Metabolic syndrome (MS) definitions predict cardiovascular events beyond traditional risk factors in patients with type 2 diabetes mellitus (DM) as well as subjects without DM. It has been shown that apolipoprotein B (apoB) and non–high-density lipoprotein cholesterol are associated with coronary artery calcification in DM. However, the relative value of MS, apoB lipoproteins, and estimates of insulin resistance is unknown in predicting atherosclerosis in DM. Cross-sectional analyses of white subjects in 2 community-based studies were performed (n = 611 patients with DM, n = 803 subjects without DM) using multivariate analysis of traditional risk factors and then adding MS, apoB, and homeostasis model assessment of insulin resistance (HOMA-IR). Incremental value was tested using likelihood ratio testing. Beyond traditional risk, HOMA-IR (tobit regression ratio 1.86, p = 0.002), apoB (tobit regression ratio 1.55, p = 0.001), and MS (tobit regression ratio 2.37, p = 0.007) were independently associated with coronary artery calcification in DM. In nested models, HOMA-IR added value to apoB (tobit regression ratio 1.72, p = 0.008), MS (tobit regression ratio 1.72, p = 0.011), and apoB and MS (tobit regression ratio 1.64, p = 0.021). ApoB showed a similar pattern when added to HOMA-IR (tobit regression ratio 1.51, p = 0.004), MS (tobit regression ratio 1.46, p = 0.005), and HOMA-IR and MS (tobit regression ratio 1.48, p = 0.006). MS added to apoB (tobit regression ratio 1.99, p = 0.032) but not HOMA-IR (tobit regression ratio 1.54, p = 0.221) or apoB and HOMA-IR (tobit regression ratio 1.32, p = 0.434). In conclusion, insulin resistance estimates add value to MS and apoB in predicting coronary artery calcification scores in DM and warrant further evaluation in clinic for identification of patients with DM at higher risk for atherosclerotic cardiovascular disease.
Levels of apolipoprotein B (apoB) lipoproteins are associated with insulin resistance and the metabolic syndrome (MS). It is unknown, however, if the presence of MS or the degree of insulin resistance represent an independent tool for cardiovascular disease risk stratification in type 2 diabetes mellitus (DM) beyond the measurement of apoB or non–high-density lipoprotein (HDL) cholesterol. Therefore, we compared the association of MS, apoB lipoproteins, and insulin resistance, estimated by homeostasis model assessment of insulin resistance (HOMA-IR), with coronary artery calcification (CAC) in a sample of patients with DM. As a comparison, we performed similar analysis in subjects without DM, in whom we have previously shown that MS and HOMA-IR predict greater CAC. We hypothesized that HOMA-IR and MS would add incremental value to apoB and non-HDL cholesterol in predicting CAC scores in subjects with and those without DM.
Methods
Details of the Penn Diabetes Heart Study (PDHS) and the Study of Inherited Risk of Coronary Atherosclerosis (SIRCA) are described elsewhere. Both are single-center, cross-sectional studies of subjects without clinical evidence of cardiovascular disease. They were recruited at the University of Pennsylvania and used the same clinical research center, research staff, electron-beam computed tomographic scanner, and biochemical laboratory. SIRCA subjects were recruited on the basis of a family history of premature cardiovascular disease, excluding DM. PDHS subjects with DM were recruited on the basis of having DM. Exclusion criteria included clinical cardiovascular disease, elevated creatinine, and, in SIRCA, the presence of DM. This report focuses on unrelated, Caucasian subjects (611 PDHS participants with DM and 803 SIRCA participants without DM).
Participants were evaluated at the Clinical and Translational Research Center at the University of Pennsylvania after a 12-hour fast. Plasma levels of lipids were measured enzymatically (Cobas Fara II; Roche Diagnostic Systems, Somerville, New Jersey) in lipoprotein fractions after ultracentrifugation (β-quantification technique) in PDHS and in whole plasma in SIRCA. For apoB and C-reactive protein (high-sensitivity), an immunoturbidimetric assay was used. Plasma insulin was measured by radioimmunoassay (Linco Research, St. Charles, Missouri). The intra- and interassay coefficients of variation for insulin were 4.1% and 11.6%, respectively. HOMA-IR, an indirect measure of insulin resistance, was calculated in a fasting state (glucose [mmol/L] × insulin [μU/ml]/22.5). The HOMA-IR2 calculation may be more robust than HOMA-IR in DM because it accounts for outlier values of glucose and insulin. Therefore, we also confirmed our findings using the HOMA-IR2 approach (Spearman’s correlation of HOMA-IR2 and HOMA-IR = 0.98) and found no differences in results (data not shown). Laboratory test results were generated by personnel blinded to the clinical characteristics and CAC scores of participants. Framingham risk scores, using total cholesterol, were calculated as described by Wilson et al. Subjects were classified as having MS using the revised National Cholesterol Education Program (NCEP) definition (glucose cut point 100 mg/dl). Global CAC scores on electron-beam computed tomography were quantified according to the method of Agatston et al.
Data are reported as median (interquartile range) or as mean ± SD for continuous variables and as proportions for categorical variables. The crude associations of apoB, non-HDL cholesterol, and HOMA-IR with lipid, metabolic, inflammatory parameters, and each component of MS were examined by Spearman’s correlation and the Kruskal-Wallis test. Multivariate analysis of CAC scores was performed using tobit conditional regression of natural logarithm (CAC + 1) because of the distribution of CAC data (many zero scores with a marked right skew). Tobit regression models the dichotomous outcome of zero versus nonzero and then assumes normality conditional on the presence of nonzero score data. The tobit model is designed to assess the relation between explanatory variables and a censored dependent variable at 1 end, where many observations are clustered. We chose this modeling because the use of ordinary least squares regression on such a non-normal distribution such as CAC would produce biased estimates and invalid inference. Tobit modeling has otherwise similar assumptions about error distributions as the linear regression model. The associations of MS (presence vs absence), HOMA-IR (1 SD), apoB (1 SD), and non-HDL cholesterol (1 SD) with CAC were assessed in tobit models with confounding risk factors: age, gender, medications, and risk factors, including hypertension (defined as systolic blood pressure >140 mm Hg or diastolic blood pressure >90 mm Hg or the use of antihypertensive therapy), dyslipidemia (defined as serum total cholesterol >5.18 mmol/L, low-density lipoprotein cholesterol >2.59 mmol/L, triglyceride >1.70 mmol/L, or the use of dyslipidemia therapy) alcohol use, exercise, and C-reactive protein. We also tested the Framingham risk score in place of hypertension, hyperlipidemia, and tobacco use and did not find differences in the results (data not shown). Finally, we applied likelihood ratio tests in nested models to assess the value of each parameter (HOMA-IR, MS, apoB, and non-HDL cholesterol) relative to one another in predicting CAC. Statistical analyses were performed using Stata version 10.0 (StataCorp LP, College Station, Texas).
Results
Table 1 lists the characteristics of PDHS participants with DM and SIRCA participants without DM. Patients with DM were older, predominantly men, and more obese. As expected, low-density lipoprotein cholesterol levels were lower in patients with DM, reflecting their higher statin use. As expected, HOMA-IR values were higher in patients with DM (15% taking insulin and were excluded from HOMA-IR calculation). NCEP-defined MS was present in 77% of patients with DM and 26% of subjects without DM. Consistent with greater cardiovascular disease risk, Framingham risk scores and CAC scores were higher in patients with DM.
| Variable | DM | |
|---|---|---|
| Yes (n = 611) | No (n = 803) | |
| Age (years) (median [full range]) | 60 (36–77) | 48 (20–73) |
| Men | 436 (71.4%) | 424 (52.8%) |
| Alcohol use | 357 (58.4%) | 544 (67.8%) |
| Current smoking | 51 (8.4%) | 91 (11.3%) |
| HDL cholesterol (mmol/L) | 1.17 (0.99–1.37) | 1.24 (1.01–1.53) |
| Men | 1.1 (0.9–1.2) | 1.1 (0.9–1.3) |
| Women | 1.3 (1.1–1.6) | 1.5 (1.2–1.8) |
| TC (mmol/L) | 4.51 (3.94–5.13) | 5.31 (4.58–5.91) |
| Triglycerides (mmol/L) | 1.51 (1.04–2.23) | 1.32 (0.98–1.80) |
| LDL cholesterol (mmol/L) | 2.51 (2.04–3.08) | 3.26 (2.67–3.83) |
| ApoB (g/L) | 0.82 (0.71–0.94) | 0.98 (0.84–1.14) |
| Blood pressure (mm Hg) | ||
| Systolic | 131 (122–140) | 126 (117–136) |
| Diastolic | 76 (71–81) | 77 (72–84) |
| BMI (kg/m 2 ) | 32 (28–36) | 27 (24–30) |
| Men | 31 (28–35) | 27 (25–30) |
| Women | 34 (29–38) | 26 (23–30) |
| Waist circumference (inches) | 42 (39–46) | 35 (32–39) |
| MS | 468 (76.6%) | 207 (25.78%) |
| Leptin (μg/L) | 11.6 (6.5–20.9) | 8.4 (4.5–16.4) |
| Adiponectin (μg/mL) | 9.1 (6.12–14.87) | 16.3 (11.58–24.55) |
| HOMA-IR | 4.38 (2.9–6.7) | 1.43 (0.9–2.1) |
| Hs-CRP (mg/dl) | 1.6 (0.8–3.4) | 1.2 (0.5–2.6) |
| IL-6 (pg/ml) | ||
| Total | 1.32 (0.8–2.1) | 1.3 (0.8–1.9) |
| Men | 1.2 (0.8–2) | 1.2 (0.7–1.8) |
| Women | 1.4 (0.7–2.3) | 1.2 (0.8–2) |
| Medications | ||
| Statins | 351 (57.5%) | 112 (13.9%) |
| Niacin | 34 (5.6%) | 23 (2.9%) |
| Fibrates | 60 (9.9%) | 9 (1.1%) |
| Insulin | 91 (14.9%) | 0 |
| Metformin | 390 (63.8%) | 0 |
| Thiazolidinediones | 167 (27.3%) | 0 |
| Sulfonylureas | 246 (40.3%) | 0 |
| HRT (women) | 276 (45.1%) | 226 (28.2%) |
| CAC | ||
| Median (IQR) | 89 (1–456) | 3 (0–45) |
| Mean ± SD | 424 ± 795 | 87 ± 266 |
| Zero score | 24.7% | 31.1% |
| 10-year Framingham LDL risk | 13% (9%–18%) | 5% (3%–7%) |
| 10-year Framingham TC risk | 13% (8%–20%) | 5% (3%–8%) |
The correlations of HOMA-IR and apoB with other risk factors were broadly similar across DM status ( Table 2 ). Interestingly, HOMA-IR had the strongest correlations with metabolic and inflammatory risk factors, while apoB showed stronger relations with atherogenic lipoproteins and the Framingham risk score. Although HOMA-IR and apoB were associated with the MS, the strength and extent of associations with individual MS components differed, such as strong associations of HOMA-IR with waist circumference, presence of MS, and HDL levels, whereas apoB had the strongest association with triglycerides (data not shown). These findings suggest that HOMA-IR and apoB may capture distinct information regarding metabolic cardiovascular disease risk. Non-HDL cholesterol was highly correlated with apoB (r ≈ 0.80). Indeed, findings were similar for non-HDL cholesterol and apoB, and therefore, only data for apoB are presented throughout this report.
| Variable | DM | |||
|---|---|---|---|---|
| Yes (n = 611) | No (n = 803) | |||
| HOMA-IR | ApoB | HOMA-IR | ApoB | |
| TC | 0.03 | 0.77 † | 0.07 ⁎ | 0.77 † |
| Triglycerides | 0.38 † | 0.47 † | 0.39 † | 0.50 † |
| LDL cholesterol | −0.01 | 0.79 † | 0.10 † | 0.78 † |
| HDL cholesterol | −0.31 † | −0.21 † | −0.32 † | −0.20 † |
| Non-HDL cholesterol | 0.16 † | 0.87 † | 0.22 † | 0.87 † |
| Systolic blood pressure | 0.14 † | 0.05 | 0.32 † | 0.20 † |
| BMI | 0.43 † | 0.07 | 0.50 † | 0.23 † |
| Waist circumference (inches) | 0.41 † | 0.08 ⁎ | 0.51 † | 0.25 † |
| FRS | 0.06 | 0.38 † | 0.27 † | 0.49 † |
| Hs-CRP | 0.22 † | 0.16 † | 0.29 † | 0.24 † |
| IL-6 | 0.26 † | 0.02 | 0.30 † | 0.12 † |
| Leptin | 0.42 † | 0.03 | 0.34 † | 0.18 † |
| Adiponectin | −0.28 † | −0.20 † | −0.27 † | −0.15 † |
| ApoB | 0.20 † | — | 0.25 † | — |
| HOMA-IR | — | 0.20 † | — | 0.25 † |
In patients with DM, MS, HOMA-IR, and apoB were associated with CAC scores after adjusting for traditional risk factors, lipid lowering, and DM medications ( Table 3 , model 1). In subjects without DM, as published, a similar pattern of association was seen for MS, HOMA-IR, and apoB.
| Variable | DM | |||
|---|---|---|---|---|
| Yes (n = 611) | No (n = 803) | |||
| Tobit Ratio (95% CI) | p Value | Tobit Ratio (95% CI) | p Value | |
| MS | ||||
| Model 1 | 2.37 (1.27–4.41) | 0.007 | 2.15 (1.43–3.24) | <0.001 |
| Model 2 (HOMA-IR) | 1.54 (0.77–3.06) | 0.221 | 1.66 (1.08–2.55) | 0.021 |
| Model 3 (apoB) | 1.99 (1.06–3.73) | 0.032 | 1.75 (1.24–2.65) | 0.009 |
| Model 4 (HOMA-IR and apoB) | 1.32 (0.66–2.64) | 0.434 | 1.40 (0.91–2.16) | 0.126 |
| HOMA-IR | ||||
| Model 1 | 1.86 (1.25–2.78) | 0.002 | 1.84 (1.40–2.42) | <0.001 |
| Model 2 (MS) | 1.72 (1.13–2.62) | 0.011 | 1.66 (1.24–2.20) | 0.001 |
| Model 3 (apoB) | 1.72 (1.15–2.57) | 0.008 | 1.66 (1.26–2.19) | <0.001 |
| Model 4 (MS and ApoB) | 1.64 (1.08–2.49) | 0.021 | 1.56 (1.17–2.07) | 0.002 |
| ApoB | ||||
| Model 1 | 1.55 (1.19–2.01) | 0.001 | 1.58 (1.33–1.90) | <0.001 |
| Model 2 (HOMA-IR) | 1.51 (1.14–1.99) | 0.004 | 1.51 (1.26–1.81) | <0.001 |
| Model 3 (MS) | 1.46 (1.12–1.91) | 0.005 | 1.50 (1.25–1.80) | <0.001 |
| Model 4 (HOMA-IR and MS) | 1.48 (1.12–1.96) | 0.006 | 1.47 (1.22–1.76) | <0.001 |
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