Transcriptome analysis based on dietary beta-carotene supplement reveals genes potentially involved in carotenoid metabolism in Crassostrea gigas.

Carotenoids are essential micronutrients for animals, and they can only be obtained from the diet for mollusk as well as other animals. In the body, carotenoids undergo processes including absorption, transport, deposition, and metabolic conversion; however, knowledge of the involved genes is still limited. To elucidate the molecular mechanisms of carotenoid processing and identify the related genes in Pacific oyster (Crassostrea gigas), we performed a comparative transcriptome analysis using digestive gland tissues of oysters on a beta-carotene supplemented diet or a normal diet. A total of 718 differentially expressed genes were obtained, including 505 upregulated and 213 downregulated genes in the beta-carotene supplemented group. Function Annotation and enrichment analyses revealed enrichment in genes possibly involved in carotenoid transport and storage (e.g., LOC105342035), carotenoid cleavage (e.g., LOC105341121), retinoid homeostasis (e.g., LOC105339597) and PPAR signaling pathway (e.g., LOC105323212). Notably, down-regulation of mRNA expressions of two apolipoprotein genes (LOC105342035 and LOC105342186) by RNA interference significantly decreased the carotenoid level in the digestive gland, supporting their role in carotenoid transport and storage. Based on these differentially expressed genes, we propose that there may be a negative feedback mechanism regulated by nuclear receptor transcription factors controlling carotenoid oxygenases. Our findings provide useful hints for elucidating the molecular basis of carotenoid metabolism and functions of carotenoid-related genes in the oyster.

Could ornithine supplementation be beneficial to prevent the formation of pro-atherogenic carbamylated low-density lipoprotein (c-LDL) particles?

Carbamylation (or carbamoylation) is a non-enzymatic post-translational modification process of lysine residues and protein N-termini, which occurs throughout the lifespan of both various plasma proteins and low-density lipoprotein (LDL) particles. Carbamylation results from the binding of isocyanates spontaneously derived from high levels of blood urea, environmental pollutants, nutritional sources and leads to the formation of potentially atherogenic carbamylated-LDL (c-LDL) particles. The carbamylation of LDL apolipoproteins is associated unfavorable downstream effects. Ornithine is a non-proteinogenic amino acid, which plays a central role at the urea cycle function. The primary use of ornithine in supplements is to support athletic performance, liver function and wound recovery. Ornithine is structurally highly similar to lysine, and is only one carbon atom shorter in its side-chain. Therefore, we hypothesize that supplemented ornithine could compete with ε-amino groups of lysine residues found in apolipoproteins of native LDL particles in their binding to isocyanates and decrease c-LDL formation. This issue still remains unresolved in current literature and needs to be elucidated in experimental studies.

Lipid composition dictates serum stability of reconstituted high-density lipoproteins: implications for in vivo applications.

Nanolipoprotein particles (NLPs) are reconstituted high-density lipoproteins, consisting of a phospholipid bilayer stabilized by an apolipoprotein scaffold protein. This class of nanoparticle has been a vital tool in the study of membrane proteins, and in recent years has been increasingly used for in vivo applications. Previous work demonstrated that the composition of the lipid bilayer component affects the stability of these particles in serum solutions. In the current study, NLPs assembled with phosphatidylcholine lipids featuring different acyl chain structures were systematically tested to understand the effect that lipid composition has on NLP stability in both neat serum and cell culture media supplemented with 10% serum by volume. The time at which 50% of the particles dissociate, as well as the fraction of the initial population that remains resistant to dissociation, were correlated to key parameters obtained from all-atom simulations of the corresponding lipid bilayers. A significant correlation was observed between the compressibility modulus of the lipid bilayer and particle stability in these complex biological milieu. These results can be used as a reference to tune the stability of these versatile biological nanoparticles for in vitro and in vivo applications.

The lipid composition of Legionella dumoffii membrane modulates the interaction with Galleria mellonella apolipophorin III.

Apolipophorin III (apoLp-III), an insect homologue of human apolipoprotein E (apoE), is a widely used model protein in studies on protein-lipid interactions, and anti-Legionella activity of Galleria mellonella apoLp-III has been documented. Interestingly, exogenous choline-cultured Legionella dumoffii cells are considerably more susceptible to apoLp-III than non-supplemented bacteria. In order to explain these differences, we performed, for the first time, a detailed analysis of L. dumoffii lipids and a comparative lipidomic analysis of membranes of bacteria grown without and in the presence of exogenous choline. (31)P NMR analysis of L. dumoffii phospholipids (PLs) revealed a considerable increase in the phosphatidylcholine (PC) content in bacteria cultured on choline medium and a decrease in the phosphatidylethanolamine (PE) content in approximately the same range. The interactions of G. mellonella apoLp-III with lipid bilayer membranes prepared from PLs extracted from non- and choline-supplemented L. dumoffii cells were examined in detail by means of attenuated total reflection- and linear dichroism-Fourier transform infrared spectroscopy. Furthermore, the kinetics of apoLp-III binding to liposomes formed from L. dumoffii PLs was analysed by fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy using fluorescently labelled G. mellonella apoLp-III. Our results indicated enhanced binding of apoLp-III to and deeper penetration into lipid membranes formed from PLs extracted from the choline-supplemented bacteria, i.e. characterized by an increased PC/PE ratio. This could explain, at least in part, the higher susceptibility of choline-cultured L. dumoffii to G. mellonella apoLp-III.

Development of a CCK1R-membrane nanoparticle as a fish-out tool for bioactive peptides.

The cholecystokinin receptor type 1 (CCK1R) is a G protein-coupled receptor (GPCR) that is involved in several biological processes including the regulation of the secretion of digestive enzymes. The peptide hormone cholecystokinin (CCK) binds to CCK1R, which is an important pharmacological target for several diseases, including obesity. Interestingly, nutritional dietary peptides also appear to activate CCK1R, and may play a role in CCK1R signaling in the gut. In this study, a novel technique to screen for CCK1R ligands based on affinity-selection is described. Functional expressed CCK1R is reconstituted into membrane nanoparticles called NABBs (nanoscale apo-lipoprotein bound bilayers). NABBs are native-like bilayer membrane systems for incorporation of GPCRs. CCK1R-NABBs were characterized using a fluorescently labeled CCK analog and can be used as a cutting-edge technology to screen for CCK1R ligands using affinity-selection mass spectrometry.

Apolipophorin III and transmission electron microscopy as toxicity indicators for harmaline and tea saponin in Spodoptera exigua (Noctuidae: Lepidoptera).

Apolipophorin III, traditionally known for lipid transport in insects is fairly established as toxicity indicator against harmaline and tea saponin during this study. Apolipophorin III expressed in the hemolymph and midgut tissues of 3rd, 4th, 5th larval instars and pupae of Spodoptera exigua. Apolipophorin III presence was further confirmed by achieving its partial cDNA (Genbank accession no. FJ606822) of 448bp. qRT PCR revealed that tea saponin resulted in significant reduction of gene expression in 3rd and 4th larval instars but increased in 5th instar as compared to control. Harmaline caused gradual increase of gene expression in 3rd, 4th and 5th instars after feeding on the treated diet. Fifth instar larvae synonymously resulted in the highest gene expressions against both the biochemicals. After the injection of harmaline and tea saponin abrupt increase in gene expression of 4th, 5th larval instar and pupae was observed as compared to control treatment. Transmission electron microscopy of midgut epithelium after being fed with harmaline and tea saponin depicted certain cytological changes. Harmaline treatment lead to cytoplasm vacuolization, mitochondrial disruption, spherocrystals with concentric layers, irregular nucleus and floating nuclei in cytoplasm. Tea saponin treatment resulted in denser cytoplasm, higher intracellular osmotic concentration and reduced complement of apical microvilli. Cells were found to have only a few mitochondria and glycogen deposits in comparison to control treatment.

Overexpression of apolipoprotein A-IV enhances lipid secretion in IPEC-1 cells by increasing chylomicron size.

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.

Hypotensive and lipid regulatory actions of 4-hydroxyderricin, a chalcone from Angelica keiskei, in stroke-prone spontaneously hypertensive rats.

1. Previously, we found that Angelica keiskei extract (ethyl acetate extract from the yellow liquid of stems) elevated serum high-density lipoprotein (HDL) levels and reduced liver triglyceride content in stroke-prone spontaneously hypertensive rats (SHRSP). To identify the active substance in A. keiskei extract, we examined the effect of 4-hydroxyderricin, a characteristic chalcone isolated from the yellow liquid of stems, on blood pressure and lipid metabolism in SHRSP. 2. Six-week-old male SHRSP were fed diets containing 0.07% 4-hydroxyderricin for 7 weeks with free access to the diet and water. Elevation of systolic blood pressure was significantly suppressed after 7 weeks treatment. Serum very low-density lipoprotein (VLDL) levels were significantly reduced, without any effect on HDL levels, and were associated with a significant decrease in the serum concentration of free fatty acids. 3. In the liver, significant decreases in relative liver weight and triglyceride content were found after treatment with 4-hydroxyderricin for 7 weeks. 4. An investigation of hepatic mRNA expression of proteins involved in lipid metabolism indicated that a significant decrease in microsomal triglyceride transferprotein may be responsible for the decrease in serum VLDL levels and that significant decreases in adipocyte determination and differentiation factor 1 and fatty acid synthase may be responsible for the decrease in hepatic triglyceride content. 5. In conclusion, dietary 4-hydroxyderricin produces suppression of the elevation of systolic blood pressure, reduction of serum VLDL levels and a decrease in hepatic triglyceride content in SHRSP.

Human apolipoprotein(a) kringle V inhibits angiogenesis in vitro and in vivo by interfering with the activation of focal adhesion kinases.

Apolipoprotein(a) [apo(a)] contains the largest numbers of kringle domains identified to date. Of these, apo(a) kringle V shows significant sequence homology with plasminogen kringle 5, which is reported to be a potent angiogenesis inhibitor. To determine the effects of apo(a) kringle V on angiogenesis, it was expressed as a soluble protein (termed rhLK8) in Pichia pastoris and its in vitro and in vivo anti-angiogenic properties were examined. rhLK8 inhibited the migration of human umbilical vein endothelial cells in vitro in a dose-dependent manner. This function was associated with the down-regulation of the activation of focal adhesion kinase and the inhibition of the consequent formation of actin stress fibers/focal adhesions. rhLK8 also inhibited new capillary formation in vivo, as assessed by the chick chorioallantoic membrane assay and the Matrigel plug assay. These results indicate that rhLK8 may be an effective angiogenesis inhibitor both in vitro and in vivo.

Helical apolipoproteins stabilize ATP-binding cassette transporter A1 by protecting it from thiol protease-mediated degradation.

ATP-binding cassette transporter (ABC) A1 was increased by apolipoprotein A-I without an increase of its message in THP-1 cells. The pulse label study demonstrated that apoA-I retarded degradation of ABCA1. Similar changes were demonstrated by apoA-II, but the effect of high density lipoprotein was almost negligible on the basis of equivalent protein concentration. Thiol protease inhibitors (leupeptin and N-acetyl-Leu-Leu-norleucinal (ALLN)) increased ABCA1 and slowed its decay in the cells, whereas none of the proteosome-specific inhibitor lactacystin, other protease inhibitors, or the lysosomal inhibitor NH(4)Cl showed such effects. The effects of apoA-I and ALLN were additive for the increase of ABCA1, and the apoA-I-mediated cellular lipid release was enhanced by ALLN. The data suggest that ABCA1 is rapidly degraded by a thiol protease(s) in the cells unless helical apolipoproteins in their lipid-free form stabilize ABCA1 by protecting it from protease-mediated degradation.

Molecular cloning of the cDNA encoding the carboxy-terminal domain of chimpanzee apolipoprotein(a): an Asp57 --> Asn mutation in kringle IV-10 is associated with poor fibrin binding.

Insight into the structural features of human lipoprotein(a) [Lp(a)] which underlie its functional implication in fibrinolysis may be gained from comparative studies of apo(a). Indeed, cloning of rhesus monkey apo(a) has shown that a Trp72 --> Arg mutation in the lysine-binding site (LBS) of KIV-10 leads to loss of lysine-binding properties of the rhesus Lp(a) particle. Consequently, comparative studies of apo(a) sequences in different Old World monkey species should further our understanding of the molecular role of Lp(a) in the fibrinolytic process. In contrast to other Old World monkeys, including rhesus monkey, cynomolgus, and baboon, the chimpanzee exhibits an elevated level of Lp(a) and a distinct isoform distribution as compared to humans [Doucet et al. J. Lipid Res. (1994) 35, 263-270]. Clearly then, the chimpanzee is an interesting animal model for study of the structure, function, and potential pathophysiological roles of Lp(a). We have cloned and sequenced the region of chimpanzee apo(a) cDNA spanning KIV-3 to the stop codon. The global organization of this region is similar to that of human apo(a) with the presence of KV, which is absent in rhesus monkey apo(a). Nucleotide sequence comparison indicates a variation of 1.4% between chimpanzee and man and 5.1% between chimpanzee and rhesus monkey. The differences concerned single base changes. An Asp57 --> Asn mutation was detected in KIV-10; this residue is critical to the LBS of KIV-10 in human apo(a). To verify that the Asp57 --> Asn substitution was specific to apo(a), we have also cloned the cDNA-encoding plasminogen, which exhibited an Asp at the corresponding position in kringle IV. Using an in vitro binding assay, we have demonstrated that chimpanzee Lp(a) exhibits poor lysine-specific interaction with both intact and plasmin-degraded fibrin as compared to its human counterpart. We propose that the Asn57 substitution in KIV-10 of chimpanzee apo(a) is responsible for this property. Chimpanzee Lp(a) therefore represents an appropriate particle with which to explore the potential effects of Lp(a) on the fibrinolytic system, such as the inhibition of plasminogen activation or inhibition of t-PA activity.

Identification of two functionally distinct lysine-binding sites in kringle 37 and in kringles 32-36 of human apolipoprotein(a).

The well documented association between high plasma levels of lipoprotein(a) (Lp(a)) and cardiovascular disease might be mediated by the lysine binding of apolipoprotein(a) (apo(a)), the plasminogen-like, multikringle glycoprotein in Lp(a). We employed a mutational analysis to localize the lysine-binding domains within human apo(a). Recombinant apo(a) (r-apo(a)) with 17 plasminogen kringle IV-like domains, one plasminogen kringle V-like domain, and a protease domain or mutants thereof were expressed in the human hepatocarcinoma cell line HepG2. The lysine binding of plasma Lp(a) and r-apo(a) in the culture supernatants of transfected HepG2 cells was analyzed by lysine-Sepharose affinity chromatography. Wild type recombinant Lp(a) (r-Lp(a)) revealed lysine binding in the range observed for human plasma Lp(a). A single accessible lysine binding site in Lp(a) is indicated by a complete loss of lysine binding observed for r-Lp(a) species that contain either a truncated r-apo(a) lacking kringle IV-37, kringle V, and the protease or a point-mutated r-apo(a) with a Trp-4174-->Arg substitution in the putative lysine-binding pocket of kringle IV-37. Evidence is also presented for additional lysine-binding sites within kringles 32-36 of apo(a) that are masked in Lp(a) as indicated by an increased lysine binding for the point mutant (Cys-4057-->Ser), which is unable to assemble into particles. An important role of these lysine-binding site(s) for Lp(a) assembly is suggested by a decreased assembly efficiency for deletion mutants lacking either kringle 32 or kringles 32-35.

Calorimetric and spectroscopic studies of the interaction of Manduca sexta apolipophorin III with zwitterionic, anionic, and nonionic lipids.

The nature of the interaction of apolipophorin III (apoLp-III) from the insect Manduca sexta with a variety of zwitterionic and anionic phospholipids and with several nonionic glycolipids was investigated by differential scanning calorimetry (DSC) and 31P-NMR spectroscopy. Monoglucosyldiacylglycerol, phosphatidylethanolamine, and phosphatidic acid (pH 7.2) appear to exhibit the weakest interaction with apoLp-III. DSC studies revealed that the gel/liquid-crystalline phase transition of these lipids is little affected by the binding of apoLp-III. Diglucosyldiacylglycerol, phosphatidylcholine (PC), phosphatidylserine, and phosphatidic acid (pH congruent to 8.8) seem to exhibit somewhat stronger interactions with apoLp-III. The binding of apoLp-III to these lipids induces the formation of lipid domains which melt less cooperatively and at higher temperatures than do the pure lipid dispersions, while having little effect on the melting enthalpy of lipid hydrocarbon chains. Phosphatidylglycerol (PG) and phosphatidic acid (pH > 9.3) appear to exhibit the strongest interactions with apoLp-III. The binding of apoLp-III to these lipids severely disrupts their bilayer structure, resulting in marked reductions in the cooperativity and enthalpy of the gel/liquid-crystalline phase transition of the lipids. Studies of binary mixtures of PC and PG indicate that such bilayer-disrupting interactions only occur in the presence of nonphysiologically high concentrations of PG. The binding of apoLp-III to binary mixtures of diacylglycerol and zwitterionic phospholipid has little effect on the chain-melting or the bilayer/nonbilayer phase transitions of these lipids, but it appears to promote the retention of water at the surface of the lipid aggregate. Our results indicate that the binding of apoLp-III to lipid bilayers is mediated primarily through polar and/or ionic interactions at the lipid bilayer surface. Our results also suggest that the interaction of apoLp-III with lipid bilayers promotes the hydration of their surfaces, a property which is consistent with the proposed in vivo functions of this protein.