN multimers to total APN concerning the determination of pro-inflammatory or antiinflammatory properties [25]. Some of these studies have stressed that HMW-APN can indeed act as a possible proinflammatory or pro-atherogenic protein as well [26,27]. For instance, HMW-APN could be shown to enhance interleukin-6 release of human monocytes, whereas LMW-APN reduced the release of this pro-inflammatory cytokine. Moreover, HMWAPN was found to be positively correlated with the severity of retinopathy and nephropathy in patients with diabetes mellitus. In fact, the results of our study showed that HMWAPN increase was associated with altered MPO. MPO acts as a potent pro-atherogenic enzyme which is increased significantly in patients with cardiovascular disease and type 2
Table 3 – Characteristics of quartiles according to their HMW-APN increase Characteristics Number of patients Age (years) Period of treatment (d) CRP (mg/dL) BMI (kg/m2) HbA1c (%) Glucose (mg/dL) Therapy Insulin Metformin Acarbose Repaglinide Glimepiride Glibenclamide Nateglinide
1
2
3
13 59.9 2.1 7.51 0.6 0.51 0.2 34.1 1.9 7.99 0.6 151.5 17.2
12 66.3 3.1 7.14 0.2 0.56 0.1 34.1 2.5 8.74 0.5 151 11.4
12 62 3.4 6.33 0.5 0.31 0.13 28.5 1.7 8.97 0.4 139 9.7
8 6 3 2 1 1 1
9 7 2 1 0 1 0
9 6 3 2 1 0 0
4 12 66.7 2.7 7.12 0.3 1.8 1.1 33.2 2.6 8.03 0.6 140.9 17 10 4 4 1 1 0 1
diabetes research and clinical practice 82 (2008) 179–184
183
Fig. 2 – Correlation of HMW-APN with MPO during anti-diabetic treatment. The study group was divided into quartiles (quartile 1: highest increase of HMW-APN after treatment; quartile 4: lowest increase of HMW-APN after treatment; MPO difference: MPOStart S MPOStop; HMW-APN difference: HMW-APNStart S HMW-APNStop).
Fig. 3 – Correlation of LMW-APN with MPO during anti-diabetic treatment. The study group was divided into quartiles (quartile 1: lowest decrease of LMW-APN after treatment; quartile 4: highest decrease of LMW-APN after treatment; MPO difference: MPOStart S MPOStop; LMW-APN difference: LMW-APNStart S LMW-APNStop).
diabetes mellitus [15,28]. This positive correlation, that did not have any dependencies in age, BMI, HbA1c, period of treatment or glucose, was still significant even after correction for BMI, CRP and leukocytes. This may indicate the importance of HMW-APN in diabetic situations. In contrast to HMW-APN, LMW-APN was correlated negatively with MPO before antidiabetic treatment. However, after treatment only a trend to a negative correlation of LMW-APN with MPO could be demonstrated, indicating that larger cohorts are necessary to verify these correlations. In conclusion, this study demonstrated that HMW-APN might be a possible pro-atherogenic protein in patients with diabetes mellitus type 2. A positive correlation of HMW with MPO was found in a hypergylcemic situation of insufficiently treated patients with insulin resistance. Anti-diabetic treatment was associated with a significant increase of HMW-APN, which was positively correlated with an increase of MPO. Thus, HMW-APN may exert possible pro-inflammatory effects in type 2 diabetes mellitus.
Acknowledgments The study was supported by grants from Ra 799/3-1 and the DFG-Sonderforschungsbereich SFB-TR19. We thank Franziska Bleis for the excellent technical assistance.
Conflict of interest The authors declare that they have no conflict of interest.
references
[1] P.J. Boyle, Diabetes mellitus and macrovascular disease: mechanisms and mediators, Am. J. Med. 120 (2007) S12–S17. [2] J. Plutzky, The vascular biology of atherosclerosis, Am. J. Med. 115 (Suppl. 8A) (2003) 55S–61S.
184
diabetes research and clinical practice 82 (2008) 179–184
[3] I. Kowalska, Role of adipose tissue in the development of vascular complications in type 2 diabetes mellitus, Diabetes Res. Clin. Pract. (2007). [4] R.S. Ahima, Adipose tissue as an endocrine organ, Obesity (Silver. Spring) 14 (Suppl. 5) (2006) 242S–249S. [5] T. Bobbert, H. Rochlitz, U. Wegewitz, S. Akpulat, K. Mai, M.O. Weickert, et al., Changes of adiponectin oligomer composition by moderate weight reduction, Diabetes 54 (2005) 2712–2719. [6] K. Hotta, T. Funahashi, N.L. Bodkin, H.K. Ortmeyer, Y. Arita, B.C. Hansen, et al., Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys, Diabetes 50 (2001) 1126–1133. [7] C. Weyer, T. Funahashi, S. Tanaka, K. Hotta, Y. Matzuzawa, R.E. Pratley, et al., Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia, J. Clin. Endocrinol. Metab. 86 (2001) 1930–1935. [8] X. Fang, G. Sweeney, Mechanisms regulating energy metabolism by adiponectin in obesity and diabetes, Biochem. Soc. Trans. 34 (2006) 798–801. [9] T. Kadowaki, T. Yamauchi, N. Kubota, K. Hara, K. Ueki, K. Tobe, Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome, J. Clin. Invest. 116 (2006) 1784–1792. [10] J.K. Hurst, W.C. Barrette, Leukocytic oxygen activation and microbicidal oxidative toxins, Crit. Rev. Biochem. Mol. Biol. 24 (1989) 271–328. [11] S.J. Klebanoff, Oxygen metabolism and the toxic properties of phagocytes, Ann. Intern. Med. 93 (1980) 480–489. [12] S. Baldus, C. Heeschen, T. Meinertz, A.M. Zeiher, J.P. Eiserich, T. Munzel, et al., Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes, Circulation 108 (2003) 1440–1445. [13] J.W. Heinecke, Oxidative stress: new approaches to diagnosis and prognosis in atherosclerosis, Am. J. Cardiol. 91 (2003) 12A–16A. [14] J.P. Eiserich, S. Baldus, M.L. Brennan, W. Ma, C. Zhang, A. Tousson, et al., Myeloperoxidase, a leukocyte-derived vascular NO oxidase, Science 296 (2002) 2391–2394. [15] M.L. Brennan, M.S. Penn, L.F. Van, V. Nambi, M.H. Shishehbor, R.J. Aviles, et al., Prognostic value of myeloperoxidase in patients with chest pain, N. Engl. J. Med. 349 (2003) 1595–1604. [16] M.C. Meuwese, E.S. Stroes, S.L. Hazen, J.N. van Miert, J.A. Kuivenhoven, R.G. Schaub, et al., Serum myeloperoxidase levels are associated with the future risk of coronary artery
[17]
[18] [19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
disease in apparently healthy individuals: the EPIC-Norfolk Prospective Population Study, J. Am. Coll. Cardiol. 50 (2007) 159–165. H. Tilg, A.R. Moschen, Adipocytokines: mediators linking adipose tissue, inflammation and immunity, Nat. Rev. Immunol. 6 (2006) 772–783. W. Aldhahi, O. Hamdy, Adipokines, inflammation, and the endothelium in diabetes, Curr. Diab. Rep. 3 (2003) 293–298. Y. Matsuzawa, T. Funahashi, S. Kihara, I. Shimomura, Adiponectin and metabolic syndrome, Arterioscler. Thromb. Vasc. Biol. 24 (2004) 29–33. K. Hotta, T. Funahashi, Y. Arita, M. Takahashi, M. Matsuda, Y. Okamoto, et al., Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients, Arterioscler. Thromb. Vasc. Biol. 20 (2000) 1595– 1599. M. Adamczak, A. Wiecek, T. Funahashi, J. Chudek, F. Kokot, Y. Matsuzawa, Decreased plasma adiponectin concentration in patients with essential hypertension, Am. J. Hypertens. 16 (2003) 72–75. T. Hara, H. Fujiwara, T. Shoji, T. Mimura, H. Nakao, S. Fujimoto, Decreased plasma adiponectin levels in young obese males, J. Atheroscler. Thromb. 10 (2003) 234–238. M. Kumada, S. Kihara, S. Sumitsuji, T. Kawamoto, S. Matsumoto, N. Ouchi, et al., Association of hypoadiponectinemia with coronary artery disease in men, Arterioscler. Thromb. Vasc. Biol. 23 (2003) 85–89. T. Inoue, N. Kotooka, T. Morooka, H. Komoda, T. Uchida, Y. Aso, et al., High molecular weight adiponectin as a predictor of long-term clinical outcome in patients with coronary artery disease, Am. J. Cardiol. 100 (2007) 569–574. K. Hara, M. Horikoshi, T. Yamauchi, H. Yago, O. Miyazaki, H. Ebinuma, et al., Measurement of the high-molecular weight form of adiponectin in plasma is useful for the prediction of insulin resistance and metabolic syndrome, Diabetes Care 29 (2006) 1357–1362. K. Kato, H. Osawa, M. Ochi, Y. Kusunoki, O. Ebisui, K. Ohno, et al., Serum total and high molecular weight adiponectin levels are correlated with the severity of diabetic retinopathy and nephropathy, Clin. Endocrinol. (Oxf.). (2007). F. Schober, M. Neumeier, J. Weigert, S. Wurm, J. Wanninger, A. Schaffler, et al., Low molecular weight adiponectin negatively correlates with the waist circumference and monocytic IL-6 release, Biochem. Biophys. Res. Commun. 361 (2007) 968–973. S.L. Hazen, Myeloperoxidase and plaque vulnerability, Arterioscler. Thromb. Vasc. Biol. 24 (2007) 1143–1146.