Structure-function analysis of naturally occurring apolipoprotein A-I L144R, A164S and L178P mutants provides insight on their role on HDL levels and cardiovascular risk.

Naturally occurring point mutations in apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), may affect plasma HDL-cholesterol levels and cardiovascular risk. Here, we evaluated the effect of human apoA-I mutations L144R (associated with low HDL-cholesterol), L178P (associated with low HDL-cholesterol and increased cardiovascular risk) and A164S (associated with increased cardiovascular risk and mortality without low HDL-cholesterol) on the structural integrity and functions of lipid-free and lipoprotein-associated apoA-I in an effort to explain the phenotypes of subjects carrying these mutations. All three mutants, in lipid-free form, presented structural and thermodynamic aberrations, with apoA-I[L178P] presenting the greatest thermodynamic destabilization. Additionally, apoA-I[L178P] displayed reduced ABCA1-mediated cholesterol efflux capacity. When in reconstituted HDL (rHDL), apoA-I[L144R] and apoA-I[L178P] were more thermodynamically destabilized compared to wild-type apoA-I, both displayed reduced SR-BI-mediated cholesterol efflux capacity and apoA-I[L144R] showed severe LCAT activation defect. ApoA-I[A164S] was thermodynamically unaffected when in rHDL, but exhibited a series of functional defects. Specifically, it had reduced ABCG1-mediated cholesterol and 7-ketocholesterol efflux capacity, failed to reduce ROS formation in endothelial cells and had reduced capacity to induce endothelial cell migration. Mechanistically, the latter was due to decreased capacity of rHDL-apoA-I[A164S] to activate Akt kinase possibly by interacting with endothelial LOX-1 receptor. The impaired capacity of rHDL-apoA-I[A164S] to preserve endothelial function may be related to the increased cardiovascular risk for this mutation. Overall, our structure-function analysis of L144R, A164S and L178P apoA-I mutants provides insights on how HDL-cholesterol levels and/or atheroprotective properties of apoA-I/HDL are impaired in carriers of these mutations.

Lipoprotein hydrophobic core lipids are partially extruded to surface in smaller HDL: "Herniated" HDL, a common feature in diabetes.

Recent studies have shown that pharmacological increases in HDL cholesterol concentrations do not necessarily translate into clinical benefits for patients, raising concerns about its predictive value for cardiovascular events. Here we hypothesize that the size-modulated lipid distribution within HDL particles is compromised in metabolic disorders that have abnormal HDL particle sizes, such as type 2 diabetes mellitus (DM2). By using NMR spectroscopy combined with a biochemical volumetric model we determined the size and spatial lipid distribution of HDL subclasses in a cohort of 26 controls and 29 DM2 patients before and after two drug treatments, one with niacin plus laropiprant and another with fenofibrate as an add-on to simvastatin. We further characterized the HDL surface properties using atomic force microscopy and fluorescent probes to show an abnormal lipid distribution within smaller HDL particles, a subclass particularly enriched in the DM2 patients. The reduction in the size, force cholesterol esters and triglycerides to emerge from the HDL core to the surface, making the outer surface of HDL more hydrophobic. Interestingly, pharmacological interventions had no effect on this undesired configuration, which may explain the lack of clinical benefits in DM2 subjects.

Elevated HDL2-paraoxonase and reduced CETP activity are associated with a dramatically lower ratio of LDL-cholesterol/total cholesterol in a hypercholesterolemic and hypertriglyceridemic patient.

A female patient (64 years of age; body mass index, 26) had a markedly and relatively low low-density lipoprotein-cholesterol (LDL-C) level (97 mg/dl) despite high serum total cholesterol (TC) (331 mg/dl) and triacylglyceride levels (307 mg/dl). Since the expected LDL-C was 222 mg/dl, there was a significant difference between the calculation and measurement based on direct enzyme assay. Only 30% of serum cholesterol was associated with LDL-C in this patient. To determine the basis for the markedly low LDL-C/TC ratio, we isolated and analyzed lipoproteins from the patient as well as age- and gender-matched controls. The patient had lowered serum CETP activity and elevated paraoxonase activity with GOT and GPT values in the normal range. The very low-density lipoprotein particles from the patient were larger than those of the controls and enriched with lipid and protein, while the LDL from the patient (LDL-P) had a lower particle number and protein content than the controls. The LDL-P was more resistant to cupric ion-mediated oxidation. HDL2 from the patient (HDL2-P) had highly enhanced paraoxonase activity and antioxidant ability. The patient had a 1.5-fold higher level of apolipoprotein (apo) A-I expression in HDL2. ApoA-I in HDL2 and HDL3 from the patient showed no fragmentation, while the control had fragmented bands (17 and 21 kDa) in the HDL. The HDL2-P also had a larger particle size and greater protein content with less lipid content. HDL3-associated cholesteryl ester transfer protein was reduced in the patient, although the particle size was similar to the controls. In conclusion, a patient who had a markedly lower LDL-C/TC ratio despite hyperlipidemia associated with higher paraoxonase activity, higher apoA-I level and lower CETP activity without fragmentation of apoA-I in the HDL fraction is presented. The enhanced antioxidant and anti-inflammatory activity of HDL might contribute to the low LDL-C/TC ratio in this patient.

Responses of LDL and HDL particle size and distribution to omega-3 fatty acid supplementation and aerobic exercise.

The purpose of this investigation was to determine the independent and combined effects of aerobic exercise and omega-3 fatty acid (n-3fa) supplementation on lipid and lipoproteins. Sedentary, normoglycemic, nonsmoking men (n = 11) were assigned to perform rest and exercise before and during n-3fa supplementation. Exercise consisted of 3 consecutive days of treadmill walking at 65% maximum O(2) consumption for 60 min. Supplementation consisted of 42 days of 4.55 g/day of n-3fa. A two-way factorial ANOVA with repeated measures revealed significant reductions in total cholesterol (P = 0.001, -9.2%) and triglyceride (P = 0.007, -32.4%) concentrations postexercise. In addition, exercise increased LDL peak particle size (P = 0.001) from 26.2 to 26.4 nm, but not HDL size. The n-3fa supplementation resulted in a significant shift in the distribution of HDL-cholesterol (HDL-C) carried by HDL(2b+2a) (P = 0.001, 14.2%) and HDL(3a+3b) (P = 0.001, -22.8%), despite no significant changes in lipid and lipoprotein-cholesterol concentrations. The majority of the shift in HDL-C was noted in HDL(2b) (P = 0.001, 20.9%) and HDL(3a) (P < 0.001, -31.0%) particles. There were no combined effects of exercise and n-3fa supplementation on lipids and lipoproteins. Three consecutive days of aerobic exercise reduced triglyceride and total cholesterol concentrations with a concomitant increase in LDL peak particle size. In contrast, n-3fa supplementation shifted HDL-C from HDL(3) particles to HDL(2) particles, despite no significant changes in HDL(2)-C and HDL(3)-C concentrations. Exercise and n-3fa supplementation do not synergistically improve serum lipids and lipoproteins, but rather independently affect the metabolism of lipids and lipoproteins.

Effect of phosphatidylethanolamine on the properties of phospholipid-apolipoprotein complexes.

Plasma high density lipoproteins (HDL) are synthesized in intestinal mucosal cells and hepatocytes and are secreted into the blood. Factors influencing the structure and function of these HDL, such as lipid and protein composition, are poorly understood. It appears, however, that intracellular, discoidal HDL are enriched, relative to plasma HDL, in phosphatidylethanolamine (PE), a phospholipid known to generate unusual, nonbilayer structures of putative physiological significance. Although incubation of dimyristoylphosphatidylcholine (DMPC) with apolipoprotein A-I at the gel-liquid crystalline phase transition temperature results in the spontaneous formation of lipid-protein complexes, the presence of proportionately small amounts of PE prevents the formation of such complexes, suggesting that PE profoundly alters the phase properties of the phospholipid bilayers. However, by using a detergent-mediated method for the formation of PE-rich model nascent HDL from phospholipids and apolipoprotein A-I, lipid-protein complexes containing as much as 75% DLPE could be formed, thus demonstrating that the presence of PE causes a kinetic, rather than a thermodynamic, barrier to spontaneous complex formation. The products contained a DLPE:DMPC molar ratio similar to that of the initial incubation mixture; however, as the mole percentage of DLPE increased, the products became less heterogeneous, the buoyant density of the products increased, and the Stokes diameter of the products decreased. Similar results were obtained when dimyristoylphosphatidylethanolamine (DMPE) and dipalmitoylphosphatidylethanolamine (DPPE) were employed in lieu of DLPE. Electron microscopy of complexes containing DLPE and DMPC at a 1:1 molar ratio showed that these particles possessed a discoidal, bilayer morphology similar to that seen with complexes containing only phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)