Measures of Insulin Resistance Add Incremental Value to the Clinical Diagnosis of Metabolic Syndrome in Association With Coronary Atherosclerosis

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1 Measures of Insulin Resistance Add Incremental Value to the Clinical Diagnosis of Metabolic Syndrome in Association With Coronary Atherosclerosis Muredach P. Reilly, MB; Megan L. Wolfe, BS; Thomas Rhodes, MSPH; Cynthia Girman, DrPH; Nehal Mehta, MD; Daniel J. Rader, MD Background Whether measures of insulin resistance provide incremental information regarding atherosclerotic cardiovascular disease beyond current National Cholesterol Education Program (NCEP) Adult Treatment Panel III metabolic syndrome (MetSyn) criteria or inflammatory markers is uncertain. Methods and Results We examined the association of insulin resistance and MetSyn with coronary artery calcification (CAC) in 840 asymptomatic nondiabetic subjects. Both NCEP and World Health Organization defined MetSyn were associated (ordinal regression odds ratio [OR] and 95% confidence intervals for NCEP-defined MetSyn) with CAC after controlling for age, non-metsyn risk factors, and plasma CRP levels (OR, 1.93 [1.43 to 2.60], P 0.001) and after further controlling for homeostasis model assessment index (HOMA) (OR, 1.56 [1.14 to 2.15], P 0.006). Conversely, HOMA was significantly associated with CAC after adjusting for age, non-metsyn risk factors, and CRP levels (OR, 1.62 [1.31 to 2.01], P 0.001) and after further adjusting for NCEP-defined MetSyn (OR, 1.45 [1.16 to 1.82], P 0.007). Addition of HOMA to the NCEP MetSyn significantly improved the association with CAC, but addition of CRP data to MetSyn or HOMA did not. Conclusions Both MetSyn and HOMA index were associated with coronary atherosclerosis independent of established risk factors, including CRP. These findings support the use of biomarkers of insulin resistance in addition to NCEP MetSyn criteria in assessing cardiovascular disease risk. (Circulation. 2004;110: ) Key Words: metabolism insulin atherosclerosis Overweight and obesity have risen dramatically in the United States, resulting in a marked increase in the metabolic syndrome (MetSyn), 1 a clustering of cardiovascular risk factors including central adiposity, insulin resistance, hypertension, dyslipidemia, and a proinflammatory state. 2 4 Because MetSyn confers risk of atherosclerotic cardiovascular disease (CVD) 5 8 even beyond that associated with the individual factors, 9 it represents a target for therapeutic interventions. 10 The National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) expert panel 10 and World Health Organization (WHO) 11 have proposed distinct definitions of MetSyn to aid in clinical practice and research. The NCEP definition, unlike the WHO, does not include measurement of insulin and therefore may fail to detect insulin resistance. 5,8,12,13 Furthermore, the association of MetSyn with incident diabetes and CVD has varied substantially when the two definitions have been applied in a single study. 5,7 9 Additional definitions of MetSyn have been proposed, 14,15 underscoring the need for unified criteria for use in clinical practice. Chronic subclinical inflammation is thought to be part of the MetSyn. 16 Inflammatory markers such as C-reactive protein (CRP) may provide independent information regarding diabetes and CVD risk beyond NCEP MetSyn criteria. 7,17 However, it remains unclear if plasma levels of CRP are predictive of cardiovascular events after adjustment for measures of insulin resistance. Recent studies of coronary artery calcification (CAC) in asymptomatic samples have shown an association of MetSyn and insulin resistance with the burden of coronary atherosclerosis Previously, we have reported on the association of established risk factors, including CRP, with CAC in the Study of Inherited Risk of Coronary Atherosclerosis (SIRCA) In this report, we examine the association of MetSyn and measures of insulin resistance with CAC and we assess the incremental value of measures of insulin resistance in predicting of CAC beyond the clinical diagnosis of the MetSyn and plasma CRP levels. Received December 23, 2003; revision received April 1, 2004; accepted April 4, From the Cardiovascular Division and Center for Experimental Therapeutics, Department of Medicine, University of Pennsylvania School of Medicine (M.P.R., M.L.W., N.M., D.J.R.), and the Department of Epidemiology, Merck Research Laboratories (T.R., C.G.), Philadelphia, Pa. The online-only Data Supplement is available at Correspondence to Muredach Reilly, Cardiovascular Division, University of Pennsylvania Medical Center, 909 BRB 2/3, 421 Curie Blvd, Philadelphia, PA muredach@spirit.gcrc.upenn.edu American Heart Association, Inc. Circulation is available at DOI: /01.CIR C 803

2 804 Circulation August 17, 2004 TABLE 1. Characteristics of the Study Sample Methods Patients SIRCA is a cross-sectional study of factors associated with CAC in asymptomatic subjects recruited on the basis of a family history of premature CVD. Study design and initial findings have been published, and a description is provided in the Appendix (see online-only Data Supplement at Subjects were eligible for SIRCA if they were men 30 to 65 years of age or women 35 to 70 years of age and were free from clinical CVD but had a family history of premature CVD. The University of Pennsylvania Institutional Review Board approved the study protocol. This report focuses on unrelated nondiabetic subjects (fasting blood glucose levels of 7.0 mmol/l) (n 840). Evaluated Parameters Study subjects were evaluated at the General Clinical Research Center at the University of Pennsylvania Medical Center after a Men (n 443) Median (IQR) Women (n 397) Median (IQR) Age, y 46 (40 to 51) 50 (44 to 57) Total cholesterol mmol/l 5.19 (4.53 to 5.82) 5.47 (4.74 to 6.11) mg/dl 200 (175 to 225) 211 (183 to 234) LDL cholesterol mmol/l 3.29 (2.68 to 3.88) 3.26 (2.64 to 3.81) mg/dl 127 (103 to 150) 126 (102 to 147) HDL cholesterol mmol/l 1.09 (0.93 to 1.29) 1.50 (1.20 to 1.76) mg/dl 42 (36 to 50) 58 (47 to 68) Triglycerides mmol/l 1.41 (1.02 to 2.01) 1.27 (0.90 to 1.66) mg/dl 125 (90 to 177) 112 (80 to 147) Glucose mmol/l 5.22 (4.83 to 5.61) 5.06 (4.78 to 5.44) mg/dl 94 (87 to 101) 91 (86 to 98) Insulin, pmol/l 44.7 (29.5 to 67.0) 37.9 (26.1 to 57.1) HOMA index 1.50 (0.95 to 2.29) 1.30 (0.85 to 1.90) Hs C-reactive protein, mg/dl 1.1 (0.5 to 2.0) 1.4 (0.6 to 3.6) Waist circumference, cm 94.0 (88.9 to 104.2) 81.3 (73.7 to 91.5) Body mass index, kg/m (25.2 to 30.3) 25.6 (22.8 to 30.2) Blood pressure, mm Hg Systolic 128 (119 to 137) 125 (113 to 136) Diastolic 79 (73 to 86) 75 (68 to 82) Smoking, % Medications Statins, % Aspirin, % Hormone replacement therapy, % N/A 28.2 Coronary artery calcification Mean score ( SD) Median (IQR) 5 (1 to 68) 1 (0 to 12) 70th Percentile, % Zero score, % IQR indicates interquartile range. 12-hour overnight fast. Plasma total cholesterol, HDL cholesterol, triglyceride, and glucose levels were measured enzymatically on a Cobas Fara II (Roche Diagnostic Systems Inc) in a Centers for Disease Control certified lipoprotein laboratory, with the use of Sigma reagents. LDL cholesterol was calculated by means of the Friedewald formula. Plasma insulin levels were measured with the use of a commercial enzyme immunoassay (Linco Research). The intra-assay coefficient of variation was % for insulin standard. The interassay coefficient of variation was 4.1% for insulin standard and 11.6% for pooled human plasma. Plasma CRP levels were assayed with the use of an ultra high-sensitivity latex turbidimetric immunoassay (Wako Ltd). 23 Subjects were classified as having MetSyn by 2 criteria, the NCEP ATP III criteria 10 and a modification of the original WHO definition as applied by Lakka et al 5 and Laaksonen et al. 12 By NCEP criteria, a person has MetSyn if 3 or more of the following are present: (1) central obesity: waist 102 cm in men and 88 cm in women, (2) hypertriglyceridemia: mmol/l (150 mg/dl), (3) low HDL

3 Reilly et al Metabolic Syndrome and Coronary Atherosclerosis 805 cholesterol: mmol/l (40 mg/dl) in men and mmol/l (50 mg/dl) in women, (4) high blood pressure: 130/85 or use of blood pressure medications, (5) high fasting glucose: 6.1 mmol/l (110 mg/dl). According to recent applications 5,12 of WHO criteria, a person has MetSyn if they have insulin resistance defined as homeostasis model assessment (HOMA) index (fasting glucose [mmol/l] fasting insulin [ U/mL]/22.5) 25 in the top quartile ( in the SIRCA sample), impaired fasting glucose ( 6.1 mmol/l), or diabetes with 2 or more of the following: (1) central obesity: waist 94 cm in men and 88 cm in women, (2) dyslipidemia: triglycerides mmol/l or HDL mmol/l in women and mmol/l (35 mg/dl) in men, (3) high blood pressure: 140/90 or use of blood pressure medications. HOMA was the primary measure of insulin sensitivity, but fasting plasma insulin levels provided comparable results. Global CAC scores were determined as described, 24 with customized software (Imatron) used according to the method of Agatston 26 from 40 continuous 3-mm-thick computed tomograms collected on an electron beam tomography (EBT) scanner (Imatron). Reproducibility studies with the use of these techniques suggest that artifact in EBT estimation of CAC accounts for a small proportion of CAC variability. 27 Statistical Analysis Data are reported as median and interquartile range or mean SD for continuous variables and as proportions for categoric variables. Analyses were performed for each sex separately, in addition to the full sample. The crude association of MetSyn with CAC was examined as an ordinal variable (number of MetSyn features) or as a binary variable (presence or absence of NCEP or WHO criteria). The crude association of HOMA with CAC was examined through the use of HOMA quartiles (0.043 to 0.90, 0.90 to 1.427, to 2.114, to 7.751). Median CAC scores were compared across ordinal MetSyn categories and HOMA quartiles by means of the Kruskal-Wallis rank test and Wilcoxon test for trend. Ordinal logistic regression is a method appropriate for the analysis of CAC data, which has a markedly nonnormal distribution and a significant proportion of subjects with no detectable CAC. 24,28 Ordinal CAC outcome categories were 0, 1 to 10, 11 to 100, 101 to 400, and 400, with published criteria used to approximate no, mild, moderate, and severe coronary atherosclerosis. 29 The association of MetSyn with CAC was assessed in sexspecific models that included (1) MetSyn and age (age and age 2 ); (2) MetSyn, CRP levels, non-metsyn risk factors, and age; and (3) HOMA data, NCEP-defined MetSyn, CRP levels, non- MetSyn risk factors, and age. Non-MetSyn risk factors included smoking, family history of premature CAD, exercise, alcohol intake, race, LDL cholesterol, use of medications (aspirin, statins, -blockers, ACE inhibitors, and hormone replacement therapy). The association of HOMA (or plasma insulin) with CAC was assessed in models adjusted for (1) age; (2) age, CRP, and non-metsyn risk factors; and (3) age, CRP, and non-metsyn risk factors and NCEP-defined MetSyn. The interaction between sex and MetSyn or HOMA data were assessed in adjusted models by means of the likelihood-ratio (LR) test. The LR test also was applied to nested models to determine if addition of HOMA to NCEP-defined MetSyn, plasma CRP levels to MetSyn, or HOMA improved the prediction of CAC. The results of ordinal logistic regression are presented as the odds ratio (OR) of being in a higher CAC category for the presence or absence of MetSyn and for a 1-U change in ln-homa. Statistical analyses were performed with the use of Stata 8.0 software (Stata Corp). Results Characteristics of SIRCA Subjects The SIRCA sample was predominantly white (95%). Women were older than men, as expected from enrollment criteria TABLE 2. Prevalence of Metabolic Syndrome Features and Coronary Artery Calcification Scores by Metabolic Syndrome Definitions Men (n 443) Women (n 397) NCEP MetSyn features Low HDL Elevated triglycerides Elevated fasting glucose High blood pressure Central obesity NCEP MetSyn definition WHO MetSyn features Insulin resistance Dyslipidemia Hypertension Central obesity WHO MetSyn definition NCEP MetSyn Absent 3 (0 to 57) 0 (0 to 6) Present 23 (3 to 93) 6 (1 to 36) 2 (P ) 19.2 ( 0.001) 27.6 ( 0.001) WHO MetSyn Absent 4 (1 to 59) 0.5 (0 to 9) Present 20 (3 to 92) 5.5 (1 to 31) 2 (P ) 8.0 ( 0.005) 17.3 ( 0.001) Values are percentages or median CAC (interquartile range). (Table 1). Consistent with a recruitment strategy based on family history of premature CVD, CAC scores were higher than in population-based studies of asymptomatic samples of similar age and demographics. 30 Approximately 27% of men and 22% of women met criteria for MetSyn according to NCEP (Table 2), roughly comparable to the age-adjusted prevalence for white subjects in the Third National Health and Nutrition Examination Survey (NHANES). 1 Estimates of the prevalence of the WHO criteria were slightly lower (Table 2). Consistent with previous reports of a lack of sensitivity of NCEP-defined MetSyn for insulin resistance, 8,12, % (19.2% of men and 10.7% of women) of subjects classified as non-metsyn by NCEP criteria (ie, expected not to be insulin resistant) had WHOdefined insulin resistance (HOMA values in the upper quartile). In fact, of subjects with HOMA values in the upper quartile, 45% were classified as not having MetSyn by the NCEP definition. Crude Association of MetSyn and Insulin Resistance With CAC There was no statistically significant interaction between sex and MetSyn or HOMA data in the association with CAC. Median CAC scores were significantly higher in both men and women with MetSyn compared with those without (Table 2) and increased with increasing prevalence of MetSyn features according to NCEP criteria (trend, P 0.001) and

4 806 Circulation August 17, 2004 Median coronary artery calcification (CAC) scores rise with increasing prevalence of metabolic syndrome features by (A) NCEP criteria (trend, P for both men and women) and (B) WHO criteria (trend, P for both men and women). CAC data are illustrated as log (CAC 1) for ease of presentation. Median (interquartile range, IQR) CAC scores and number of subjects in each group are shown beneath each plot. WHO criteria (trend, P 0.001) (Figure). Median (interquartile range) CAC scores increased across HOMA quartiles (P 0.001) [(men: 2.0 (0 to 53), 2.5 (0 to 37.5), 6.0 (1.5 to 75.5), 20 (3 to 92); women: 0 (0 to 4), 1 (0 to 9), 1 (0 to 29), 4 (0 to 22)]. Multivariable Analysis of the Association of MetSyn and Insulin Resistance With CAC MetSyn definitions, individual MetSyn features, and HOMA index were associated with CAC in age-adjusted analyses, although the strength of these associations varied by sex and

5 Reilly et al Metabolic Syndrome and Coronary Atherosclerosis 807 TABLE 3. Age-Adjusted Association of Metabolic Syndrome Features, HOMA Index, and Plasma Insulin Levels With Coronary Artery Calcification Men Women Odds Ratio (CI) P Odds Ratio (CI) P NCEP MetSyn features Elevated fasting glucose 1.28 (0.70 to 2.34) (1.01 to 4.85) Low HDL cholesterol 1.64 (1.17 to 2.34) (0.91 to 2.00) 0.1 Elevated triglycerides 1.44 (1.01 to 2.05) (1.20 to 2.86) High blood pressure 1.98 (1.38 to 2.83) (1.58 to 3.49) Central obesity 1.92 (1.34 to 2.76) (2.18 to 4.81) NCEP MetSyn present 2.14 (1.46 to 3.13) (1.72 to 4.10) WHO MetSyn features Insulin resistance 1.63 (1.14 to 2.34) (1.30 to 3.07) Dyslipidemia 1.66 (1.17 to 2.34) (1.29 to 2.90) High blood pressure 1.68 (1.18 to 2.39) (1.20 to 2.62) Central obesity 1.81 (1.28 to 2.56) (2.53 to 5.75) WHO MetSyn present 1.55 (1.06 to 2.27) (1.65 to 4.22) HOMA (1 log unit) 1.51 (1.15 to 1.96) (1.65 to 3.06) Insulin levels (1 SD) 1.36 (1.15 to 1.62) (1.33 to 2.00) The odds ratio for increase in CAC category is shown for 1 log unit change in HOMA index and for 1 SD change in plasma insulin levels (31.50 pmol/l). There were no statistically significant interactions between sex and MetSyn definitions, HOMA index, or plasma insulin levels in the association with CAC. the criteria used (Table 3). Waist circumference was the strongest single predictor of CAC. Both MetSyn and HOMA index were independently associated with CAC after controlling for age, non-metsyn risk factors, and plasma CRP (Table 4). Notably, adding ln-homa to a model with NCEP-defined MetSyn significantly strengthened (LR test P 0.001) the association with CAC, and adding MetSyn to a model containing ln-homa also improved (LR test P 0.006) prediction of CAC scores. In contrast, CRP did not add significantly to models that already contained MetSyn (LR test P 0.15) or ln-homa (LR test P 0.48). Results were similar when plasma insulin levels were used in place of HOMA data (Tables 3 and 4). TABLE 4. Association of the Metabolic Syndrome, HOMA Index, and Plasma Insulin Levels With Coronary Artery Calcification in Multivariable Ordinal Logistic Regression Men Women Total* Adjusted for Odds Ratio (CI) P Odds Ratio (CI) P Odds Ratio (CI) P NCEP MetSyn Age, RF, CRP 1.92 (1.46 to 3.13) (1.23 to 3.25) (1.43 to 2.60) Age, RF, CRP, HOMA 1.67 (1.10 to 2.53) (0.94 to 2.59) (1.14 to 2.15) WHO MetSyn Age, RF, CRP 1.42 (0.95 to 2.10) (1.19 to 3.29) (1.18 to 2.19) HOMA (1 log unit) Age, RF, CRP 1.46 (1.11 to 1.92) (1.31 to 2.59) (1.31 to 2.01) Age, RF, CRP, NCEP-MetSyn 1.30 (0.97 to 1.80) (1.14 to 2.36) (1.16 to 1.82) Insulin level (1 SD) Age, RF, CRP 1.39 (1.16 to 1.67) (1.18 to 1.83) (1.22 to 1.61) Age, RF, CRP, NCEP-MetSyn 1.31 (1.09 to 1.58) (1.09 to 1.73) (1.15 to 1.53) *Models that include both men and women were adjusted for sex in addition to other risk factors. Non-MetSyn risk factors (RF) included LDL cholesterol, cigarette smoking, exercise, alcohol, race, family history of premature coronary artery disease, and medications (aspirin, statins, ACE inhibitors, and hormone replacement therapy). Natural log-transformed HOMA. The odds ratio for increase in CAC category is shown for 1 log unit change in HOMA index and for 1 SD change in plasma insulin levels (31.50 pmol/l). There were no statistically significant interactions between sex and MetSyn definitions, HOMA index, or plasma insulin levels in the association with CAC.

6 808 Circulation August 17, 2004 Discussion We found that MetSyn, with the use of either NCEP or WHO criteria, was associated with CAC in an asymptomatic nondiabetic sample even after controlling for multiple established CVD risk factors and plasma levels of CRP. Furthermore, we found that measures of insulin resistance but not plasma CRP levels provided independent and additive value to NCEPdefined MetSyn in predicting CAC scores. The insulin resistance syndrome or MetSyn 2 is characterized by a clustering of atherosclerotic CVD risk factors. 3,4,16 Insulin resistance is thought to be the most prominent pathophysiological process underlying MetSyn. 2,14,15 The WHO definition of MetSyn 11 requires the presence of insulin resistance plus 2 other of central obesity, hypertension, or dyslipidemia, whereas the NCEP definition does not require a laboratory measure of insulin. 10 Despite this, the NCEP criteria, based on features typically correlated with insulin resistance, should identify insulin-resistant subjects. However, analysis of NHANES data suggests that the NCEP criteria fail to detect a significant proportion of patients with insulin resistance, particularly in ethnic minorities. 8 The NCEP definition was less sensitive than the WHO definition in predicting both type 2 diabetes 12,15 and the development of CVD in men. 5,8,9 In our nondiabetic sample, the NCEP definition failed to detect 15% of subjects defined as insulin-resistant by WHO criteria. Evidence continues to emerge supporting the increased risk of CVD events in patients with MetSyn. 5 9,17 Lakka et al 5 found a 2- to 4-fold increased risk of cardiovascular death with MetSyn in a sample of 1209 Finnish men free from diabetes and CVD at baseline. MetSyn predicted atherosclerosis progression and CVD events in 888 subjects in the Bruneck study, whereas most individual components of the syndrome were not significantly associated with CVD outcomes, 9 supporting the concept that MetSyn provides information that is more than the sum of its parts. 31 Studies demonstrating an increased burden of subclinical atherosclerosis in subjects with insulin resistance and MetSyn suggest that promotion of atherosclerosis is a basis, at least in part, for the link to clinical events. 9,18 21,32 Arad et al 18 found a positive association between CAC and the number of MetSyn features, HOMA, and visceral adiposity in the St Francis Heart Study (n 1160). In 1000 asymptomatic subjects recruited to the Prospective Army Coronary Calcification study, there was a graded association between CAC and the number of NCEP MetSyn features or the presence of MetSyn, 20 but this failed to meet statistical significance for MetSyn after adjusting for LDL cholesterol (OR, 1.54 [0.91 to 2.6]). Wong et al 21 found that the NCEP MetSyn (OR, 1.40 [1.05 to 1.87]) and the number of MetSyn features were associated with CAC after controlling for traditional risk factors in 1823 asymptomatic subjects. Chronic inflammation may represent a common pathophysiological basis for insulin resistance, MetSyn, and atherosclerotic CVD. 16,33 However, current definitions of MetSyn do not incorporate measures of inflammation. Recent studies have shown that CRP levels predict CVD even in subjects with NCEP-defined MetSyn. 7,17 However, in SIRCA, CRP did not add value to the association with CAC beyond MetSyn or insulin resistance. Thus, an association between CRP and plaque rupture or thrombosis, rather than atherosclerotic burden, may form the basis for the relation of CRP to clinical events. 23 Alternatively, CRP levels may not be predictive of CVD if laboratory measures of insulin resistance are included in the analyses. A unique and important finding in our study was that insulin resistance and NCEP-defined MetSyn were additive in the association with CAC, independent of plasma levels of CRP. This study has several limitations. SIRCA is a crosssectional, non population- based study that is predominantly white and may not be generalizable to minority groups. 8 We used surrogate measures of insulin resistance and therefore may have underestimated its prevalence. There remains controversy regarding the type of atherosclerotic plaques detected at EBT and the utility of CAC in predicting clinical events. However, CAC exhibits strong correlation with both histopathological and angiographic measures of atherosclerosis, 34,35 and prospective data support its ability to predict future events. 36 The results of our study need to be confirmed in diverse samples with the use of alternative measures of atherosclerosis and, ultimately, in prospective studies of CVD. Conclusions We found that MetSyn is an important predictor of subclinical coronary atherosclerosis in a sample of asymptomatic nondiabetic individuals with a family history of premature CVD. Furthermore, laboratory measures of insulin resistance provided additional value when added to NCEP MetSyn definition in the association with CAC independent of plasma CRP. We suggest that the development of standardized insulin assays or alternative biomarkers of insulin resistance may facilitate CVD risk prediction in MetSyn. Acknowledgments This study was funded in part by grant M01-RR00040 from the NCRR/NIH supporting the University of Pennsylvania General Clinical Research Center (GCRC), by the Penn Diabetes and Endocrinology Research Center (DK19525), and by Merck & Co, Inc. Dr Reilly is supported by a Mentored Patient-Oriented Research Career Development Award from the National Center for Research Resources (NIH-RR ), by (RO1-HL ) from the NIH, and by the W.W. Smith Charitable Trust (No. H0204). Dr Rader is supported by grants from the NHLBI, HIDDK, and NCRR and is a recipient of the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research and a recipient of a Doris Duke Distinguished Clinical Investigator Award. We are indebted to the nursing staff of the University of Pennsylvania GCRC. References 1. Ford ES, Giles WH, Dietz WH. Prevalence of the metabolic syndrome among US adults: findings from the third National Health and Nutrition Examination Survey. JAMA. 2002;287: Reaven GM. 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