Cholesteryl ester diffusion, location and self-association constraints determine CETP activity with discoidal HDL: excimer probe study.

The transfer of cholesteryl ester by recombinant cholesteryl ester transfer protein (CETP) between reconstituted discoidal high-density lipoprotein (rHDL) was studied. Particles contained apolipoprotein A-I, unsaturated POPC or saturated DPPC and cholesteryl ester as cholesteryl 1-pyrenedecanoate (CPD) or cholesteryl laurate (CL) in donor and acceptor rHDL, respectively. Probe dynamics fulfilled the quenching sphere-of-action model. The cholesteryl ester exchange between donor and acceptor particles was characterized by a heterogeneous kinetics; the fast exchanging CPD pool was much higher in a case of POPC compared to DPPC complexes. Probe fraction accessible to CETP increased with temperature, suggesting a more homogeneous probe distribution. Noncompetitive inhibition of probe transfer by acceptor particles was observed. The values of Vmax (0.063µMmin(-1)) and catalytic rate constant kcat (0.42s(-1)) together with a similarity of Km (0.9µM CPD) and KI (2.8µM CL) values for POPC-containing rHDL suggest the efficient cholesteryl ester transfer between nascent HDL with unsaturated phosphatidylcholine in vivo. The phospholipid matrix in discoidal HDL may underlie CETP activity through the self-association, diffusivity and location of cholesteryl ester in the bilayer, the accessibility of cholesteryl ester to cholesterol-binding site in apoA-I structure and the binding of cholesteryl ester, positionable by apoA-I, to CETP.

Insights into the molecular mechanisms of the anti-atherogenic actions of flavonoids in normal and obese mice.

Obesity is a major and independent risk factor for cardiovascular disease and it is strongly associated with the development of dyslipidemia, insulin resistance and type 2 diabetes. Flavonoids, a diverse group of polyphenol compounds of plant origin widely distributed in human diet, have been reported to have numerous health benefits, although the mechanisms underlying these effects have remained obscure. We analyzed the effects of chronic dietary supplementation with flavonoids extracted from cranberry (FLS) in normal and obese C57/BL6 mice compared to mice maintained on the same diets lacking FLS. Obese mice supplemented with flavonoids showed an amelioration of insulin resistance and plasma lipid profile, and a reduction of visceral fat mass. We provide evidence that the adiponectin-AMPK pathway is the main mediator of the improvement of these metabolic disorders. In contrast, the reduced plasma atherogenic cholesterol observed in normal mice under FLS seems to be due to a downregulation of the hepatic cholesterol synthesis pathway. Overall, we demonstrate for the first time that the molecular mechanisms underlying the beneficial effects of flavonoids are determined by the metabolic state.

Composition, structure and substrate properties of reconstituted discoidal HDL with apolipoprotein A-I and cholesteryl ester.

To investigate the influence of lipid unsaturation and neutral lipid on the maturation of high density lipoproteins, the discoidal complexes of apoA-I, phosphatidylcholine and cholesteryl ester (CE) were prepared. Saturated dipalmitoylphosphatidylcholine (DPPC) and unsaturated palmitoyllinoleoylphosphatidylcholine (PLPC), palmitoyloleoylphosphatidylcholine (POPC), and fluorescent probe cholesteryl 1-pyrenedecanoate (CPD) that forms in a diffusion- and concentration-dependent manner short-lived dimer of unexcited and excited molecules (excimer) were used. The apoA-I/DPPC/CPD complexes were heterogeneous by size, composition and probe location. CPD molecules incorporated more efficiently into larger complexes and accumulated in a central part of the discs. The apoA-I/POPC(PLPC)/CPD were also heterogeneous, however, probe molecules distributed preferentially into smaller complexes and accumulated at disc periphery. The kinetics of CPD transfer by recombinant cholesteryl ester transfer protein (CETP) to human plasma LDL is well described by two-exponential decay, the fast component with a shorter transfer time being more populated in PLPC compared to DPPC complexes. The presence of CE molecules in discoidal HDL results in particle heterogeneity. ApoA-I influences the CETP activity modulating the properties of apolipoprotein-phospholipid interface. This may include CE molecules accumulation in the boundary lipid in unsaturated phosphatidylcholine and cluster formation in the bulk bilayer in saturated phosphatidylcholine.

Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids.

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.