Resistin Is an Inflammatory Marker of Atherosclerosis in Humans
http://www.100md.com
循环学杂志 2005年第2期
the Divisions of Cardiology (M.P.R., D.J.R.) and Endocrinology, Diabetes, and Metabolism (M.L., M.A.L.)
Department of Medicine (M.P.R., M.L., M.L.W., A.R., M.A.L., D.J.R.)
Center for Experimental Therapeutics, and the Penn Diabetes Center (M.P.R., M.L.W., D.J.R.)
University of Pennsylvania School of Medicine, Philadelphia, Pa.
Abstract
Background— Resistin, a plasma protein, induces insulin resistance in rodents. Recent reports suggest that circulating levels of resistin are elevated in obese and insulin-resistant rodents and humans. Whereas rodent resistin is made in adipocytes, macrophages are a major source of human resistin. Given the convergence of adipocyte and macrophage function, resistin may provide unique insight into links between obesity, inflammation, and atherosclerosis in humans.
Methods and Results— We examined whether plasma resistin levels were associated with metabolic and inflammatory markers, as well as with coronary artery calcification (CAC), a quantitative index of atherosclerosis, in 879 asymptomatic subjects in the Study of Inherited Risk of Coronary Atherosclerosis. Resistin levels were positively associated with levels of inflammatory markers, including soluble tumor necrosis factor- receptor-2 (P<0.001), interleukin-6 (P=0.04), and lipoprotein-associated phospholipase A2 (P=0.002), but not measures of insulin resistance in multivariable analysis. Resistin levels also were associated (odds ratio and 95% confidence interval in ordinal regression) with increasing CAC after adjustment for age, sex, and established risk factors (OR, 1.23 [CI, 1.03 to 1.52], P=0.03) and further control for metabolic syndrome and plasma C-reactive protein (CRP) levels (OR, 1.25 [CI, 1.04 to 1.50], P=0.01). In subjects with metabolic syndrome, resistin levels further predicted CAC, whereas CRP levels did not.
Conclusions— Plasma resistin levels are correlated with markers of inflammation and are predictive of coronary atherosclerosis in humans, independent of CRP. Resistin may represent a novel link between metabolic signals, inflammation, and atherosclerosis. Further studies are needed to define the relationship of resistin to clinical cardiovascular disease.
Key Words: resistin ; inflammation ; diabetes ; atherosclerosis
Introduction
Obesity and atherosclerosis are increasingly viewed as inflammatory states. Biomarkers that integrate metabolic and inflammatory signals are attractive candidates for defining risk of atherosclerotic cardiovascular disease (CVD).1 Resistin belongs to a family of cysteine-rich secretory proteins called resistin-like molecules2,3 or FIZZ (found in inflammatory zones) proteins.4 In rodents, resistin is derived almost exclusively from fat tissue, and adipose expression and serum levels are elevated in models of obesity and insulin resistance.2,5,6 Hyperresistinemia impairs glucose tolerance2 and induces hepatic insulin resistance in rodents,7 whereas mice deficient in resistin are protected from obesity-associated insulin resistance.8
Although assays for human resistin are in their infancy, in the past year several small studies have reported that circulating resistin levels are increased in human obesity9–12 and diabetes,13–17 although not all reports have been consistent in this regard.18–21 In contrast to rodents, in humans, resistin is expressed primarily in inflammatory cells.22–25 Resistin expression in human monocytes was markedly increased by treatment with endotoxin and proinflammatory cytokines.25,26 Recombinant resistin upregulates cytokines and adhesion molecule expression on human endothelial cells,27,28 suggesting a potential role in atherosclerosis. However, the relationship of resistin to inflammation, insulin resistance, and atherosclerosis in humans remains largely unexplored.
We examined whether plasma levels of resistin were associated with inflammatory markers, metabolic parameters, and coronary artery calcification (CAC), a measure of coronary atherosclerosis, in the 879 asymptomatic, nondiabetic subjects in the Study of Inherited Risk of Coronary Atherosclerosis (SIRCA). We also compared resistin levels with inflammatory markers in a type 2 diabetic sample (n=215) and examined short-term variation in plasma levels by repeated sampling in young, healthy control subjects. Our results indicate that resistin is an independent inflammatory marker of atherosclerosis.
Methods
Study Subjects
The primary focus of this article is on subjects enrolled into SIRCA in a cross-sectional study of factors associated with CAC in a community-based sample of asymptomatic subjects and their families. The study design and initial findings have been published.29–31 Subjects were included if they were healthy men 30 to 65 years old or women 35 to 70 years old who had a family history of premature coronary artery disease (CAD) (before the age of 60 in male and 70 in female first-degree relative). Exclusions included evidence of clinical CAD (myocardial infarction, coronary revascularization, angiographic evidence of CAD, or ischemia at cardiac stress test) and serum creatinine >3.0 mg/dL. We focused, for this study, on unrelated nondiabetic subjects recruited to SIRCA (n=879).
We also measured plasma resistin levels, during the same time period as for SIRCA, in 2 additional clinical research studies.32,33 First, we measured plasma resistin in a cross-sectional study of cardiovascular risk factors in asymptomatic type 2 diabetic subjects (n=215; 167 male and 48 female; 59% white and 35% black) recruited through the diabetic clinics of the University of Pennsylvania Medical Center and the Veterans Affairs Medical Center, Philadelphia, Pa. Further characteristics of the study sample are provided in Table I and in Reference 32. Second, we assessed baseline variability in plasma resistin over a 24-hour period in healthy, young volunteers (n=6; 3 male and 3 female; age 24 to 34 years; body mass index [BMI] 24.3±1.07 kg/m2) without any past medical history and on no medications. These subjects were recruited to a 60-hour inpatient, General Clinical Research Center (GCRC) protocol designed to assess the metabolic responses to an inflammatory stimulus. Plasma resistin levels were determined in serial blood samples collected at 8 time points over 24 hours before the intravenous administration of human-research-grade endotoxin (3 ng/kg) as described in more detail in Reference 33. The University of Pennsylvania Institutional Review Board approved all 3 study protocols. All subjects gave informed consent.
Evaluated Parameters
SIRCA and diabetic study subjects were evaluated at the GCRC at the University of Pennsylvania Medical Center after a 12-hour overnight fast. Study procedures, including questionnaire, physical examination, ECG, and blood collection, were performed as described previously.29–31 Plasma total and 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. LDL cholesterol was calculated by use of the Friedewald formula. Young, healthy participants in the endotoxin protocol had 8 blood draws (at 6 AM, 8 AM, 12 noon, 2 PM, 6 PM, 10 PM, 2 AM, and 6 AM) during 24 hours of constant routine in the GCRC before endotoxin administration.
Plasma resistin levels were measured by enzyme immunoassay (Linco Research) as also described in recent reports.34 Monoclonal antibodies raised against recombinant full-length Flag-tagged resistin protein were generated by Mitch A. Lazar and made available to Linco through the University of Pennsylvania. This antibody does not react with human resistin–like molecule-;, the other member of this gene family found in humans. The average correlation coefficient for standards was 0.99. The average intra-assay coefficient of variation (CV) was 4.6% for low and 1.7% for high resistin standards and 4.3% for fresh aliquots of pooled human plasma, included in duplicate on all plates. Results for plasma samples across different assay plates, for SIRCA, diabetic, and healthy, young controls, were standardized by use of the ratio of individual plate pooled plasma to the average pooled plasma value for all plates combined. A direct comparison of the Linco assay with kit with another commercially available resistin ELISA (Biovendor) yielded high correlation (R=0.99, P<0.001), and details are provided in the Data Supplement.
Plasma levels of interleukin-6 (IL-6), soluble tumor necrosis factor (TNF) receptor 2 (sol TNF-R2), and soluble intercellular adhesion molecule-1 (sol ICAM-1) were measured by use of commercially available enzyme immunoassays (ELISAs) according to the manufacturer’s guidelines (R+D Systems). The intra-assay and interassay CVs for pooled human plasma were 8.7% and 10.9%, respectively, for IL-6, 5.3% and 12.1% for sol TNF R2, and 1.4% and 10.4% for sol ICAM-1. Plasma C-reactive protein (CRP) levels were assayed by use of an ultra-high-sensitivity latex turbidimetric immunoassay (Wako Ltd) as described previously.29 Plasma levels of lipoprotein-associated phospholipase A2 (LpPLA2) were measured by use of a commercial ELISA (PLAC test; diaDexus). Intra-assay and interassay CVs for pooled plasma were 6.6% and 8.9%, respectively. Plasma insulin levels were measured by ELISA (Linco Research). The intra-assay and interassay CVs were 2.9% and 11.6%, respectively, for pooled human plasma.
Subjects were classified as having the metabolic syndrome by use of the National Cholesterol Education Program (NCEP) criteria35 as described previously in the SIRCA sample.30 The homeostasis model (HOMA index = fasting glucose [mmol/L] x fasting insulin [μU/mL] / 22.5)36 was used as a measure of insulin sensitivity. Global CAC scores were determined by use of customized software (Imatron) according to the method of Agatston et al37 from 40 continuous 3-mm-thick computed tomograms collected on an EBT scanner (Imatron).
Statistical Analysis
Data are reported as median and interquartile range (IQR) or mean±SD for continuous variables and as proportions for categorical variables. Spearman correlations of plasma resistin levels with other continuous variables are presented. The association of resistin levels with categorical variables was examined by use of the Kruskal-Wallis rank test and the Wilcoxon test for trend. Multivariable linear regression modeling was used to identify factors associated logarithmically transformed resistin levels (ln-resistin). Sex interaction with other variables in the association with plasma resistin levels was assessed by use of the likelihood-ratio test. To explore the range of resistin values in different human samples, we examined plasma levels in (1) SIRCA subgroups: (a) subjects with BMI >35 kg/m2 (n=72) and (b) subjects with NCEP-defined metabolic syndrome (n=249); (2) our type 2 diabetic sample; and (3) young healthy subjects with repeated blood sampling. Changes in plasma resistin levels in young healthy subjects were analyzed by repeated-measures ANOVA.
Median CAC scores were compared across plasma resistin quartiles (1.66 to <4.13, 4.13 to <5.46, 5.46 to <7.28, and >7.28 ng/mL) by use of the Wilcoxon test for trend. Ordinal logistic regression is a method appropriate for the analysis of CAC data, which have a markedly nonnormal distribution and a significant proportion of subjects with no detectable CAC.29,31 CAC scores were divided into 4 ordered outcome categories (0, 1 to 10, 11 to 100, >100) by use of published criteria used to approximate no, mild, and moderate coronary atherosclerosis.38
The association of plasma resistin with CAC was assessed in regression models that included (1) resistin, sex, and age (age and age2); (2) resistin, established risk factors, sex, and age; (3) resistin, metabolic syndrome, non–metabolic syndrome factors, sex, and age; and (4) resistin, plasma CRP levels, metabolic syndrome, non–metabolic syndrome factors, sex, and age. Established risk factors included total (or LDL) and HDL cholesterol, plasma glucose, systolic blood pressure, smoking (current versus never and ex-smokers), race, exercise (none versus any), alcohol intake (drinks per week), and use of medications (aspirin, statins, ACE inhibitors, and hormone replacement therapy in women). In models that contained metabolic syndrome, non–metabolic syndrome factors were smoking, exercise, alcohol intake, race, LDL cholesterol, and use of medications. Recently, CRP levels were shown to predict CVD in subjects with the metabolic syndrome.39,40 Because additional biomarkers are being sought to refine CVD risk prediction in the metabolic syndrome,41 we compared plasma resistin with CRP in their association with CAC in metabolic syndrome subgroups.
The interaction between sex and plasma resistin levels in the association with CAC was assessed in adjusted models by use of the likelihood-ratio test. The likelihood-ratio test also was applied to nested models to determine whether the addition of resistin to CRP levels, or CRP to resistin levels, improved the prediction of CAC. The results of ordinal logistic regression are presented as the OR of being in higher CAC category for a 5-ng/mL increase in plasma resistin. The proportional-odds assumption of ordinal regression, assessed by the Brant test, was satisfied for resistin in all models. Statistical analyses were performed by use of Stata 8.0 software (Stata Corp).
Results
Characteristics of SIRCA Subjects
As described previously,29–31 the SIRCA sample was predominantly white (95%); women were older than men, as expected from enrollment criteria (Table 1), and more than 70% of these asymptomatic subjects had detectable CAC consistent with prevalent subclinical atherosclerosis and a recruitment strategy based on family history of premature heart disease (Table 1). Plasma resistin levels (median [IQR], ng/mL) were modestly but significantly higher in women than men (5.88 [4.42 to 7.84] versus 5.20 [3.87 to 6.90] ng/mL; P<0.001) (Table 1).
Association of Plasma Resistin With Inflammatory Factors in SIRCA
Plasma resistin levels were highly correlated with levels of diverse inflammatory markers, particularly sol TNF-R2, but also IL-6 and LpPLA2, and to a lesser degree with sol ICAM-1 and CRP (Figure 1 and Table 2). Levels of sol TNF-R2 (P<0.001), LpPLA2 (P=0.002), and IL-6 (P=0.04), but not CRP (P=0.2), remained positively associated with resistin in fully adjusted models: sol TNF-R2 levels were the strongest single predictor and accounted for 10% of variability in circulating resistin (Table II).
Notably, resistin levels did not correlate with insulin resistance as defined by the HOMA index (Figure 1 and Table 2). In this regard, it is important to note that this study focuses on nondiabetic subjects of relatively normal weight (73% with BMI <30 kg/m2). However, consistent with previous reports,9–12 SIRCA subjects with marked obesity (BMI >35 kg/m2; n=72) had modest but significant increases in resistin levels compared with subjects with BMI <35 kg/m2 (6.32 [4.38 to 8.76] versus 5.44 [4.12 to 7.23] ng/mL; P=0.04). Similarly, SIRCA subjects with NCEP-defined metabolic syndrome (n=249) had slightly higher levels than subjects without the metabolic syndrome (5.72 [4.44 to 7.75] versus 5.41 [4.04 to 7.14] ng/mL; P=0.03). Resistin levels also correlated inversely with HDL cholesterol in women (Table 2), but this was not significant in adjusted analysis. Despite a trend toward sex differences in the strength of association with plasma resistin, there was no significant interaction of sex with inflammatory or metabolic factors in the relationship with resistin.
Plasma Resistin Levels in Type 2 Diabetics and Young, Healthy Subjects
In the type 2 diabetic sample, resistin levels (median [IQR], ng/mL) tended to be higher in women (5.98 [3.42 to 7.89]) than men (5.76 [4.29 to 7.95] in men) and tended to be higher than in our SIRCA sample. Remarkably, as in SIRCA, resistin levels were strongly associated with plasma sol TNF-R2 (P<0.001) but were not significantly correlated with measures of adiposity and insulin resistance (Table 3). In fact, in multivariable analysis, only plasma levels of sol TNF-R2 (P<0.001) and the white cell count (P=0.013) were independent predictors of log-transformed plasma resistin levels.
In young, healthy subjects, plasma resistin levels (eg, at 6 AM, 3.73 [2.50 to 4.58]; at 12 noon, 3.65 [2.10 to 3.94]; at 6 PM, 3.22 [2.27 to 4.24]; and at 6 AM next morning, 3.15 [2.27 to 3.59]) tended to be lower than in SIRCA and were remarkably stable over a 24-hour period (repeated-measures ANOVA F statistic for time=1.15, P=0.36).
Association of Plasma Resistin Levels With CAC in SIRCA
Risk factors that are associated with CAC in the SIRCA sample have been published31 and include age, sex, adiposity, LDL cholesterol, HDL cholesterol, smoking, systolic blood pressure, plasma glucose, and use of statins. The metabolic syndrome,30 but not CRP levels,29 is strongly associated with CAC in this sample.
Median (IQR) CAC scores increased across plasma resistin quartiles in men (P=0.01) and women (P=0.05) (Figure 2). There was no significant interaction (likelihood-ratio test, P=0.8) between sex and plasma resistin levels in the association with CAC. Therefore, results of multivariable analyses are presented for men and women combined. Resistin levels were associated with CAC after control for age, sex, and established risk factors and even with further adjustment for the metabolic syndrome and CRP levels (Table 4). Addition of plasma resistin levels to a fully adjusted multivariable model containing plasma CRP levels (likelihood-ratio test, P=0.04) strengthened the association with CAC scores, whereas CRP did not add significantly to a model that already contained plasma resistin levels (likelihood-ratio test, P=0.2). In multivariable models adjusted for age, sex, and non–metabolic syndrome risk factors, plasma levels of resistin were significantly associated with CAC in subjects with the metabolic syndrome (P=0.003) (Table 5). By contrast, in this sample, CRP levels were not predictive of CAC independent of metabolic syndrome (P=0.65).
Discussion
We present the first large study in humans to examine the relationship of circulating resistin with diverse inflammatory markers, as well as with coronary atherosclerosis. We found that plasma resistin levels were associated with markers of inflammation, but not insulin resistance, both in SIRCA, a study of asymptomatic nondiabetic subjects, and in a type 2 diabetic sample. Furthermore, we found that resistin levels were significantly associated with coronary atherosclerosis in SIRCA even after control for multiple established risk factors and the presence of the metabolic syndrome. In fact, plasma levels of resistin, unlike those of CRP, provided incremental value in the association with CAC in subjects with the metabolic syndrome.
The convergence of insulin resistance and inflammation in the pathogenesis of atherosclerotic CVD has been recognized over the past decade.35,42–44 Metabolic syndrome definitions and markers of inflammation, such as CRP, have been proposed for use in clinical practice to aid in the identification of asymptomatic patients at high risk for CVD. However, there remains uncertainty as to the most appropriate definition of metabolic syndrome and the optimal inflammatory marker for use in clinical practice.41
Resistin has emerged as a novel secreted protein with links to both insulin resistance and inflammation.1,7,23,45 In rodents, resistin is expressed in adipose tissue and regulates glucose metabolism and insulin sensitivity.2,7,8 Although resistin mRNA is detectable in human adipocytes,46 levels are much higher in human inflammatory cells.23,25,26 Recently, we have found that acute endotoxemia dramatically (>7-fold) elevates plasma levels of resistin in humans.33 Consistent with recent small clinical studies,47,48 these findings suggest that, in contrast to those of other adipokines, expression and secretion of resistin in humans may be regulated by innate inflammatory signals. Endotoxemia is known to produce a state of insulin resistance in humans,49 but it remains to be determined whether the marked endotoxemia-induced hyperresistinemia plays a critical role.
In SIRCA, plasma resistin levels were strongly and independently correlated with sol TNF-R2, an index of TNF- system activation,50 and IL-6. Both TNF- and IL-6 are derived from adipose tissue as well as macrophages, and increased levels of these inflammatory cytokines have been linked to obesity, insulin resistance, and atherosclerotic CVD.51 We found that resistin levels also correlated significantly with sol ICAM-1 and LpPLA2, plasma markers thought to derive from monocytes and the endothelium rather than adipose tissue. Notably, plasma CRP, which is secreted largely by the liver in response to circulating cytokines, was not associated with resistin independently of TNF-R2 or IL-6 in adjusted analysis. The contribution of innate inflammatory cells to the circulating resistin levels, versus that of adipocytes, is uncertain but may be greater in our relatively lean, nondiabetic SIRCA population than in other studies that have focused on obesity9–11,47 or type 2 diabetes.13–17
Therefore, we examined resistin levels in SIRCA subgroups, in our type 2 diabetic sample, and in healthy volunteers. Although these studies were recruited separately and were not designed to compare levels across study samples, our findings are consistent with modest increases in resistin in overweight and type 2 diabetic subjects, as has been published in small studies.11,12,47 Obesity and type 2 diabetes are associated with activation of innate immune pathways and chronic inflammation.52 The consistent correlation of resistin with sol TNF-R2 in both SIRCA and diabetic subjects and the increase in circulating resistin during endotoxemia in healthy humans strongly support our mechanistic studies33 defining resistin as an inflammatory adipokine across a variety of settings in humans. The finding of stable resistin levels in healthy subjects over a 24-hour period in the GCRC also suggests that measurement of plasma levels of resistin in cross-sectional studies will be useful in gaining further insight into the role of resistin in human pathophysiology.
Plasma resistin levels were significantly associated with CAC in the SIRCA sample. Although not a direct measure of coronary atherosclerosis, autopsy studies have shown that CAC is a quantitative measure of coronary atherosclerosis,54 and recent studies support its usefulness as a predictor of CVD events in asymptomatic samples, even at relatively low scores.55,56 The association of resistin with CAC was maintained even after control for established risk factors, as well as the presence of the metabolic syndrome and plasma levels of CRP. Because the metabolic syndrome is a strong risk factor for atherosclerotic CVD but the optimal definition for use in practice remains unclear, additional biomarkers are being sought to refine CVD risk prediction. CRP is promising in this regard,39,40 and therefore, we compared plasma resistin with CRP in their association with CAC in metabolic syndrome subgroups. Notably, in metabolic syndrome subjects, resistin levels further predicted increased CAC, whereas CRP levels did not. These clinical correlations are consistent with recent reports showing that recombinant resistin induced cytokine, chemokine, and adhesion molecule expression in human endothelial cells,27,28 whereas adiponectin opposed the effect of resistin on adhesion molecules.28 Although much needs to be learned about the relationship between resistin, inflammation, and the cardiovascular system, plasma resistin may provide incremental value in cardiovascular risk prediction beyond current approaches. These novel findings need to be confirmed in ethnically diverse samples by use of alternative measures of atherosclerosis and, ultimately, in large prospective studies of cardiovascular events.
In conclusion, we found that plasma levels of resistin were associated with inflammatory markers in a large, nondiabetic sample as well as in type 2 diabetes. Resistin also was associated with CAC, a measure of coronary atherosclerosis, even after control for established risk factors, metabolic syndrome, and CRP levels. Whether resistin plays a pathophysiological role in insulin resistance or atherosclerosis in humans remains to be determined.
Acknowledgments
This study was funded in part by grant M01-RR00040 from the National Center for Research Resources (NCRR)/National Institutes of Health (NIH) supporting the University of Pennsylvania General Clinical Research Center (GCRC) and by the Penn Diabetes and Endocrinology Research Center (DK-19525). Dr Reilly is supported by NIH grants K23-RR15532-04 and RO1-HL73278-01 and by the W.W. Smith Charitable Trust (No. H0204). Dr Lehrke is supported by a grant from the German Scientific Foundation (Deutsche Forschungsgemeinschaft), LE 1350/1-1. Dr Lazar is supported by NIH grants RO1-DK-49780 and RO1-DK-49210 and an unrestricted Bristol Myers-Squibb Freedom to Discover Award for Metabolic Research. Dr Rader is supported by grants from the National Heart, Lung, and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, and NCRR and is a recipient of the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research and of a Doris Duke Distinguished Clinical Investigator Award. We are indebted to the nursing staff of the University of Pennsylvania GCRC and to Jennifer Dykhouse, BS, and Kimberly McMahon, BS, for expert technical assistance. The sponsors played no role in the design of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or writing of the manuscript.
Disclosure
Dr Reilly is in receipt of research funding or honoraria from GlaxoSmithKline, Merck & Co, Ely Lilly Inc, and KOS Pharmaceuticals. Dr Rader is involved as a consultant to or in receipt of research funding or honoraria from AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, GlaxoSmithKline, KOS Pharmaceuticals, Merck & Co, Merck/Schering-Plough, Pfizer, Schering-Plough, and Takeda. Dr Lazar is a consultant to Abbott and receives grant support from GlaxoSmithKline and Bristol Myers Squibb Research Institute. Dr Lazar and the University of Pennsylvania have licensed reagents used in the human resistin assay to Linco. Dr Lehrke and Ms Wolfe have no conflict of interest.
Footnotes
The first 2 authors contributed equally to this work.
The online-only Data Supplement, which contains Table I and Table II, can be found with this article at http://www.circulationaha.org.
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Reilly MP, Iqbal N, Schutta M, Wolfe ML, Scally M, Localio AR, Rader DJ, Kimmel SE. Plasma leptin levels are associated with coronary atherosclerosis in type 2 diabetes. J Clin Endocrinol Metab. 2004; 89: 3872–3878.
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Deleted in proof.
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Pletcher MJ, Tice JA, Pignone M, Browner WS. Using the coronary artery calcium score to predict coronary heart disease events: a systematic review and meta-analysis. Arch Intern Med. 2004; 164: 1285–1292.(Muredach P. Reilly, MB; M)
Department of Medicine (M.P.R., M.L., M.L.W., A.R., M.A.L., D.J.R.)
Center for Experimental Therapeutics, and the Penn Diabetes Center (M.P.R., M.L.W., D.J.R.)
University of Pennsylvania School of Medicine, Philadelphia, Pa.
Abstract
Background— Resistin, a plasma protein, induces insulin resistance in rodents. Recent reports suggest that circulating levels of resistin are elevated in obese and insulin-resistant rodents and humans. Whereas rodent resistin is made in adipocytes, macrophages are a major source of human resistin. Given the convergence of adipocyte and macrophage function, resistin may provide unique insight into links between obesity, inflammation, and atherosclerosis in humans.
Methods and Results— We examined whether plasma resistin levels were associated with metabolic and inflammatory markers, as well as with coronary artery calcification (CAC), a quantitative index of atherosclerosis, in 879 asymptomatic subjects in the Study of Inherited Risk of Coronary Atherosclerosis. Resistin levels were positively associated with levels of inflammatory markers, including soluble tumor necrosis factor- receptor-2 (P<0.001), interleukin-6 (P=0.04), and lipoprotein-associated phospholipase A2 (P=0.002), but not measures of insulin resistance in multivariable analysis. Resistin levels also were associated (odds ratio and 95% confidence interval in ordinal regression) with increasing CAC after adjustment for age, sex, and established risk factors (OR, 1.23 [CI, 1.03 to 1.52], P=0.03) and further control for metabolic syndrome and plasma C-reactive protein (CRP) levels (OR, 1.25 [CI, 1.04 to 1.50], P=0.01). In subjects with metabolic syndrome, resistin levels further predicted CAC, whereas CRP levels did not.
Conclusions— Plasma resistin levels are correlated with markers of inflammation and are predictive of coronary atherosclerosis in humans, independent of CRP. Resistin may represent a novel link between metabolic signals, inflammation, and atherosclerosis. Further studies are needed to define the relationship of resistin to clinical cardiovascular disease.
Key Words: resistin ; inflammation ; diabetes ; atherosclerosis
Introduction
Obesity and atherosclerosis are increasingly viewed as inflammatory states. Biomarkers that integrate metabolic and inflammatory signals are attractive candidates for defining risk of atherosclerotic cardiovascular disease (CVD).1 Resistin belongs to a family of cysteine-rich secretory proteins called resistin-like molecules2,3 or FIZZ (found in inflammatory zones) proteins.4 In rodents, resistin is derived almost exclusively from fat tissue, and adipose expression and serum levels are elevated in models of obesity and insulin resistance.2,5,6 Hyperresistinemia impairs glucose tolerance2 and induces hepatic insulin resistance in rodents,7 whereas mice deficient in resistin are protected from obesity-associated insulin resistance.8
Although assays for human resistin are in their infancy, in the past year several small studies have reported that circulating resistin levels are increased in human obesity9–12 and diabetes,13–17 although not all reports have been consistent in this regard.18–21 In contrast to rodents, in humans, resistin is expressed primarily in inflammatory cells.22–25 Resistin expression in human monocytes was markedly increased by treatment with endotoxin and proinflammatory cytokines.25,26 Recombinant resistin upregulates cytokines and adhesion molecule expression on human endothelial cells,27,28 suggesting a potential role in atherosclerosis. However, the relationship of resistin to inflammation, insulin resistance, and atherosclerosis in humans remains largely unexplored.
We examined whether plasma levels of resistin were associated with inflammatory markers, metabolic parameters, and coronary artery calcification (CAC), a measure of coronary atherosclerosis, in the 879 asymptomatic, nondiabetic subjects in the Study of Inherited Risk of Coronary Atherosclerosis (SIRCA). We also compared resistin levels with inflammatory markers in a type 2 diabetic sample (n=215) and examined short-term variation in plasma levels by repeated sampling in young, healthy control subjects. Our results indicate that resistin is an independent inflammatory marker of atherosclerosis.
Methods
Study Subjects
The primary focus of this article is on subjects enrolled into SIRCA in a cross-sectional study of factors associated with CAC in a community-based sample of asymptomatic subjects and their families. The study design and initial findings have been published.29–31 Subjects were included if they were healthy men 30 to 65 years old or women 35 to 70 years old who had a family history of premature coronary artery disease (CAD) (before the age of 60 in male and 70 in female first-degree relative). Exclusions included evidence of clinical CAD (myocardial infarction, coronary revascularization, angiographic evidence of CAD, or ischemia at cardiac stress test) and serum creatinine >3.0 mg/dL. We focused, for this study, on unrelated nondiabetic subjects recruited to SIRCA (n=879).
We also measured plasma resistin levels, during the same time period as for SIRCA, in 2 additional clinical research studies.32,33 First, we measured plasma resistin in a cross-sectional study of cardiovascular risk factors in asymptomatic type 2 diabetic subjects (n=215; 167 male and 48 female; 59% white and 35% black) recruited through the diabetic clinics of the University of Pennsylvania Medical Center and the Veterans Affairs Medical Center, Philadelphia, Pa. Further characteristics of the study sample are provided in Table I and in Reference 32. Second, we assessed baseline variability in plasma resistin over a 24-hour period in healthy, young volunteers (n=6; 3 male and 3 female; age 24 to 34 years; body mass index [BMI] 24.3±1.07 kg/m2) without any past medical history and on no medications. These subjects were recruited to a 60-hour inpatient, General Clinical Research Center (GCRC) protocol designed to assess the metabolic responses to an inflammatory stimulus. Plasma resistin levels were determined in serial blood samples collected at 8 time points over 24 hours before the intravenous administration of human-research-grade endotoxin (3 ng/kg) as described in more detail in Reference 33. The University of Pennsylvania Institutional Review Board approved all 3 study protocols. All subjects gave informed consent.
Evaluated Parameters
SIRCA and diabetic study subjects were evaluated at the GCRC at the University of Pennsylvania Medical Center after a 12-hour overnight fast. Study procedures, including questionnaire, physical examination, ECG, and blood collection, were performed as described previously.29–31 Plasma total and 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. LDL cholesterol was calculated by use of the Friedewald formula. Young, healthy participants in the endotoxin protocol had 8 blood draws (at 6 AM, 8 AM, 12 noon, 2 PM, 6 PM, 10 PM, 2 AM, and 6 AM) during 24 hours of constant routine in the GCRC before endotoxin administration.
Plasma resistin levels were measured by enzyme immunoassay (Linco Research) as also described in recent reports.34 Monoclonal antibodies raised against recombinant full-length Flag-tagged resistin protein were generated by Mitch A. Lazar and made available to Linco through the University of Pennsylvania. This antibody does not react with human resistin–like molecule-;, the other member of this gene family found in humans. The average correlation coefficient for standards was 0.99. The average intra-assay coefficient of variation (CV) was 4.6% for low and 1.7% for high resistin standards and 4.3% for fresh aliquots of pooled human plasma, included in duplicate on all plates. Results for plasma samples across different assay plates, for SIRCA, diabetic, and healthy, young controls, were standardized by use of the ratio of individual plate pooled plasma to the average pooled plasma value for all plates combined. A direct comparison of the Linco assay with kit with another commercially available resistin ELISA (Biovendor) yielded high correlation (R=0.99, P<0.001), and details are provided in the Data Supplement.
Plasma levels of interleukin-6 (IL-6), soluble tumor necrosis factor (TNF) receptor 2 (sol TNF-R2), and soluble intercellular adhesion molecule-1 (sol ICAM-1) were measured by use of commercially available enzyme immunoassays (ELISAs) according to the manufacturer’s guidelines (R+D Systems). The intra-assay and interassay CVs for pooled human plasma were 8.7% and 10.9%, respectively, for IL-6, 5.3% and 12.1% for sol TNF R2, and 1.4% and 10.4% for sol ICAM-1. Plasma C-reactive protein (CRP) levels were assayed by use of an ultra-high-sensitivity latex turbidimetric immunoassay (Wako Ltd) as described previously.29 Plasma levels of lipoprotein-associated phospholipase A2 (LpPLA2) were measured by use of a commercial ELISA (PLAC test; diaDexus). Intra-assay and interassay CVs for pooled plasma were 6.6% and 8.9%, respectively. Plasma insulin levels were measured by ELISA (Linco Research). The intra-assay and interassay CVs were 2.9% and 11.6%, respectively, for pooled human plasma.
Subjects were classified as having the metabolic syndrome by use of the National Cholesterol Education Program (NCEP) criteria35 as described previously in the SIRCA sample.30 The homeostasis model (HOMA index = fasting glucose [mmol/L] x fasting insulin [μU/mL] / 22.5)36 was used as a measure of insulin sensitivity. Global CAC scores were determined by use of customized software (Imatron) according to the method of Agatston et al37 from 40 continuous 3-mm-thick computed tomograms collected on an EBT scanner (Imatron).
Statistical Analysis
Data are reported as median and interquartile range (IQR) or mean±SD for continuous variables and as proportions for categorical variables. Spearman correlations of plasma resistin levels with other continuous variables are presented. The association of resistin levels with categorical variables was examined by use of the Kruskal-Wallis rank test and the Wilcoxon test for trend. Multivariable linear regression modeling was used to identify factors associated logarithmically transformed resistin levels (ln-resistin). Sex interaction with other variables in the association with plasma resistin levels was assessed by use of the likelihood-ratio test. To explore the range of resistin values in different human samples, we examined plasma levels in (1) SIRCA subgroups: (a) subjects with BMI >35 kg/m2 (n=72) and (b) subjects with NCEP-defined metabolic syndrome (n=249); (2) our type 2 diabetic sample; and (3) young healthy subjects with repeated blood sampling. Changes in plasma resistin levels in young healthy subjects were analyzed by repeated-measures ANOVA.
Median CAC scores were compared across plasma resistin quartiles (1.66 to <4.13, 4.13 to <5.46, 5.46 to <7.28, and >7.28 ng/mL) by use of the Wilcoxon test for trend. Ordinal logistic regression is a method appropriate for the analysis of CAC data, which have a markedly nonnormal distribution and a significant proportion of subjects with no detectable CAC.29,31 CAC scores were divided into 4 ordered outcome categories (0, 1 to 10, 11 to 100, >100) by use of published criteria used to approximate no, mild, and moderate coronary atherosclerosis.38
The association of plasma resistin with CAC was assessed in regression models that included (1) resistin, sex, and age (age and age2); (2) resistin, established risk factors, sex, and age; (3) resistin, metabolic syndrome, non–metabolic syndrome factors, sex, and age; and (4) resistin, plasma CRP levels, metabolic syndrome, non–metabolic syndrome factors, sex, and age. Established risk factors included total (or LDL) and HDL cholesterol, plasma glucose, systolic blood pressure, smoking (current versus never and ex-smokers), race, exercise (none versus any), alcohol intake (drinks per week), and use of medications (aspirin, statins, ACE inhibitors, and hormone replacement therapy in women). In models that contained metabolic syndrome, non–metabolic syndrome factors were smoking, exercise, alcohol intake, race, LDL cholesterol, and use of medications. Recently, CRP levels were shown to predict CVD in subjects with the metabolic syndrome.39,40 Because additional biomarkers are being sought to refine CVD risk prediction in the metabolic syndrome,41 we compared plasma resistin with CRP in their association with CAC in metabolic syndrome subgroups.
The interaction between sex and plasma resistin levels in the association with CAC was assessed in adjusted models by use of the likelihood-ratio test. The likelihood-ratio test also was applied to nested models to determine whether the addition of resistin to CRP levels, or CRP to resistin levels, improved the prediction of CAC. The results of ordinal logistic regression are presented as the OR of being in higher CAC category for a 5-ng/mL increase in plasma resistin. The proportional-odds assumption of ordinal regression, assessed by the Brant test, was satisfied for resistin in all models. Statistical analyses were performed by use of Stata 8.0 software (Stata Corp).
Results
Characteristics of SIRCA Subjects
As described previously,29–31 the SIRCA sample was predominantly white (95%); women were older than men, as expected from enrollment criteria (Table 1), and more than 70% of these asymptomatic subjects had detectable CAC consistent with prevalent subclinical atherosclerosis and a recruitment strategy based on family history of premature heart disease (Table 1). Plasma resistin levels (median [IQR], ng/mL) were modestly but significantly higher in women than men (5.88 [4.42 to 7.84] versus 5.20 [3.87 to 6.90] ng/mL; P<0.001) (Table 1).
Association of Plasma Resistin With Inflammatory Factors in SIRCA
Plasma resistin levels were highly correlated with levels of diverse inflammatory markers, particularly sol TNF-R2, but also IL-6 and LpPLA2, and to a lesser degree with sol ICAM-1 and CRP (Figure 1 and Table 2). Levels of sol TNF-R2 (P<0.001), LpPLA2 (P=0.002), and IL-6 (P=0.04), but not CRP (P=0.2), remained positively associated with resistin in fully adjusted models: sol TNF-R2 levels were the strongest single predictor and accounted for 10% of variability in circulating resistin (Table II).
Notably, resistin levels did not correlate with insulin resistance as defined by the HOMA index (Figure 1 and Table 2). In this regard, it is important to note that this study focuses on nondiabetic subjects of relatively normal weight (73% with BMI <30 kg/m2). However, consistent with previous reports,9–12 SIRCA subjects with marked obesity (BMI >35 kg/m2; n=72) had modest but significant increases in resistin levels compared with subjects with BMI <35 kg/m2 (6.32 [4.38 to 8.76] versus 5.44 [4.12 to 7.23] ng/mL; P=0.04). Similarly, SIRCA subjects with NCEP-defined metabolic syndrome (n=249) had slightly higher levels than subjects without the metabolic syndrome (5.72 [4.44 to 7.75] versus 5.41 [4.04 to 7.14] ng/mL; P=0.03). Resistin levels also correlated inversely with HDL cholesterol in women (Table 2), but this was not significant in adjusted analysis. Despite a trend toward sex differences in the strength of association with plasma resistin, there was no significant interaction of sex with inflammatory or metabolic factors in the relationship with resistin.
Plasma Resistin Levels in Type 2 Diabetics and Young, Healthy Subjects
In the type 2 diabetic sample, resistin levels (median [IQR], ng/mL) tended to be higher in women (5.98 [3.42 to 7.89]) than men (5.76 [4.29 to 7.95] in men) and tended to be higher than in our SIRCA sample. Remarkably, as in SIRCA, resistin levels were strongly associated with plasma sol TNF-R2 (P<0.001) but were not significantly correlated with measures of adiposity and insulin resistance (Table 3). In fact, in multivariable analysis, only plasma levels of sol TNF-R2 (P<0.001) and the white cell count (P=0.013) were independent predictors of log-transformed plasma resistin levels.
In young, healthy subjects, plasma resistin levels (eg, at 6 AM, 3.73 [2.50 to 4.58]; at 12 noon, 3.65 [2.10 to 3.94]; at 6 PM, 3.22 [2.27 to 4.24]; and at 6 AM next morning, 3.15 [2.27 to 3.59]) tended to be lower than in SIRCA and were remarkably stable over a 24-hour period (repeated-measures ANOVA F statistic for time=1.15, P=0.36).
Association of Plasma Resistin Levels With CAC in SIRCA
Risk factors that are associated with CAC in the SIRCA sample have been published31 and include age, sex, adiposity, LDL cholesterol, HDL cholesterol, smoking, systolic blood pressure, plasma glucose, and use of statins. The metabolic syndrome,30 but not CRP levels,29 is strongly associated with CAC in this sample.
Median (IQR) CAC scores increased across plasma resistin quartiles in men (P=0.01) and women (P=0.05) (Figure 2). There was no significant interaction (likelihood-ratio test, P=0.8) between sex and plasma resistin levels in the association with CAC. Therefore, results of multivariable analyses are presented for men and women combined. Resistin levels were associated with CAC after control for age, sex, and established risk factors and even with further adjustment for the metabolic syndrome and CRP levels (Table 4). Addition of plasma resistin levels to a fully adjusted multivariable model containing plasma CRP levels (likelihood-ratio test, P=0.04) strengthened the association with CAC scores, whereas CRP did not add significantly to a model that already contained plasma resistin levels (likelihood-ratio test, P=0.2). In multivariable models adjusted for age, sex, and non–metabolic syndrome risk factors, plasma levels of resistin were significantly associated with CAC in subjects with the metabolic syndrome (P=0.003) (Table 5). By contrast, in this sample, CRP levels were not predictive of CAC independent of metabolic syndrome (P=0.65).
Discussion
We present the first large study in humans to examine the relationship of circulating resistin with diverse inflammatory markers, as well as with coronary atherosclerosis. We found that plasma resistin levels were associated with markers of inflammation, but not insulin resistance, both in SIRCA, a study of asymptomatic nondiabetic subjects, and in a type 2 diabetic sample. Furthermore, we found that resistin levels were significantly associated with coronary atherosclerosis in SIRCA even after control for multiple established risk factors and the presence of the metabolic syndrome. In fact, plasma levels of resistin, unlike those of CRP, provided incremental value in the association with CAC in subjects with the metabolic syndrome.
The convergence of insulin resistance and inflammation in the pathogenesis of atherosclerotic CVD has been recognized over the past decade.35,42–44 Metabolic syndrome definitions and markers of inflammation, such as CRP, have been proposed for use in clinical practice to aid in the identification of asymptomatic patients at high risk for CVD. However, there remains uncertainty as to the most appropriate definition of metabolic syndrome and the optimal inflammatory marker for use in clinical practice.41
Resistin has emerged as a novel secreted protein with links to both insulin resistance and inflammation.1,7,23,45 In rodents, resistin is expressed in adipose tissue and regulates glucose metabolism and insulin sensitivity.2,7,8 Although resistin mRNA is detectable in human adipocytes,46 levels are much higher in human inflammatory cells.23,25,26 Recently, we have found that acute endotoxemia dramatically (>7-fold) elevates plasma levels of resistin in humans.33 Consistent with recent small clinical studies,47,48 these findings suggest that, in contrast to those of other adipokines, expression and secretion of resistin in humans may be regulated by innate inflammatory signals. Endotoxemia is known to produce a state of insulin resistance in humans,49 but it remains to be determined whether the marked endotoxemia-induced hyperresistinemia plays a critical role.
In SIRCA, plasma resistin levels were strongly and independently correlated with sol TNF-R2, an index of TNF- system activation,50 and IL-6. Both TNF- and IL-6 are derived from adipose tissue as well as macrophages, and increased levels of these inflammatory cytokines have been linked to obesity, insulin resistance, and atherosclerotic CVD.51 We found that resistin levels also correlated significantly with sol ICAM-1 and LpPLA2, plasma markers thought to derive from monocytes and the endothelium rather than adipose tissue. Notably, plasma CRP, which is secreted largely by the liver in response to circulating cytokines, was not associated with resistin independently of TNF-R2 or IL-6 in adjusted analysis. The contribution of innate inflammatory cells to the circulating resistin levels, versus that of adipocytes, is uncertain but may be greater in our relatively lean, nondiabetic SIRCA population than in other studies that have focused on obesity9–11,47 or type 2 diabetes.13–17
Therefore, we examined resistin levels in SIRCA subgroups, in our type 2 diabetic sample, and in healthy volunteers. Although these studies were recruited separately and were not designed to compare levels across study samples, our findings are consistent with modest increases in resistin in overweight and type 2 diabetic subjects, as has been published in small studies.11,12,47 Obesity and type 2 diabetes are associated with activation of innate immune pathways and chronic inflammation.52 The consistent correlation of resistin with sol TNF-R2 in both SIRCA and diabetic subjects and the increase in circulating resistin during endotoxemia in healthy humans strongly support our mechanistic studies33 defining resistin as an inflammatory adipokine across a variety of settings in humans. The finding of stable resistin levels in healthy subjects over a 24-hour period in the GCRC also suggests that measurement of plasma levels of resistin in cross-sectional studies will be useful in gaining further insight into the role of resistin in human pathophysiology.
Plasma resistin levels were significantly associated with CAC in the SIRCA sample. Although not a direct measure of coronary atherosclerosis, autopsy studies have shown that CAC is a quantitative measure of coronary atherosclerosis,54 and recent studies support its usefulness as a predictor of CVD events in asymptomatic samples, even at relatively low scores.55,56 The association of resistin with CAC was maintained even after control for established risk factors, as well as the presence of the metabolic syndrome and plasma levels of CRP. Because the metabolic syndrome is a strong risk factor for atherosclerotic CVD but the optimal definition for use in practice remains unclear, additional biomarkers are being sought to refine CVD risk prediction. CRP is promising in this regard,39,40 and therefore, we compared plasma resistin with CRP in their association with CAC in metabolic syndrome subgroups. Notably, in metabolic syndrome subjects, resistin levels further predicted increased CAC, whereas CRP levels did not. These clinical correlations are consistent with recent reports showing that recombinant resistin induced cytokine, chemokine, and adhesion molecule expression in human endothelial cells,27,28 whereas adiponectin opposed the effect of resistin on adhesion molecules.28 Although much needs to be learned about the relationship between resistin, inflammation, and the cardiovascular system, plasma resistin may provide incremental value in cardiovascular risk prediction beyond current approaches. These novel findings need to be confirmed in ethnically diverse samples by use of alternative measures of atherosclerosis and, ultimately, in large prospective studies of cardiovascular events.
In conclusion, we found that plasma levels of resistin were associated with inflammatory markers in a large, nondiabetic sample as well as in type 2 diabetes. Resistin also was associated with CAC, a measure of coronary atherosclerosis, even after control for established risk factors, metabolic syndrome, and CRP levels. Whether resistin plays a pathophysiological role in insulin resistance or atherosclerosis in humans remains to be determined.
Acknowledgments
This study was funded in part by grant M01-RR00040 from the National Center for Research Resources (NCRR)/National Institutes of Health (NIH) supporting the University of Pennsylvania General Clinical Research Center (GCRC) and by the Penn Diabetes and Endocrinology Research Center (DK-19525). Dr Reilly is supported by NIH grants K23-RR15532-04 and RO1-HL73278-01 and by the W.W. Smith Charitable Trust (No. H0204). Dr Lehrke is supported by a grant from the German Scientific Foundation (Deutsche Forschungsgemeinschaft), LE 1350/1-1. Dr Lazar is supported by NIH grants RO1-DK-49780 and RO1-DK-49210 and an unrestricted Bristol Myers-Squibb Freedom to Discover Award for Metabolic Research. Dr Rader is supported by grants from the National Heart, Lung, and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, and NCRR and is a recipient of the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research and of a Doris Duke Distinguished Clinical Investigator Award. We are indebted to the nursing staff of the University of Pennsylvania GCRC and to Jennifer Dykhouse, BS, and Kimberly McMahon, BS, for expert technical assistance. The sponsors played no role in the design of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or writing of the manuscript.
Disclosure
Dr Reilly is in receipt of research funding or honoraria from GlaxoSmithKline, Merck & Co, Ely Lilly Inc, and KOS Pharmaceuticals. Dr Rader is involved as a consultant to or in receipt of research funding or honoraria from AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, GlaxoSmithKline, KOS Pharmaceuticals, Merck & Co, Merck/Schering-Plough, Pfizer, Schering-Plough, and Takeda. Dr Lazar is a consultant to Abbott and receives grant support from GlaxoSmithKline and Bristol Myers Squibb Research Institute. Dr Lazar and the University of Pennsylvania have licensed reagents used in the human resistin assay to Linco. Dr Lehrke and Ms Wolfe have no conflict of interest.
Footnotes
The first 2 authors contributed equally to this work.
The online-only Data Supplement, which contains Table I and Table II, can be found with this article at http://www.circulationaha.org.
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