ATHEROSCLEROTIC cardiovascular complications are the leading cause of. Diabetes Mellitus Has an Additional Effect on Coronary Artery Disease

Diabetes Mellitus Has an Additional Effect on Coronary Artery Disease To Decrease Plasma Adiponectin Levels Kuei-Chuan CHAN, 1 MD, Hsi-Hsien CHOU, 1 PhD, Der-Jinn WU, 1 PhD, Yi-Liang WU, 1 MD, and Chien-Ning HUANG, 1 MD SUMMARY We investigated whether plasma levels of adiponectin in patients with both coronary artery disease (CAD) and diabetes mellitus (DM) are lower than in patients with CAD alone. We examined plasma adiponectin levels in 113 patients, 82 with CAD (40 of whom had both CAD and type 2 DM) and 31 normal controls. We found differences in plasma adiponectin levels between CAD patients with and without DM (7.8 ± 4.75 versus 12.1 ± 6.87 µg/ml, P = 0.002), between patients with CAD and controls (10.0 ± 6.27 versus 15.3 ± 5.38 µg/ml, P ), and between men and women (10.2 ± 6.41 versus 13.1 ± 6.22 µg/ml, P = 0.017). Plasma adiponectin levels were correlated negatively with body mass index, triglyceride, total cholesterol, hemoglobin A1c, and fibrinogen levels (r = -0.456, P ; r = -0.355, P ; r = -0.286, P = 0.002; r = -0.299, P < 0.0001 ; r = -0.400, P, respectively), but were not significantly correlated with high sensitivity C-reactive protein or low density lipoprotein levels (r = -0.088, P = 0.352; r = -0.167, P = 0.077, respectively). Plasma adiponectin levels correlated positively with high density lipoprotein levels (r = 0.410, P ). Our study demonstrates that plasma adiponectin levels in patients with both CAD and DM are lower than in patients with CAD alone. We speculate that people who have very low plasma adiponectin levels may be at increased risk of developing both CAD and DM. (Jpn Heart J 2004; 45: 921-927) Key words: Adiponectin, Coronary artery disease, Diabetes mellitus ATHEROSCLEROTIC cardiovascular complications are the leading cause of mortality and morbidity among patients with type 2 diabetes. The precise mechanism underlying the development of atherosclerotic vascular disease in diabetic patients has not been fully clarified. Insulin resistance, abnormalities in lipid metabolism and hemostatic factors may contribute to the development of cardiovascular complications in diabetes. 1) It was found recently that adipose tissue is From the 1 Department of Internal Medicine, Institute of Medicine, Chung-Shan Medical University Hospital, Taichung, Taiwan. Address for correspondence: Chien-Ning Huang, MD, Department of Internal Medicine, Chung-Shan Medical University Hospital, No. 110, Sec. 1, Chien-Kuo N. Road, Taichung 402, Taiwan. Received for publication March 30, 2004. Revised and accepted May 31, 2004. 921

922 CHAN, ET AL Jpn Heart J November 2004 not only an energy storage organ, but also plays an important role in the development of obesity-related disease, including insulin resistance, diabetes mellitus (DM), hyperlipidemia, and vascular diseases through the secretion of various bioactive substances conceptualized as adipocytokines. 2,3) Adiponectin, a 244-amino acid adipocyte-derived protein, accounting for approximately 0.01% of total plasma protein and with concentrations ranging between 4.8 and 34.7µg/mL, was recently revealed to have anti-inflammatory, antidiabetic, and antiatherogenic properties. 4-13) Plasma adiponectin concentrations were found to decrease in individuals with obesity, type 2 DM, and CAD, while an improvement of insulin sensitivity by either weight reduction 8) or administration of thiazolidinediones was associated with increased adiponectin concentrations. 14-16) Adiponectin levels have also been found to be lower in adult men than women, and downregulation of adiponectin by androgens, which has been observed in animal models, may explain these differences. 7) People who have high plasma adiponectin concentrations are less likely to develop type 2 diabetes than those with low concentrations. 11,12) It has been reported that plasma levels of adiponectin in diabetic patients with CAD were lower than those in diabetic patients without CAD. 1) That is to say, decreased plasma adiponectin concentrations in diabetes may be an indicator of CAD. However, it also has been reported that the differences in the adiponectin levels between CAD patients with and without DM were relatively small. 17) In this study, we investigated the differences in plasma adiponectin levels between CAD patients with and without DM to determine if they are similar or different. METHODS Patient groups: One hundred and thirteen Taiwanese patients (63 males and 50 females, age range, 23 to 83 years) were recruited in this study. Of these 113 patients, 82 had coronary artery disease (40 had both coronary artery disease and type 2 diabetes mellitus). As a control group, 31 patients without coronary artery disease or diabetes mellitus were included. The criteria for coronary artery disease were 75% or greater stenosis of at least 1 segment of a major coronary artery confirmed by quantitative coronary angiography by perpendicular projections. Diagnosis of diabetes mellitus was in accordance with the criteria adapted from the American Diabetes Association, 2000. Patients who took any antihyperlipidemic drugs, antibiotics, or thiazolidinediones were excluded. Informed consent was obtained from all subjects. Blood sampling and measurement of adiponectin: Blood samples from coronary artery disease patients were obtained after an overnight fast and immediately after coronary intervention. Blood samples for the measurement of fasting plasma adi-

Vol 45 No 6 ADIPONECTIN AND DIABETIC CORONARY ARTERY DISEASE 923 ponectin concentrations were drawn into prechilled EDTA-containing tubes and immediately placed on ice. All tubes were centrifuged within several minutes of collection and stored at -70 C until assay. Fasting plasma adiponectin concentrations were measured using a radioimmunoassay kit specific for human adiponectin (HADP-61 HK; LINCO Research, St. Charles, Missouri, USA) after 1:500 dilution. Samples were analysed in two batches, with an intra-assay coefficient of variation (CV) < 5% and inter-assay CV < 8% for duplicates over a range of adiponectin concentrations. Statistical analysis: A commercially available software package (Statistical Package for Social Sciences, SPSS 10.0, for Windows) was used for analyzing the data, which is presented as the mean ± standard deviation. The statistical significance of intergroup differences was analyzed by the chi-square test and by Student s t test. ANOVA analysis was used to compare differences in continuous variables between more than two groups. Correlations between 2 parameters were determined by the Pearson correlation coefficient. A P < 0.05 was considered statistically significant. RESULTS The demographic data for the study patients are presented in Table I. One hundred and thirteen patients, 82 with CAD (40 of whom had both CAD and type 2 DM) and 31 normal controls, were enrolled in the study. Patients with both CAD and DM had the highest BMI, hs-crp, triglyceride, total cholesterol, hemoglobin A1c and fibrinogen levels, and the lowest levels of adiponectin and high Table I. Demographic Data Variable CAD (+)/DM (+) (n = 40) CAD (+)/DM (-) (n = 42) Control (n = 31) P-value (ANOVA) Age (years) Gender (male) BMI (kg/m 2 ) Adiponectin level (µg/ml) hs-crp (mg/l) Triglycerides (mg/dl) Total cholesterol (mg/dl) High density lipoprotein (mg/dl) Low density lipoprotein (mg/dl) Hemoglobin A1c (%) Fibrinogen (mg/dl) 61.7 ± 11.0 22 (55.0%) 25.9 ± 3.40 7.8 ± 4.75 2.2 ± 4.44 218.7 ± 139.65 197.6 ± 43.44 36.2 ± 7.49 139.9 ± 44.20 8.5 ± 1.75 377.5 ± 104.12 67.2 ± 10.8 30 (71.4%) 25.0 ± 3.19 12.1 ± 6.87 1.0 ± 2.05 154.31 ± 113.40 189.9 ± 45.13 41.1 ± 9.82 122.0 ± 35.37 5.9 ± 0.63 346.3 ± 91.42 45.0 ± 16.2 11 (35.5%) 21.9 ± 3.10 15.3 ± 5.38 0.02 ± 0.03 101.7 ± 58.20 165.5 ± 39.92 54.8 ± 10.63 131.3 ± 29.18 5.7 ± 0.48 257.1 ± 44.55 0.009* 0.009* 0.007* 0.099 Data are mean ± standard deviation. CAD (+) = patients with coronary artery disease; DM (+) = patients with diabetes mellitus; DM (-) = patients without diabetes mellitus; BMI = body mass index; hs-crp = high sensitivity C-reactive protein.

924 CHAN, ET AL Jpn Heart J November 2004 density lipoprotein. The BMI values for CAD with and without DM were similar. As shown in Table II, there were statistically significant differences in adiponectin levels between the patients with both CAD and DM and the patients with CAD alone (7.8 ± 4.75 versus 12.1 ± 6.87 µg/ml, P = 0.002), between patients with CAD and normal controls (10.0 ± 6.27 versus 15.3 ± 5.38 µg/ml, P ), and between males and females (10.2 ± 6.41 versus 13.1 ± 6.22 µg/ml, P = 0.017). Table III shows that there were significant negative correlations between (a) the adiponectin level and (b) body mass index, triglyceride, total cholesterol, hemoglobin A1c, and fibrinogen levels (r = -0.456, P ; r = -0.355, P < 0.0001 ; r = -0.286, P = 0.002; r = -0.299, P ; r = -0.400, P, respectively), but there were no significant negative correlations between the adiponectin level and high sensitivity C-reactive protein or low density lipoprotein level (r = -0.088, P = 0.352; r = -0.167, P = 0.077, respectively). There was a positive correlation between adiponectin levels and high density lipoprotein levels (r = 0.410, P ). As shown in Table IV, compared with normal controls, patients with CAD have higher BMIs and higher levels of high sensitivity C-reactive protein, triglycerides, total cholesterol, hemoglobin A1c, and fibrinogen. Table II. Differences in Adiponectin Levels Between Subgroups CAD (+), DM (+) (n = 40) CAD (+), DM (-) (n = 42) CAD (+) (n = 82) Normal controls (n = 31) Male (n = 63) Female (n = 50) Adiponectin 7.8 ± 4.75 12.1 ± 6.87 10.0 ± 6.27 15.3 ± 5.38 10.2 ± 6.41 13.1 ± 6.22 P-value 0.002* 0.017* Table III. Correlations Between Adiponectin Level and Other Cardiovascular Risk Factors BMI (kg/m 2 ) hs-crp (mg/l) Triglycerides (mg/dl) Total cholesterol (mg/dl) High density lipoprotein (mg/dl) Low density lipoprotein (mg/dl) Hemoglobin A1c (%) Fibrinogen (mg/dl) Pearson -0.456** -0.088-0.355** -0.286** 0.410** -0.167-0.299** -0.400** P-value 0.352 0.002 0.077

Vol 45 No 6 ADIPONECTIN AND DIABETIC CORONARY ARTERY DISEASE 925 Table IV. Differences Between CAD Patients and Normal Controls Variable CAD (+) (n = 82) Normal Controls (n = 31) P value Gender (male) BMI (kg/m 2 ) Adiponectin (µg/ml) hs-crp (mg/l) Triglycerides (mg/dl) Total cholesterol (mg/dl) High density lipoprotein (mg/dl) Low density lipoprotein (mg/dl) Hemoglobin A1c (%) Fibrinogen (mg/dl) 52 (63.4%) 25.4 ± 3.71 10.0 ± 6.27 1.6 ± 3.46 185.7 ± 130.19 193.6 ± 44.21 38.7 ± 9.04 130.7 ± 40.68 7.2 ± 1.81 361.6 ± 98.47 11 (35.5%) 21.8 ± 3.10 15.3 ± 5.38 0.02 ± 0.03 101.7 ± 58.20 165.5 ± 39.92 54.8 ± 10.63 131.3 ± 29.18 5.7 ± 0.48 257.1 ± 44.55 0.008* 0.014* 0.001* 0.002* 0.943 However, these patients have lower levels of adiponectin and high density lipoprotein. DISCUSSION Plasma adiponectin accumulates in the vascular wall when the endothelial barrier is injured. 18) It has also been reported that adiponectin inhibits monocyte adhesion to endothelial cells and macrophage-to-foam cell transformation, and attenuates TNF-α-induced expression of adhesion molecules in endothelial cells, which is the initial step of atherosclerosis. 1,5,8,9) This study has demonstrated that plasma adiponectin levels in patients with both CAD and DM are lower than in patients with CAD alone and that plasma levels of adiponectin are also associated with some coronary risk factors in patients with CAD. Previous studies showed that adiponectin levels were associated with gender, BMI, CAD, DM, insulin resistance, triglyceride levels, low density lipoprotein (LDL) levels, high density lipoprotein (HDL) levels, 1-3,5-13) and high sensitivity C-reactive protein (hs-crp) levels. 19) In our study, the levels of adiponectin were significantly correlated with gender, BMI, triglyceride levels, total cholesterol levels, HDL levels, fibrinogen levels, and hemoglobin A1c levels, but not significantly correlated with hs-crp or low density lipoprotein levels. Fibrinogen levels have been found to have a significant association with premature CAD. The mechanism by which fibrinogen promotes CAD is not well understood but may involve, at least in part, its effects on plasma viscosity. 20) The percent of glycated hemoglobin (hemoglobin A1c) gives an estimate of blood sugar control for the preceding 3-month period. In our study, we found that plasma levels of adiponectin were correlated negatively and significantly with hemoglobin A1c

926 CHAN, ET AL Jpn Heart J November 2004 levels. This suggests that patients who have poor glycemic control will have lower plasma adiponectin levels than those with good glycemic control. Our study included only a small sample size, and larger sample sizes in further studies are needed. Although the small sample size does not enable us to make a definitive conclusion, ours is the first study to reveal that there are significant differences in plasma levels of adiponectin between CAD patients with and without DM. DM has an additional effect on CAD, that of decreasing plasma adiponectin levels. We speculate that people who have very low plasma adiponectin levels may be at increased risk of developing both CAD and DM. ACKNOWLEDGEMENTS This work was supported by a research fund from Chung Shan Medical University (CSMC 87-OM-B-041) to Chien-Ning Huang. REFERENCES 1. Hotta K, Funahashi T, Arita Y, et al. Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients. Arterioscler Thromb Vasc Biol 2000; 20: 1595-9. 2. Stefan N, Stumvoll M. Adiponectin - its role in metabolism and beyond. Horm Metab Res 2002; 34: 469-74. (Review) 3. Yamamoto Y, Hirose H, Saito I, Nishikai K, Saruta T. Adiponectin, an adipocyte-derived protein, predicts future insulin resistance: two-year follow-up study in Japanese population. J Clin Endocrinol Metab 2004; 89: 87-90. 4. Lindsay RS, Walker JD, Havel PJ, et al. Adiponectin is present in cord blood but is unrelated to birth weight. Diabetes Care 2003; 26: 2244-9. 5. Ouchi N, Kihara S, Arita Y, et al. Novel modulator for endothelial adhesion molecules: adipocyte-derived plasma protein adiponectin. Circulation 1999; 100: 2473-6. 6. Zoccali C, Mallamaci F, Tripepi G, et al. Adiponectin, metabolic risk factors, and cardiovascular events among patients with end-stage renal disease. J Am Soc Nephrol 2002; 13: 134-41. 7. Nishizawa H, Shimomura I, Kishida K, et al. Androgens decrease plasma adiponectin, an insulin-sensitizing adipocyte-derived protein. Diabetes 2002; 51: 2734-41. 8. Yang WS, Lee WJ, Funahashi T, et al. Weight reduction increases plasma levels of an adipose-derived antiinflammatory protein, adiponectin. J Clin Endocrinol Metab 2001; 86: 3815-9. 9. Kumada M, Kihara S, Sumitsuji S, et al. Association of hypoadiponectinemia with coronary artery disease in men. Arterioscler Thromb Vasc Biol 2003; 23: 85-9. 10. Weyer C, Funahashi T, Tanaka S, et al. Hypoadiponectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperinsulinemia. J Clin Endocrinol Metab 2001; 86: 1930-5. 11. Spranger J, Kroke A, Mohlig M, et al. Adiponectin and protection against type 2 diabetes mellitus. Lancet 2003; 361: 226-8. 12. Lindsay RS, Funahashi T, Hanson RL, et al. Adiponectin and development of type 2 diabetes in the Pima India population. Lancet 2002; 360: 57-8. 13. Arita Y, Kihara S, Ouchi N, et al. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun 1999; 257: 79-83. 14. Maeda N, Takahashi M, Funahashi T, et al. PPARγ ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein. Diabetes 2001; 50: 2094-9.

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