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Randomized Controlled Trial
. 2008 Oct;57(10):2794-800.
doi: 10.2337/db08-0450. Epub 2008 Jul 3.

Correction of HDL dysfunction in individuals with diabetes and the haptoglobin 2-2 genotype

Rabea Asleh  1 , Shany Blum, Shiri Kalet-Litman, Jonia Alshiek, Rachel Miller-Lotan, Roy Asaf, Wasseem Rock, Michael Aviram, Uzi Milman, Chen Shapira, Zaid Abassi, Andrew P Levy
Affiliations
Randomized Controlled Trial

Correction of HDL dysfunction in individuals with diabetes and the haptoglobin 2-2 genotype

Rabea Asleh et al. Diabetes. 2008 Oct.

Abstract

Objective: Pharmacogenomics is a key component of personalized medicine. The Israel Cardiovascular Events Reduction with Vitamin E Study, a prospective placebo-controlled study, recently demonstrated that vitamin E could dramatically reduce CVD in individuals with diabetes and the haptoglobin (Hp) 2-2 genotype (40% of diabetic individuals). However, because of the large number of clinical trials that failed to demonstrate benefit from vitamin E coupled with the lack of a mechanistic explanation for why vitamin E should be beneficial only in diabetic individuals with the Hp 2-2 genotype, enthusiasm for this pharmacogenomic paradigm has been limited. In this study, we sought to provide such a mechanistic explanation based on the hypothesis that the Hp 2-2 genotype and diabetes interact to promote HDL oxidative modification and dysfunction.

Research design and methods: Hb and lipid peroxides were assessed in HDL isolated from diabetic individuals or mice with the Hp 1-1 or Hp 2-2 genotypes. HDL function was assessed based on its ability to promote cholesterol efflux from macrophages. A crossover placebo-controlled study in Hp 2-2 diabetic humans and in Hp 1-1 and Hp 2-2 diabetic mice assessed the ability of vitamin E to favorably modify these structural and functional parameters. RESULTS-Hb and lipid peroxides associated with HDL were increased and HDL function was impaired in Hp 2-2 diabetic individuals and mice. Vitamin E decreased oxidative modification of HDL and improved HDL function in Hp 2-2 diabetes but had no effect in Hp 1-1 diabetes.

Conclusions: Vitamin E significantly improves the quality of HDL in Hp 2-2 diabetic individuals.

Trial registration: ClinicalTrials.gov NCT00314379.

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Figures

FIG. 1.
FIG. 1.
Hp is an HDL-associated protein. A: Hp is an HDL-associated protein in humans. One microgram of HDL prepared from three different individuals with diabetes by ultracentrifugation (UC) or immunoprecipitation (IP) was subjected to Western blot analysis for Hp. Purified Hp 2-1 protein was run as a control to indicate the location of the Hp α-chains. M, MW marker. An immunoreactive band for Hp is seen only in HDL prepared by immunoprecipitation. To confirm equal loading of protein in all lanes, the same blot was subsequently developed with an anti-ApoA1 antibody. B: Increased amount of Hp 2-2 protein associated with human HDL. Hp was assessed in the HDL immunoprecipitate by Western blotting. Hp α-chains detected by Western blotting are shown. Purified Hp 2-1 protein (2-1) was run as control to indicate the location of Hp 2-α (17 kDa) and Hp 1-α chain (11 kDa). Samples denoted 2-2 or 1-1 represent the HDL immunoprecipitate from six different individuals with either the Hp 2-2 or Hp 1-1 genotype.
FIG. 2.
FIG. 2.
The association of 125Hp-Hb and 125Hb with HDL is Hp genotype- and diabetes-dependent. A: Increased association of injected Hp-Hb with HDL in Hp 2-2 diabetic mice. 125I-Hp-Hb complex (one million cpm) was injected in the tail vein. The percentage of the injected cpm that coimmunoprecipitated with HDL at all time points after the injection (1–180 min) is shown (n = 5 for Hp 1-1 and Hp 2-2 nondiabetes and n = 6 for Hp 1-1 and Hp 2-2 diabetes). There was a significant increase in cpm in the HDL immunoprecipitate of Hp 2-2 diabetes (P < 0.0001 compared with Hp 2-2 nondiabetes). There was no significant difference in cpm in the HDL immunoprecipitate of Hp 1-1 diabetes compared with Hp 1-1 nondiabetes (P = 0.24). B: The ability of 125I-Hb to bind to human HDL in vitro is increased in Hp 2-2 and decreased in Hp 0. 125I-Hb was incubated with serum from individuals with Hp 1-1, Hp 2-2, or Hp 0. 125I-Hb associating with HDL was assessed by immunoprecipitation, and the mean ± SE for 10 individuals from each of the three groups is shown. There was significantly more 125I-Hb associated with HDL using serum from Hp 2-2 individuals compared with Hp 1-1 individuals (P < 0.0001). The amount of 125I-Hb associating with HDL using Hp 0 serum was significantly less than that observed in Hp 1-1 serum (P < 0.002). Note that Hp 0 does not indicate that these individuals lack Hp, but rather that the level of Hp is below the level of detection by gel electrophoresis.
FIG. 3.
FIG. 3.
Hb is an HDL-associated protein in Hp 2-2 diabetic humans and mice. A: The amount of Hb associated with HDL is increased in Hp 2-2 diabetic individuals. Western blot for Hb of HDL immunoprecipitate of serum of Hp 1-1 or Hp 2-2 diabetic individuals. Hb was identifiable in 14 of 15 diabetic individuals with the Hp 2-2 genotype and in 0 of 15 of the diabetic individuals with the Hp 1-1 genotype. Hb was not found associated with HDL from nondiabetic Hp 1-1 or Hp 2-2 individuals (not shown). Hb indicates purified Hb used as positive control. B: The amount of Hb associated with HDL is increased in Hp 2-2 diabetic mice. Western blot for Hb of HDL immunoprecipitate of serum of Hp 1-1 or Hp 2-2 mice with or without diabetes (D). Hb indicates purified Hb used as positive control.
FIG. 4.
FIG. 4.
HDL function is impaired in Hp 2-2 diabetic mice and humans. Cholesterol efflux from macrophages incubated with serum from Hp 2-2 diabetic mice and Hp 2-2 humans with type 1 diabetes and with type 2 diabetes is significantly decreased compared with Hp 1-1 diabetic mice and Hp 1-1 type 1 and type 2 diabetic humans (P = 0.0001, n = 10 comparing Hp 1-1 vs. Hp 2-2 diabetic mice; P < 0.0006, n = 15 comparing Hp 1-1 vs. Hp 2-2 type 1 diabetic individuals; P < 0.001, n = 30 comparing Hp 1-1 vs. Hp 2-2 type 2 diabetic individuals). Efflux is expressed as the percentage of that obtained for Hp 1-1 diabetic mice, type 1 diabetic, and type 2 diabetic individuals, respectively.
FIG. 5.
FIG. 5.
Vitamin E improves HDL function and reduces HDL oxidative modification in Hp 2-2 diabetic mice but not in Hp 1-1 diabetic mice. A: Vitamin E improves the ability of serum of Hp 2-2 diabetic mice, but not Hp 1-1 diabetic mice, to promote cholesterol efflux from macrophages. There was a significant difference in efflux elicited by serum from Hp 1-1 and Hp 2-2 diabetic mice (P = 0.002 comparing placebo groups). Vitamin E significantly improved cholesterol efflux in Hp 2-2 diabetic mice (P = 0.0006 comparing Hp 2-2 placebo vs. Hp 2-2 vitamin E). Efflux elicited by the serum of Hp 2-2 diabetic mice treated with vitamin E was not significantly different from that elicited by Hp 1-1 diabetic mice. Vitamin E had no effect on efflux in Hp 1-1 diabetic mice (P = 0.29). B: Vitamin E reduces HDL-associated lipid peroxides in Hp 2-2 diabetic mice but not in Hp 1-1 diabetic mice. There was a significant difference in HDL-associated lipid peroxides between Hp 1-1 and Hp 2-2 diabetic mice (P = 0.0001). Vitamin E significantly reduced lipid peroxides in Hp 2-2 diabetic mice (P = 0.001 comparing Hp 2-2 placebo vs. Hp 2-2 vitamin E) but had no effect on efflux in Hp 1-1 diabetic mice (P = 0.74 comparing Hp 1-1 placebo vs. Hp 1-1 vitamin E).
FIG. 6.
FIG. 6.
Vitamin E improves HDL function and reduces HDL oxidative modification in Hp 2-2 diabetic humans. Crossover-design, placebo-controlled, double-blind trial. Eighteen Hp 2-2 diabetic individuals divided into two cohorts were randomized to either vitamin E or placebo and treated for 2 months. After a 2-week washout, patients were crossed over to the other treatment and treated for an additional 2 months. Blood samples were taken at baseline (test 1), after 2 months of the initial treatment (test 2), and after 2 months with the second treatment (test 3). A: Improvement in cholesterol efflux stimulated by Hp 2-2 serum with vitamin E in humans. There was a significant improvement in efflux with vitamin E treatment (test 1-test 2 in cohort 1, P = 0.004; test 2-test 3 in cohort 2, P = 0.04) and no change with placebo treatment (test 1-test 2 in cohort 2, P = 0.33). Of note in cohort 1, test 3 is not significantly different from the baseline value, demonstrating that even though vitamin E improved HDL function (compare test 1-test 2), after a 2-month period without vitamin E, HDL function deteriorated to baseline levels (P = 0.13 comparing test 1-test 3 in cohort 1). B: Reduction in HDL-associated lipid peroxides with vitamin E. There was a significant reduction in lipid peroxides with vitamin E treatment (test 1-test 2 in cohort 1, P = 0.03; test 2-test 3 in cohort 2, P = 0.01) and no change with placebo treatment (test 1-test 2 in cohort 2, P = 0.35). Of note in cohort 1, test 3 was not significantly different from the baseline value, demonstrating that even though vitamin E reduced lipid peroxides (compare test 1-test 2), 2 months after the vitamin E was stopped, lipid peroxides returned to baseline levels (P = 0.31 comparing test 1-test 3 in cohort 1).
FIG. 7.
FIG. 7.
Hb released intravascularly from erythrocytes (RBC) is rapidly bound by Hp protein to form an Hp-Hb complex. In Hp 2-2 diabetic individuals, the complex is cleared more slowly than in Hp 1-1 diabetic individuals by the scavenger receptor CD163. The Hp-Hb complex can bind to Apo A1 in HDL, with increased binding of Hp 2-2–Hb occurring due its increased avidity for HDL and its increased plasma concentration. The Hp 2-2–Hb complex, but not the Hp 1-1–Hb complex, when bound to HDL can produce reactive oxygen species that can oxidize protein (i.e., ApoA1, glutathione peroxidase [GPx], and LCAT) and lipid components (cholesterol) of HDL and render the HDL dysfunctional (because of decreased reversed cholesterol transport [RCT] and antioxidant activity), proatherogenic, and prothrombotic.
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