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. Author manuscript; available in PMC: 2011 Dec 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2010 Sep 16;30(12):2528–2534. doi: 10.1161/ATVBAHA.110.212894

Low Clusterin Levels in High Density Lipoprotein Associate with Insulin Resistance, Obesity, and Dyslipoproteinemia

Andrew N Hoofnagle 1,2,3, Mingyuan Wu 5, Albina K Gosmanova 5, Jessica O Becker 1, Ellen M Wijsman 2,4, John D Brunzell 2, Steven E Kahn 2,6, Robert H Knopp 2,7, Timothy J Lyons 5, Jay W Heinecke 2,3
PMCID: PMC2988100  NIHMSID: NIHMS237104  PMID: 20847305

Abstract

Objective

Insulin resistance and obesity are hallmarks of type 2 diabetes and the metabolic syndrome, which confer an increased risk of cardiovascular disease. Recent studies suggest that the protein cargo of high density lipoprotein (HDL) makes important contributions to the lipoprotein’s cardioprotective effects. We used targeted proteomics to determine if obesity and insulin resistance associate with changes in HDL’s protein content in two different groups of men.

Methods and Results

In a discovery study, we used isotope dilution mass spectrometry to quantify the relative concentrations of five proteins previously implicated in HDL’s cardioprotective effects in three groups of healthy subjects: lean insulin-sensitive, lean insulin-resistant, and obese insulin-resistant. We validated our findings in a different group of subjects. Clusterin concentration in HDL strongly and negatively associated with insulin resistance and body mass index in both populations. HDL clusterin levels were lower in subjects with low HDL and high triglycerides, key components of the metabolic syndrome. There was an inverse correlation between clusterin levels in HDL and VLDL/LDL.

Conclusions

Clusterin levels in HDL are lower in men with reduced insulin sensitivity, higher body mass index, and an unfavorable lipid profile. Our observations raise the possibility that clusterin depletion contributes to the loss of HDL’s cardioprotective properties.

Keywords: High density lipoprotein, atherosclerosis, apolipoprotein J, inflammation, intra-abdominal fat

Many environmental and genetic factors contribute to the metabolic syndrome, whose hallmark is reduced insulin sensitivity accompanied by hypertension, hypertriglyceridemia, and obesity.1,2 Obesity and insulin resistance are also components of type 2 diabetes.3,4 These factors in turn contribute to the initiation and progression of atherosclerotic cardiovascular disease (CVD).3,4 Another cardinal feature of the metabolic syndrome is a low level of high density lipoprotein (HDL), which strongly associates with an increased risk of atherosclerotic vascular disease.

One important cardioprotective function of HDL is to remove cholesterol from cholesteryl ester-laden macrophages in the artery wall.5–7 The anti-inflammatory properties of HDL may also contribute to its anti-atherogenic effects.8,9 It has been proposed that HDL’s cardioprotective ability depends on the types of particles generated metabolically and that HDL in humans with established CVD is dysfunctional.8–12 Indeed, animal studies convincingly demonstrate that changes in HDL’s protein composition can promote atherosclerosis, even when plasma levels of HDL cholesterol (HDL-C) are normal or even elevated.13–15

To explore the potential clinical relevance of protein composition in HDL’s cardioprotective effects, we used targeted proteomics to determine the relationship between the relative concentration of five different proteins in HDL and two key features of the metabolic syndrome: obesity and insulin resistance. We quantified proteins that have been previously implicated in HDL’s cardioprotective effects: apolipoprotein (apo) A-I,6,7 clusterin,5,16–20 apoE,21 apoM,22,23 and complement component C3.20,24–26 Our observations indicate strong positive correlations between clusterin levels in HDL and insulin sensitivity and strong negative correlations with increased obesity and dyslipoproteinemia. In contrast, levels of the other proteins were not associated with these features of the metabolic syndrome. These results raise the possibility that clusterin depletion indicates the presence of dysfunctional HDL.

Methods

Study populations

All studies were approved by the Institutional Review Boards of the University of Washington and the University of Oklahoma, and subjects gave written informed consent. The discovery study (University of Washington) involved 30 healthy non-smoking men with no history of atherosclerosis (Table 1). Subjects were distributed into three groups: lean insulin-sensitive, lean insulin-resistant, and obese insulin-resistant (N=10 per group).27 Body habitus was based on body mass index (BMI; lean subject, BMI< 27.5 kg/m2).28 Insulin sensitivity was quantified by the sensitivity index (SI), using the frequently sampled tolbutamide-modified intravenous-glucose tolerance test (insulin-sensitive subject, SI ≥4.2 × 10−4 mi−1 per μU/mL).27 Intra-abdominal and subcutaneous fat areas were determined by computed tomography.29 The validation study (University of Oklahoma) involved 23 healthy men age-matched to 16 men with type 2 diabetes mellitus (Table 3) and 6 (15%) of the subjects were smokers at the time of the study. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as previously described (fasting glucose (mmol/L) × fasting insulin (μU/mL) ÷ 22.5).30 Additional details on the two populations of subjects, including how subjects were diagnosed with metabolic syndrome, are provided in Supplemental Methods.

Table 1.

Characteristics of the discovery population.

Lean insulin- sensitive (N=10) Lean insulin- resistant (N=10) Obese insulin- resistant (N=10) p value
Age (years)* 44.6 (6.7) 46.6 (5.9) 46.9 (5.0) 0.61
BMI (kg/m2)† 23.8 (3.8) 25.4 (1.3) 30.5 (2.4) <0.001
SI (×1−4 min−1 per μU/mL)† 6.3 (3.6) 2.8 (1.5) 2.1 (1.0) <0.001
HOMA-IR (mmol/L × μU/mL)† 1.50 (1.06) 1.89 (0.51) 3.17 (1.58) 0.012
Waist-to-hip ratio* 0.83 (0.06) 0.89 (0.02) 0.96 (0.06) <0.001
Intra-abdominal fat area (cm2)† 56.1 (66.5) 92.9 (30.1) 200.9 (55.2) <0.001
Subcutaneous fat area (cm2)† 67.0 (71.3) 152.3 (78.3) 257.3 (137.2) <0.001
Total cholesterol (mmol/L)* 4.32 (1.00) 5.14 (0.77) 4.93 (1.31) 0.26
HDL cholesterol (mmol/L)* 1.16 (0.18) 1.03 (0.17) 0.98 (0.16) 0.076
Triglycerides (mmol/L)† 0.77 (0.29) 1.30 (1.15) 1.16 (0.91) 0.009
LDL cholesterol (mmol/L)* 2.75 (0.78) 3.41 (0.70) 3.13 (0.96) 0.25
Mean arterial blood pressure (mmHg) 86.9 (6.4) 87.4 (8.0) 93.9 (6.4) 0.067
HDL clusterin (arbitrary units)*‡ 1.18 (0.20) 0.97 (0.22) 0.85 (0.27) 0.014
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*

Mean group values (SD). Significance assessed by ANOVA.

†

Median group values (25th–75th interquartile range). Significance assessed by the Kruskal-Wallis test.

‡

N= 9, 9, and 10 for lean insulin sensitive, lean insulin resistant, and obese insulin resistant groups. To quantify the relative amount of each protein in HDL, we normalized the relative content of each of the five proteins (as monitored by two different isotope-labeled peptides for each protein) to a value of 1 for the entire study population. Thus, a sample in which clusterin has a value >1 is enriched in that protein relative to the population average, while values <1 represent depletion.

Table 3.

Characteristics of the validation population.

Normal Diabetic p value
Age† 45.8 (13.0) 49.8 (9.2) 0.28
BMI‡ 26.4 (8.0) 32.1 (9.3) 0.004
HOMA-IR‡ 1.02 (1.38) 2.77 (4.40) 0.037
Hemoglobin A1c† 5.2 (0.3) 7.5 (1.5) 0.008
Total cholesterol† 4.65 (0.96) 5.12 (0.71) 0.09
HDL cholesterol† 1.06 (0.26) 0.97 (0.21) 0.28
Triglycerides‡ 0.99 (0.65) 1.25 (1.50) 0.23
LDL cholesterol† 3.03 (0.94) 3.40 (0.64) 0.16
Mean arterial blood pressure† 92.5 (21.9) 89.5 (26.3) 0.70
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†

Mean group values (SD). Significance assessed by Student’s t-test.

‡

Median group values (25th–75th interquartile range). Significance assessed by the Wilcoxon rank sum test.

Lipoprotein isolation

HDL(d=1.063–1.210 g/mL) and VLDL/LDL particles (d<1.063 g/mL) were isolated by sequential density gradient ultracentrifugation from EDTA-anticoagulated plasma collected after an overnight fast.20 Plasma was brought to a density of 1.21 g/mL with solid KBr, loaded into an ultracentrifuge tube, and gently overlaid with normal saline adjusted with KBr to a density of 1.21 g/mL. After ultracentrifugation at 146,000 × g for 41 h, lipoproteins in the top one-fourth of the tube were collected, the density of the solution was adjusted to 1.063 g/mL, and the samples were centrifuged at 146,000 × g for 16 h. HDL from the bottom two-thirds and VLDL/LDL from the top one-third of each tube was iteratively concentrated and diluted 7 times with 20 mM potassium phosphate, 0.1 mM diethylenetriamine pentaacetic acid, pH 7, using a 50 kDa centrifuge filter (Millipore). Protein concentrations were determined by the Bradford assay (Pierce). Lipoproteins were stored at −80°C until analysis.

Liquid chromatography-tandem mass spectrometric quantification of proteins in lipoproteins

Complete details are provided in Supplemental Methods. Briefly, tryptic digests of reduced, alkylated HDL or VLDL/LDL (10 μg protein) were supplemented with isotope-labeled internal standard peptides for 5 proteins (Supplemental Table I). This approach controls for sample-specific suppression of ionization and variability in mass spectrometer performance.31 Peptides were desalted on a C18 trapping column and subsequently eluted and separated with a C18 analytical column using a linear gradient of acetonitrile. Peptides were detected by selected reaction monitoring on a triple quadrupole tandem mass spectrometer. Peptides were quantified by isotope dilution, using the ratio of endogenous peptide ion current to the corresponding internal standard peptide ion current. Relative concentrations for each protein were normalized across each population (discovery or validation) such that a relative protein concentration >1 indicates that the protein is enriched compared with the average of the population and <1 indicates that the protein is depleted. The method was validated by comparing liquid chromatography-tandem mass spectrometric quantification of apoA-I with a nephelometric assay (Supplemental Fig. I). Absolute concentrations of clusterin in HDL were not calculated because a suitable matrix-matched calibrator was not available.

Statistical analyses

BMI, triglycerides, intra-abdominal fat, subcutaneous fat, and insulin sensitivity (SI and HOMA-IR) were modeled using loge-transformed data, based on the log-normal distribution of the larger population (N=186 to 275 observations for each variable) from which the subjects of the discovery population were drawn.27 Two HDL samples in the discovery study with very low apoA-I levels were excluded from the analysis. Means of groups were compared using one-way ANOVA or Student’s t-test. For log-normal data, medians were compared using Kruskal-Wallis or Wilcoxon rank sum tests. All tests of significance were two-sided.

Results

We used a two-tiered strategy to determine whether the relative concentration of five important proteins in HDL associated with features of the metabolic syndrome and other risk factors for CVD. First, in the discovery study, we used isotope dilution mass spectrometry with selected reaction monitoring to measure the concentrations of the five proteins in HDL isolated from healthy subjects enrolled in a metabolic study (N=30). We validated our observations in a different group of subjects recruited from an outpatient medical clinic (N=39).

Characteristics of the discovery group subjects

For the discovery study we isolated HDL from three groups of age-matched, healthy men: lean insulin-sensitive; lean insulin-resistant; and obese insulin-resistant (N=10 per group; Table 1).27–29 None of the subjects was using lipid-lowering therapy16 or had diabetes. Insulin sensitivity was determined from the intravenous glucose tolerance test that was used to categorize the subjects in the original metabolic study. As expected, other measures of insulin sensitivity (HOMA-IR) and obesity (waist-to-hip ratio, intra-abdominal fat, and subcutaneous fat) were statistically different among the three groups. Plasma HDL cholesterol levels were slightly but not significantly lower and plasma triglycerides were higher in the insulin-resistant groups. Mean arterial pressure was not different between the groups.

Clusterin levels in HDL correlate with features of the metabolic syndrome

HDL was proteolytically digested, and the resulting peptides were analyzed by tandem mass spectrometry. The relative amounts of five HDL proteins (per total HDL protein) were quantified by isotope dilution, using two synthetic internal standard peptides. By ANOVA, the amount of clusterin in HDL differed significantly among the three groups of subjects (p=0.014; Table 1). However, the amount of the other four proteins measured, apoA-I, apoE, apoM, and complement component C3, were not statistically different between the three groups (Supplemental Table II).

We next determined if the abundance of clusterin in HDL associated with obesity in the discovery population (Table 2). Univariate linear regression revealed that clusterin correlated negatively with BMI (r=−0.50, p=0.007) as well as with intra-abdominal and subcutaneous fat content as assessed by computed tomography (r=−0.58 and −0.48; p=0.001 and p=0.010, respectively). The correlation was higher for intra-abdominal fat area, suggesting that this specific depot might influence clusterin levels in HDL.

Table 2.

Correlation of subject characteristics with HDL clusterin levels in the discovery population.

Correlation with HDL clusterin by LC-MS/MS
Age −0.31
BMI* −0.50†
SI* 0.52†
HOMA-IR* −0.54†
Waist-to-hip ratio −0.56†
Intra-abdominal fat area* −0.58†
Subcutaneous fat area* −0.51†
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*

For BMI, SI, HOMA-IR, intra-abdominal fat, and subcutaneous fat, loge-transformed data were used.

†

p<0.01

HDL clusterin also correlated with insulin sensitivity, as assessed by both the SI and HOMA-IR. It is important to note that the direction of change (and units) associated with insulin sensitivity as assessed by the SI and HOMA-IR are different; thus, increased insulin sensitivity increases the SI but decreases HOMA-IR. For both measures of insulin sensitivity, the correlation with HDL clusterin was significant (r=0.52, p=0.004 for SI and r=−0.54, p=0.003 for HOMA-IR; Table 2).

Importantly, none of the other four quantified proteins, including apoA-I, correlated with obesity or insulin resistance (Supplemental Table III).

Characteristics of the validation group subjects

To validate the correlations between HDL clusterin concentration and BMI and insulin sensitivity, we analyzed a second population of apparently healthy control (N=23) and type 2 diabetic men (N=16) who lived in a different city. We selected these subjects because the samples were collected and stored at a different institution, thus eliminating the possibility that factors related to sample collection at the first site could explain our observations. All diabetic subjects were receiving oral hypoglycemic agents and/or insulin therapy (one subject was on insulin therapy). Importantly, none of the subjects was on lipid-lowering therapy.16 The BMI of the validation group was significantly greater than that of the discovery group (median 30.4 and 26.9, respectively; p=0.015). Within the validation population, the diabetic subjects were more obese, were less insulin sensitive, and had higher hemoglobin A1c levels than normal subjects (Table 3).

HDL clusterin level correlates negatively with BMI and insulin resistance in control and type 2 diabetic subjects

HDL clusterin content was quantified in the validation population by isotope dilution mass spectrometry. Again, clusterin levels in HDL correlated negatively with BMI and reduced insulin sensitivity (Fig. 1A,B), and the correlations remained significant after smokers were removed from the analysis (15% of the validation population). In contrast, HDL clusterin did not correlate with mean arterial blood pressure (Fig. 1C), another risk factor for CVD and an important component of the metabolic syndrome. The correlations of clusterin with BMI and HOMA-IR were similar in the validation and discovery populations (r=−0.55 vs. −0.50 for loge transformed BMI and r=−0.61 vs. −0.54 for loge transformed HOMA-IR, respectively), and both were significant (p<0.001). Importantly, the slopes of the linear regression analysis of HDL clusterin and HOMA-IR were also very similar despite the significantly different median BMIs of the validation and discovery populations (standardized coefficient=−0.63 and −0.54, respectively), suggesting that the relationship is valid over a wide range of body sizes (Fig. 1A,B).

Figure 1. Relationships between relative HDL clusterin concentration and clinical characteristics.

Figure 1

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Relative HDL clusterin concentration (per g total HDL protein) is plotted against loge-transformed BMI (A), loge-transformed HOMA-IR (B), and mean arterial blood pressure (C). Results represent the discovery population (open circles) and validation population (closed circles). Lines represent linear regression analysis of the relationship between HDL clusterin and clinical parameters for the discovery (dashed lines), validation (dotted lines), and combined populations (solid lines). Pearson correlation coefficients (r) and associated p values are presented for the combined population.

To further investigate the relationships among BMI, insulin resistance, and the relative concentration of clusterin in HDL, we combined the discovery and validation populations (Fig. 1). The correlations and slopes between HDL clusterin and BMI (r=−0.51) or HOMA-IR (r=−0.57) were similar to those for the discovery and validation populations. These observations indicate that in two distinct groups of male subjects, clusterin levels in HDL correlate negatively with insulin resistance and BMI.

HDL clusterin is depleted in subjects with the metabolic syndrome

By immunoassay, plasma concentrations of clusterin are elevated in subjects with diabetes.32,33 Using isotope dilution mass spectrometry, we also found an increased plasma concentration of clusterin in diabetic subjects, which was statistically significant even after removing 3 outlying data points (Supplemental Fig. II). However, when quantifying the clusterin in HDL, we observed no significant differences between diabetic and control subjects, though there was trend toward a lower level in diabetic subjects (Fig. 2A; p=0.14).

Figure 2. Relative concentration of HDL clusterin in control subjects, and subjects with diabetes, the metabolic syndrome, or dyslipoproteinemia.

Figure 2

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Diabetic subjects were compared with age-matched normal subjects in the validation population (A). Subjects with metabolic syndrome were compared with normal subjects in the discovery population (B) as described in Supplemental Methods. Also shown are subjects categorized by plasma HDL-C (C; normal ≥40 mg/dL), plasma triglycerides (D; normal <150 mg/dL), and plasma LDL cholesterol (E; normal <130 mg/dL). Results and error bars are mean and SEM, respectively. The number of subjects in each group is indicated.

Because our observations suggested that HDL clusterin levels associate with insulin resistance and obesity, we next determined if subjects with the metabolic syndrome had lower HDL clusterin levels. Indeed, we found that subjects with the metabolic syndrome had significantly lower HDL clusterin levels than the control subjects (p=0.004; Fig. 2B).

HDL clusterin is depleted in subjects with dyslipoproteinemia

Because high triglycerides and low HDL-C are characteristic features of the metabolic syndrome, we investigated the relationships between dyslipoproteinemia and the concentration of clusterin in HDL. When the data from the discovery and validation populations were combined (Fig. 2), levels of HDL clusterin were significantly lower in subjects with high plasma levels of triglycerides (>150 mg/dL; p<0.0001), low levels of HDL-C (<40 mg/dL; p<0.0001), or elevated levels of LDL cholesterol (>130 mg/dL; p=0.024).

We used linear regression analysis to examine the overall relationships between HDL clusterin and triglycerides or HDL-C in our combined study population. HDL clusterin concentration strongly correlated with plasma HDL-C (r=0.64; p<0.0001; Fig. 3A), and negatively correlated with loge-transformed triglycerides (r=−0.59, p<0.0001; Fig. 3B), LDL-C (r=−0.30; p=0.015; Fig. 3C), and total cholesterol (r=−0.37; p=0.002; not shown). These observations suggest that plasma lipid composition is one important factor that affects the concentration of clusterin in HDL. Importantly, HDL triglyceride concentration (per g HDL protein) had no relationship with HDL clusterin levels (Supplemental Fig. III).

Figure 3. Correlations of lipid parameters with relative HDL clusterin concentration.

Figure 3

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Linear regression analysis is shown for HDL clusterin and plasma HDL-C (A), plasma triglycerides (B), and clusterin concentration in VLDL/LDL particles (C). Data are presented as in Fig. 1, except that the regression line presented in C is for the validation population only.

HDL clusterin is negatively associated with VLDL/LDL clusterin levels

Although plasma concentrations of clusterin are elevated in subjects with insulin resistance and diabetes,32,33 the relative distribution of clusterin between HDL and the apoB-containing lipoproteins has not been investigated. We therefore isolated total VLDL/LDL from plasma of the validation subjects and quantified clusterin by isotope dilution mass spectrometry. In contrast to HDL, clusterin in the non-HDL lipoprotein fraction was directly proportional to BMI (r = 0.61, p=0.0005; not shown) and HOMA-IR (r = 0.46, p=0.009; not shown). In addition, there was a strong inverse relationship between clusterin in HDL and clusterin in LDL/VLDL (r = −0.66, p<0.0001; Fig. 3D).

Discussion

Insulin resistance, obesity, and dyslipidemia are hallmarks of the metabolic syndrome and type 2 diabetes, which increase the risk of death from CVD.1–4 We demonstrated a strong negative correlation between the concentration of clusterin in HDL and both insulin sensitivity and BMI in two different populations of male subjects. HDL clusterin was unrelated to elevated blood pressure, another component of the metabolic syndrome, but there was a significant decrease in HDL clusterin levels in subjects with metabolic syndrome. None of the other four HDL proteins that we investigated correlated with obesity or reduced insulin sensitivity. Collectively, our observations indicate that insulin resistance and the metabolic syndrome are associated with low clusterin levels in HDL.

Circulating clusterin is generally believed to be predominantly associated with HDL and has been shown to be enriched in the dense HDL3 subfraction.34 Based on peptide peak areas in this study, ~22% of clusterin is present on HDL and ~9% is present on LDL/VLDL. Previous studies have found that clusterin levels increase in the serum of patients with diabetes or CVD,32,33 leading to the suggestion that levels of clusterin in HDL increase in subjects with an increased risk of vascular disease. However, none of these studies reported the concentration of clusterin in apoB-containing lipoproteins. In contrast, mass spectrometry-based studies suggest that the concentrations of clusterin in HDL3 are apparently decreased in subjects with established CVD.20 Moreover, clusterin levels in HDL3 increase when newly diagnosed CVD subjects are treated with statin and niacin therapy,16 raising the possibility that there is interplay between the levels of clusterin in HDL, VLDL, and LDL.

To explore the factors that affect the levels of clusterin in HDL in metabolic syndrome and other disorders, we determined the relationship between levels of clusterin in HDL and triglycerides, total cholesterol, LDL-C, and HDL-C. Clusterin in HDL correlated negatively with total cholesterol, LDL-C, and triglycerides, but was positively associated with HDL-C.

Our observations suggest a model in which alterations in lipid metabolism that are secondary to insulin resistance and the metabolic syndrome diminish HDL clusterin levels. Thus, we observed an inverse association between clusterin in HDL and clusterin in non-HDL lipoproteins (VLDL and LDL). This finding, combined with the direct correlation between clusterin in apoB-containing lipoproteins and insulin resistance, raises the possibility that insulin resistance might shift proteins from HDL to VLDL and LDL. It is well-established that certain lipoprotein-associated proteins can move from one particle type to another, which is related in part to the lipid composition of HDL and the apoB-containing lipoproteins.35 Insulin resistance, obesity, and diabetes alter the lipid content and composition of lipoproteins,36,37 raising the possibility that such alterations modulate the distribution of clusterin between different classes of lipoprotein particles. However, there was no association between HDL clusterin levels and HDL triglyceride levels (Supplemental Fig. III), indicating that another class of lipids or that particular triglycerides with specific fatty acyl side chains might be involved.

Decreased levels of clusterin might contribute to the loss of HDL’s cardioprotective properties in subjects with insulin resistance and metabolic syndrome. For example, synthetic peptides based on clusterin are potently atheroprotective in animal models.17 Clusterin is also an inhibitor of complement activation, which is implicated in atherogenesis.38–41 Recent studies indicate that complement activation plays a key role in promoting tissue injury in a rodent model of myocardial infarction,42 raising the possibility that clusterin in HDL modulates tissue damage. Indeed, mice deficient in clusterin exhibit increased tissue damage in a model of cerebral ischemia43 and clusterin prevents an age-related progressive glomerulopathy in mice.44

Major strengths of our study include the use of isotope dilution mass spectrometry (an intrinsically precise, quantitative, and multiplex approach that is transferable across laboratories31) to investigate two diverse clinical populations that included both healthy subjects and type 2 diabetic subjects. Important limitations include the relatively small number of subjects, the cross-sectional design, and the focus on male subjects. In future studies, it will be important to extend our observations to larger groups of subjects, to women, and to younger and older subjects at increased risk of CVD.

In summary, we showed that clusterin levels in HDL correlate negatively with obesity and insulin resistance and that they are lower in subjects with the metabolic syndrome. Because clusterin is protective in mouse models of tissue injury, and because obesity and insulin resistance are important risk factors for CVD, our results raise the possibility that insufficient clusterin in HDL might impair the lipoprotein’s cardioprotective functions.

Supplementary Material

Supp1

Acknowledgments

We thank Dr. Alan Chait for helpful comments on the manuscript.

Funding Sources

This research was supported by the National Institutes of Health (DK002456, HL030086, HL086798), the Clinical Nutrition Research Unit (University of Washington, DK035816), the Diabetes and Endocrinology Research Center (University of Washington, DK017047), the Clinical Mass Spectrometry Facility (University of Washington), the Proteome Resource (University of Washington), the General Clinical Research Center (University of Oklahoma Health Sciences Center), and the United States Department of Veterans Affairs.

Footnotes

Disclosures

Andrew Hoofnagle has been a consultant for Thermo Fisher. Jay Heinecke is on the Merck/Schering Plough speaker bureau and serves as a consultant for Merck, Corcept, and Insilicos.

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