Obesity is associated with inflammation, insulin resistance, and type 2 diabetes, which are major risk factors for CVD. One dietary component of ruminant animal foods, 10,12-conjugated linoleic acid (10,12 CLA), has been shown to promote weight loss in humans. Previous work has shown that 10,12 CLA is atheroprotective in mice by a mechanism that may be distinct from its weight loss effects, but this exact mechanism is unclear. To investigate this, we evaluated HDL composition and function in obese LDL receptor (Ldlr-/-) mice that were losing weight because of 10,12 CLA supplementation or caloric restriction (CR; weight-matched control group) and in an obese control group consuming a high-fat high-sucrose diet. We show that 10,12 CLA-HDL exerted a stronger anti-inflammatory effect than CR- or high-fat high-sucrose-HDL in cultured adipocytes. Furthermore, the 10,12 CLA-HDL particle (HDL-P) concentration was higher, attributed to more medium- and large-sized HDL-Ps. Passive cholesterol efflux capacity of 10,12 CLA-HDL was elevated, as was expression of HDL receptor scavenger receptor class B type 1 in the aortic arch. Murine macrophages treated with 10,12 CLA in vitro exhibited increased expression of cholesterol transporters Abca1 and Abcg1, suggesting increased cholesterol efflux potential of these cells. Finally, proteomics analysis revealed elevated Apoa1 content in 10,12 CLA-HDL-Ps, consistent with a higher particle concentration, and particles were also enriched with alpha-1-antitrypsin, an emerging anti-inflammatory and antiatherosclerotic HDL-associated protein. We conclude that 10,12 CLA may therefore exert its atheroprotective effects by increasing HDL-P concentration, HDL anti-inflammatory potential, and promoting beneficial effects on cholesterol efflux.

Human high-density lipoproteins (HDLs) are a complex mixture of structurally related nanoparticles that perform distinct physiological functions. We previously showed that human HDL containing apolipoprotein A-I (APOA1) but not apolipoprotein A-II (APOA2), designated LpA-I, is composed primarily of two discretely sized populations. Here, we isolated these particles directly from human plasma by antibody affinity chromatography, separated them by high-resolution size-exclusion chromatography and performed a deep molecular characterization of each species. The large and small LpA-I populations were spherical with mean diameters of 109 Å and 91 Å, respectively. Unexpectedly, isotope dilution MS/MS with [15N]-APOA1 in concert with quantitation of particle concentration by calibrated ion mobility analysis demonstrated that the large particles contained fewer APOA1 molecules than the small particles; the stoichiometries were 3.0 and 3.7 molecules of APOA1 per particle, respectively. MS/MS experiments showed that the protein cargo of large LpA-I particles was more diverse. Human HDL and isolated particles containing both APOA1 and APOA2 exhibit a much wider range and variation of particle sizes than LpA-I, indicating that APOA2 is likely the major contributor to HDL size heterogeneity. We propose a ratchet model based on the trefoil structure of APOA1 whereby the helical cage maintaining particle structure has two "settings"-large and small-that accounts for these findings. This understanding of the determinants of HDL particle size and protein cargo distribution serves as a basis for determining the roles of HDL subpopulations in metabolism and disease states.

Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL's size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL's content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL's content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL's ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL's APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages.

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