Abstract
Low levels of high-density lipoprotein cholesterol (HDL-C) and high triglyceride levels contribute to the excess rate of cardiovascular events seen in subjects with type 2 diabetes. Fenofibrate treatment partially reverses dyslipidemia in these subjects. However, a paradoxical marked reduction in HDL-C and HDL's major protein, apolipoprotein A-I, is a complication of fenofibrate in combination with rosiglitazone, an insulin-sensitizing agent. Risk factors for this condition, termed hypoalphalipoproteinemia, have yet to be identified. Using a case-control study design with subjects enrolled in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial, we tested the hypothesis that alterations in HDL's protein cargo predispose diabetic subjects to fenofibrate/rosiglitazone-induced hypoalphalipoproteinemia. HDL was isolated from blood obtained from controls (no decreases or increase in HDL-C while receiving fenofibrate/rosiglitazone therapy) and cases (developed hypoalphalipoproteinemia after fenofibrate/rosiglitazone treatment) participating in the ACCORD study before they began fenofibrate/rosiglitazone treatment. HDL proteins were quantified by targeted parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) with isotope dilution. This approach demonstrated marked increases in the relative concentrations of paraoxonase/arylesterase 1 (PON1), apolipoprotein C-II (APOC2), apolipoprotein C-I, and apolipoprotein H in the HDL of subjects who developed hypoalphalipoproteinemia. The case and control subjects did not differ significantly in baseline HDL-C levels or other traditional lipid risk factors. We used orthogonal biochemical techniques to confirm increased levels of PON1 and APOC2. Our observations suggest that an imbalance in HDL proteins predisposes diabetic subjects to develop hypoalphalipoproteinemia on fenofibrate/rosiglitazone therapy.
Highlights
From the ‡Department of Medicine, University of Washington, Seattle, WA, 98109; § Columbia University College of Physicians and Surgeons, Department of Medicine, New York, NY 10032; ¶Children’s Hospital Oakland Research Institute, Oakland, CA 94609
HDL was isolated from fasting plasma samples obtained while the 60 subjects were on simvastatin therapy [11] but before they were on combination therapy with fenofibrate/rosiglitazone
This approach confirmed that paraoxonase/arylesterase 1 (PON1), APOC2, APOC1, and APOH were elevated in HDL of subjects that later developed fenofibrate/rosiglitazone-induced hypoalphalipoproteinemia
Summary
Materials and Reagents—Unless otherwise specified, all reagents were obtained from Sigma Aldrich We prespecified that of the two [15N]APOA1 peptides with the best r, the one with the larger integrated area would be used as internal standard for protein quantification. Immunoblotting Analysis for Serum Paraoxonase/Arylesterase 1 (PON1)—Fourteen HDLs that contained a wide range of PON1 concentrations (as quantified by PRM) were selected at random (case or control designation was ignored) for confirmation by immunoblotting. The obtained CVs for PRM (29%) and SRM (12%) are in accordance with the precision recommended for research use assays for quantifying proteins and peptides (20 –35%)(37). The areas obtained for the [15N]APOA1 peptide by PRM and SRM are provided in Supplemental Table 4. For proteins differing significantly in expression between controls and cases, multiple linear regression analyses controlling for HDL-C and triglycerides (log scale) were performed. All statistical analyses were performed with STATA software version 12 (Stata Corp, College Park, TX)
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