Supplementation with two probiotic strains, Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032, reduces fasting triglycerides and enhances apolipoprotein A-V levels in non-diabetic subjects with hypertriglyceridemia.

OBJECTIVE: Previous studies have indicated that supplementation with probiotics might improve lipid metabolism. The objective of the study was to evaluate the effect of supplementation with probiotic strains Lactobacillus curvatus (L. curvatus) HY7601 and Lactobacillus plantarum (L. plantarum) KY1032 on triglyceride (TG) and apolipoprotein A-V (apo A-V) levels. METHODS: A randomized, double-blinded, placebo-controlled study was conducted with 128 non-diabetic subjects with hypertriglyceridemia. Over a 12-week test period, the probiotic group consumed 2 g/day of a powdered supplement containing L. curvatus HY7601 and L. plantarum KY1032, whereas the placebo group consumed a powder lacking probiotics. RESULTS: After the treatment, the probiotic group showed an 18.3% (P < 0.001) reduction in TGs and increases of 21.1% (P = 0.001) and 15.6% (P < 0.001) in the apo A-V and LDL particle size, respectively. The probiotic group had a significant reduction in TGs (P = 0.040) and increases in the plasma apo A-V (P = 0.003) and LDL particle size (P < 0.001) compared with the placebo group. In the probiotic group, the reduction in the TG levels was negatively correlated with changes in the apo A-V and baseline TGs, regardless of the APOA5 -1131T > C genotype. CONCLUSION: The consumption of two probiotic strains for 12 weeks reduced TGs and increased the apo A-V and LDL particle size in hypertriglyceridemic subjects. This effect was more pronounced in subjects with higher levels of fasting TGs regardless of their APOA5 -1131T > C genotype.

Accelerated atherosclerosis in apolipoprotein E-deficient mice fed Western diets containing palm oil compared with extra virgin olive oils: a role for small, dense high-density lipoproteins.

To test the hypothesis that extra virgin olive oils from different cultivars added to Western diets might behave differently than palm oil in the development of atherosclerosis, apoE-deficient mice were fed diets containing different cultivars of olive oil for 10 weeks. Female mice were assigned randomly to one of the following five groups: (1-4) fed chow diets supplemented with 0.15% (w/w) cholesterol and 20% (w/w) extra virgin olive oil from the Arbequina, Picual, Cornicabra, or Empeltre cultivars, and (5) fed a chow diet supplemented with 0.15% cholesterol and 20% palm oil. Compared to diets containing palm oil, a Western diet supplemented with one of several varieties of extra virgin olive oil decreased atherosclerosis lesions, reduced plaque size, and decreased macrophage recruitment. Unexpectedly, total plasma paraoxonase activity, apoA-I, plasma triglycerides, and cholesterol played minor roles in the regulation of differential aortic lesion development. Extra virgin olive oil induced a cholesterol-poor, apoA-IV-enriched lipoparticle that has enhanced arylesterase and antioxidant activities, which is closely associated with reductions in atherosclerotic lesions. Given the anti-atherogenic properties of extra virgin olive oil evident in animal models fed a Western diet, clinical trials are needed to establish whether these oils are a safe and effective means of treating atherosclerosis.

Lysine-phosphatidylcholine adducts in kringle V impart unique immunological and potential pro-inflammatory properties to human apolipoprotein(a).

Lipoprotein(a), Lp(a), an athero-thrombotic risk factor, reacts with EO6, a natural monoclonal autoantibody that recognizes the phophorylcholine (PC) group of oxidized phosphatidylcholine (oxPtdPC) either as a lipid or linked by a Schiff base to lysine residues of peptides/proteins. Here we show that EO6 reacts with free apolipoprotein(a) apo(a), its C-terminal domain, F2 (but not the N-terminal F1), kringle V-containing fragments obtained by the enzymatic digestion of apo(a) and also kringle V-containing apo(a) recombinants. The evidence that kringle V is critical for EO6 reactivity is supported by the finding that apo(a) of rhesus monkeys lacking kringle V did not react with EO6. Based on the previously established EO6 specificity requirements, we hypothesized that all or some of the six lysines in human kringle V are involved in Schiff base linkage with oxPtdPC. To test this hypothesis, we made use of a recombinant lysine-containing apo(a) fragment, rIII, containing kringle V but not the protease domain. EO6 reacted with rIII before and after reduction to stabilize the Schiff base and also after extensive ethanol/ether extraction that yielded no lipids. On the other hand, delipidation of the saponified product yielded an average of two mol of phospholipids/mol of protein consistent with direct analysis of inorganic phosphorous on the non-saponified rIII. Moreover, only two of the six theoretical free lysine amino groups per mol of rIII were unavailable to chemical modification by 2,4,6-trinitrobenzene sulfonic acid. Finally, rIII, like human apo(a), stimulated the production of interleukin 8 in THP-1 macrophages in culture. Together, our studies provide evidence that in human apo(a), kringle V is the site that reacts with EO6 via lysine-oxPtdPC adducts that may also be involved in the previously reported pro-inflammatory effect of apo(a) in cultured human macrophages.

Apolipoprotein A-V: a novel apolipoprotein associated with an early phase of liver regeneration.

Liver regeneration in response to various forms of liver injury is a complex process, which ultimately results in restoration of the original liver mass and function. Because the underlying mechanisms that initiate this response are still incompletely defined, this study was aimed to identify novel factors. Liver genes that were up-regulated 6 h after 70% hepatectomy (PHx) in the rat were selected by cDNA subtractive hybridization. Besides known genes associated with cell proliferation, several novel genes were isolated. The novel gene that was most up-regulated was further studied. Its mRNA showed a liver-specific expression and encoded a protein comprising 367 amino acids. The mouse and human cDNA analogues were also isolated and appeared to be highly homologous. The human gene analogue was located at an apolipoprotein gene cluster on chromosome 11q23. The protein encoded by this gene had appreciable homology with apolipoproteins A-I and A-IV. Maximal expression of the gene in the rat liver and its gene product in rat plasma was observed 6 h after PHx. The protein was present in plasma fractions containing high density lipoprotein particles. Therefore, we have identified a novel apolipoprotein, designated apolipoprotein A-V, that is associated with an early phase of liver regeneration.

Structural organization of the N-terminal domain of apolipoprotein A-I: studies of tryptophan mutants.

Site-directed mutagenesis and detailed fluorescence studies were used to study the structure and dynamics of recombinant human proapolipoprotein (proapo) A-I in the lipid free state and in reconstituted high-density lipoprotein (rHDL) particles. Five different mutants of proapoA-I, each containing a single tryptophan residue, were produced in bacteria corresponding to each of the naturally occurring Trp residues (position -3 in the pro-segment, 8, 50, 72, and 108) in the N-terminal half of the protein. Structural analyses indicated that the conservative Phe-Trp substitutions did not perturb the conformation of the mutants with respect to the wild-type protein. Steady-state fluorescence studies indicated that all of the Trp residues exist in nonpolar environments that are highly protected from solvent in both the lipid-free and lipid-bound forms. Time-resolved lifetime and anisotropy studies indicated that the shape of the monomeric form of proapoA-I is a prolate ellipsoid with an axial ratio of about 6:1. In addition, the region surrounding Trp 108 appears to be more mobile than the rest of the protein in the lipid-free state. However, in rHDL particles, no significant domain motion was detected for any of the Trp residues. The results presented in this work are consistent with a model for monomeric lipid-free proapoA-I in which the N-terminal half of the molecule is organized into a bundle of helices.

The role of apo-(a) kringle-IVs in the assembly of lipoprotein-(a).

Lipoprotein-(a) [Lp(a)] is a highly atherogenic lipoprotein with unknown function, consisting of a low-density lipoprotein (LDL) core and the apo(a) glycoprotein. The characteristic structural feature of apo(a) is the presence of multiple so called "kringle' repeats which are in part identical and in part exhibit slight sequence differences. The assembly of apo(a) and LDL, which is determinant for plasma Lp(a) levels, takes place extracellularly and requires specific structural motifs in apo(a) and apoB. Here we studied the structural features in apo(a) necessary for high-efficient assembly. Thirteen recombinant apo(a) glycoproteins, which differed in the set of kringle-IV (K-IV) motifs, were expressed in COS-7 cells and incubated with LDL. The rate of total and disulfide-stabilized Lp(a) complex formation was measured by an immunochemical assay. Constructs containing K-IV T(type)5-T10 yielded almost 100% total and 80% stable complexes, respectively. Deletion or replacement of the different kringles revealed that K-IV T6 and T7 were responsible for the high-yield assembly and that K-IV T5 had an amplifying effect. Increasing the absolute number of K-IV repeats had an additional amplifying effect. The rate of Lp(a) assembly correlated strongly with the affinity of these constructs to Lys-Sepharose. Our results have implications for understanding the metabolism of Lp(a) and may help to design strategies for searching natural apo(a) mutants with aberrant plasma Lp(a) levels.

Analysis of structure--function relationships in human apolipoprotein(a).

Elevated levels of lipoprotein(a) (Lp(a)) have been strongly correlated with the development of atherosclerosis in human populations. Lp(a) is distinguishable from low density lipoprotein by the presence of the unique protein component apolipoprotein(a) (apo(a)), which contains repeated domains that closely resemble that of plasminogen kringle IV. Using human embryonic kidney cells, we have expressed a recombinant form of apo(a) (r-apo(a)) containing 17 kringle IV-like domains. We have utilized this recombinant expression system to study the assembly of Lp(a) particles. We have demonstrated that Lp(a) particles containing r-apo(a) can be assembled extracellularly in plasma by covalent linkage to low density lipoprotein. Using site-directed mutagenesis, we have demonstrated that a cysteine residue present at position 4057 of the apo(a) protein (i.e., in the penultimate kringle IV repeat) mediates this covalent linkage. Using polymerase chain reaction amplification of liver apo(a) complementary DNA, we have demonstrated the presence of a polymorphism in apo(a) kringle IV type 10, which results in the substitution of a threonine for a methionine. Preliminary studies indicate that the presence of a threonine at this position may enhance the interaction of Lp(a) with lysine-Sepharose.