BBA - Molecular and Cell Biology of Lipids (v.1583, #2)

Role of hepatic lipase and scavenger receptor BI in clearing phospholipid/free cholesterol-rich lipoproteins in PLTP-deficient mice by Koichi Kawano; Shucun Qin; Claude Vieu; Xavier Collet; Xian-Cheng Jiang (133-140).
Phospholipid transfer protein knock-out (PLTP0) mice have defective transfer of phospholipids (PL) from triglyceride-rich lipoproteins (TRL) into high-density lipoproteins (HDL). In this study, we examined the role of diet, hepatic lipase (HL) and scavenger receptor BI (SRBI) in determining the accumulation of excess PL and free cholesterol (FC, “surface remnants”) in plasma of PLTP0 mice. PL and FC accumulated in the very low-density lipoprotein (VLDL)–LDL region of PLTP0 mice on a highly saturated, coconut oil-based diet, but not on chow or milk-fat based Western diets. Accumulation of PL and FC was dramatically increased in PLTP0/HL0 mice, compared to PLTP0 mice, but only on the coconut oil diet. Turnover studies indicated that the coconut oil diet was associated with delayed catabolism of PL of PL/FC-rich particles. Incubation of these particles with primary hepatocytes in the presence of SRBI neutralizing antibody indicated that SRBI was primarily responsible for removal of FC and PL on the Western diet. In hepatocytes of coconut oil-fed mice, removal of FC and PL from these particles by SRBI was markedly reduced, even though SRBI protein expression levels were unchanged. These studies indicate that HL and SRBI both have major role in the clearance of PL and FC of surface remnants in PLTP0 mice. SRBI appears to be dysfunctional in coconut oil diet-fed animals, possibly related to changes in hepatocyte membrane fatty acid composition.
Keywords: Hepatic lipase; Scavenger receptor BI; Phospholipid transfer protein;

High density lipoprotein (HDL) promotes reverse cholesterol transport from peripheral tissues to the liver where its cholesterol is secreted preferentially into bile. The scavenger receptor class B type I (SR-BI) is believed to play a pivotal role in unloading HDL cholesterol and its ester to hepatocytes. Here, using male SR-BI “att” mice with a dysfunctional mutation in the Sr-b1 promoter, we studied whether ∼50% of normal SR-BI expression influences gallstone susceptibility in these mice fed a lithogenic diet containing 1% cholesterol, 0.5% cholic acid and 15% butterfat. Our results showed that the disruption of SR-BI expression reduced cholesterol secretion by 37% in the chow-fed state and 10% on the lithogenic diet, and while delaying incidence slightly, did not influence cumulative susceptibility to cholesterol gallstones. The lithogenic diet induced marked increases in biliary cholesterol and phospholipid secretion rates but not of bile salts. Basal expression of hepatic SR-BI protein was dissimilar in both wild-type and SR-BI mice, and remained unaltered in response to the lithogenic diet. By two independent dual isotope methods, intestinal cholesterol absorption was unimpaired by attenuation of the SR-BI which also displays low-density expression on small intestinal enterocytes. We conclude that although HDL cholesterol is a principal source of biliary cholesterol in the basal state, uptake of cholesterol from chylomicron remnants appears to be the major contributor to biliary cholesterol hypersecretion during diet-induced cholelithogenesis in the mouse.
Keywords: Bile flow; Bile salt; Chylomicron; Intestinal cholesterol absorption; Lipoprotein; Phospholipid;

Use of vitamin D4 analogs to investigate differences in hepatic and target cell metabolism of vitamins D2 and D3 by Valarie Byford; Stephen Strugnell; Ruth Coldwell; Neil Schroeder; Hugh L.J Makin; Joyce C Knutson; Charles W Bishop; Glenville Jones (151-166).
In this study, we used molecules with either of the structural differences in the side chains of vitamin D2 and vitamin D3 to investigate which feature is responsible for the significant differences in their respective metabolism, pharmacokinetics and toxicity. We used two cell model systems—HepG2 and HPK1A-ras—to study hepatic and target cell metabolism, respectively. Studies with HepG2 revealed that the pattern of 24- and 26-hydroxylation of the side chain reported for 1α-hydroxyvitamin D2 (1α-OH-D2) but not for 1α-OH-D3 is also observed in both 1α-OH-D4 and Δ22-1α-OH-D3 metabolism. This suggests that the structural feature responsible for targeting the enzyme to the C24 or C26 site could be either the C24 methyl group or the 22–23 double bond. In HPK1A-ras cells, the pattern of metabolism observed for the 24-methylated derivative, 1α,25-(OH)2D4, was the same pattern of multiple hydroxylations at C24, C26 and C28 seen for vitamin D2 compounds without evidence of side chain cleavage observed for vitamin D3 derivatives, suggesting that the C24 methyl group plays a major role in this difference in target cell metabolism of D2 and D3 compounds. Novel vitamin D4 compounds were tested and found to be active in a variety of in vitro biological assays. We conclude that vitamin D4 analogs and their metabolites offer valuable insights into vitamin D analog design, metabolic enzymes and maybe useful clinically.
Keywords: 1α-hydroxyvitamin D2; 1α-hydroxyvitamin D4; Cytochrome P450; Vitamin D-dependent genes; 1α,25-dihydroxyvitamin D3;

Pro-oxidant and antioxidant potential of catecholestrogens against ferrylmyoglobin-induced oxidative stress by Rosa Martı́nez; Kristina Quintana; Rosaura Navarro; César Martı́n; M.Luisa Hernández; Igor Aurrekoetxea; José Ignacio Ruiz-Sanz; Mercedes Lacort; M.Begoña Ruiz-Larrea (167-175).
Ferryl heme proteins may play a major role in vivo under certain pathological conditions. Catecholestrogens, the estradiol-derived metabolites, can act either as antioxidants or pro-oxidants in iron-dependent systems. The aim of the present work was (1) to determine the effects of ferrylmyoglobin on hepatocyte cytotoxicity, and (2) to assess the pro/antioxidant potential of a series of estrogens (phenolic, catecholic and stilbene-derived) against ferrylmyoglobin induced lipid peroxidation in rat hepatocytes. Cells were exposed to metmyoglobin plus hydrogen peroxide to form ferrylmyoglobin in the presence of the transition metal chelator diethylentriaminepentaacetic acid. Results showed that ferrylmyoglobin induced an initial oxidative stress, mainly reflected in an early lipid peroxidation and further decrease in GSH and ATP. However, cells gradually adapted to this situation, by recovering the endogenous ATP and GSH levels at longer incubation times. Phenolic and stilbene-derived estrogens inhibited ferrylmyoglobin-induced lipid peroxidation to different degrees: diethylstilbestrol>estradiol>resveratrol. Catecholestrogens at concentrations higher than 1 μM also inhibited lipid peroxidation with similar efficacy. The ability of estrogens to reduce ferrylmyoglobin to metmyoglobin may account for their antioxidant activity. In contrast, physiological concentrations (100 pM–100 nM) of the catecholestrogens exerted pro-oxidant activities, 4-hydroxyestradiol being more potent than 2-hydroxyestradiol. The implications of these interactions should be considered in situations where local myoglobin or hemoglobin microbleeding takes place.
Keywords: Toxicity; Ferrylmyoglobin; Catecholestrogen; Lipid peroxidation; Antioxidant; Rat hepatocyte;

On-line EPR study of free radicals induced by peroxidase/H2O2 in human low-density lipoprotein by Donatella Pietraforte; Laura Turco; Elena Azzini; Maurizio Minetti (176-184).
The aim of this study was to use direct electron paramagnetic resonance (EPR) spectroscopy at 37 °C and spin trapping techniques to study radical species formed during horseradish peroxidase/H2O2-initiated low-density lipoprotein (LDL) oxidation. Using direct EPR, we obtained evidence for the formation not only of the α-tocopheroxyl radical but also of a protein radical(s), assigned to a tyrosyl radical(s) of apolipoprotein B-100 (apo B-100). Spin trapping with 2-methyl-2-nitrosopropane revealed (i) the formation of a mobile adduct with β-hydrogen coupling assigned to a lipid radical and (ii) a partially immobilised adduct detected in LDL as well as in apo B-100, assigned after proteolytic digestion to the trapping of a radical centred on a tertiary carbon atom of an aromatic residue, probably tyrosine. Our results support the hypothesis that radicals are initiators of the oxidative process, and show that their formation is an early event in peroxidase-mediated oxidation. We also tested the effects of resveratrol (RSV), a polyphenolic antioxidant present in red wine. Our data indicate that 1–10 μM RSV is able to accelerate α-tocopherol consumption, conjugated dienes formation and the decay kinetics of LDL-centred radicals. Since phenols are substrates for peroxidases, this result may be ascribed to a RSV-mediated catalysis of peroxidase activity.
Keywords: LDL; Horseradish peroxidase; Free radical; EPR; Resveratrol;

Phospholipase C inhibitors and prostaglandins differentially regulate phosphatidylcholine synthesis in rat renal papilla by Marı́a del Carmen Fernández-Tomé; Emir H.S Speziale; Norma B Sterin-Speziale (185-194).
Phosphatidylcholine (PC) is the most abundant phospholipid in mammalian cell membranes. Several lines of evidence support that PC homeostasis is preserved by the equilibrium between PC biosynthetic enzymes and phospholipases catabolic activities. We have previously shown that papillary synthesis of PC depends on prostaglandins (PGs) that modulate biosynthetic enzymes. In papillary tissue, under bradikynin stimulus, arachidonic acid (AA) mobilization (the substrate for PG synthesis) requires a previous phospholipase C (PLC) activation. Thus, in the present work, we study the possible involvement of PLC in PC biosynthesis and its relationship with PG biosynthetic pathway on the maintenance of phospholipid renewal in papillary membranes; we also evaluated the relevance of CDP-choline pathway enzymes compartmentalization. To this end, neomycin, U-73122 and dibutiryl cyclic AMP, reported as PLC inhibitors, were used to study PC synthesis in rat renal papilla. All the PLC inhibitors assayed impaired PC synthesis. PG synthesis was also blocked by PLC inhibitors without affecting cyclooxygenase activity, indicating a metabolic connection between both pathways. However, we found that PC biosynthesis decrease in the presence of PLC inhibitors was not a consequence of PG decreased synthesis, suggesting that basal PLC activity and PGs exert their effect on different targets of PC biosynthetic pathway. The study of PC biosynthetic enzymes showed that PLC inhibitors affect CTP:phosphocholine cytidylyltransferase (CCT) activity while PGD2 operates on CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT), both activities associated to papillary enriched-nuclei fraction. The present results suggest that renal papillary PC synthesis is a highly regulated process under basal conditions. Such regulation might occur at least at two different levels of the CDP-choline pathway: on the one hand, PLC operates on CCT activity; on the other, while PGs regulate CPT activity.
Keywords: Prostaglandin; Phospholipase C inhibitor; Neomycin; U-73122; Dibutiryl cyclic AMP; Phosphatidylcholine (PC); CTP:phosphocholine cytidylyltransferase (CCT); CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT);

The extent of mitochondrial and peroxisomal contribution to β-oxidation of 18-, 20- and 24-carbon n-3 and n-6 polyunsaturated fatty acids (PUFAs) in intact rat hepatocytes is not fully clear. In this study, we analyzed radiolabeled acid soluble oxidation products by HPLC to identify mitochondrial and peroxisomal oxidation of 24:5n-3, 18- and 20-carbon n-3 and n-6 PUFAs. Mitochondrial fatty acid oxidation produced high levels of ketone bodies, tricarboxylic acid cycle intermediates and CO2, while peroxisomal β-oxidation released acetate. Inhibition of mitochondrial fatty acid oxidation with 2-tetradecylglycidic acid (TDGA), high amounts of [14C]acetate from oxidation of 24:5n-3, 18- and 20-carbon PUFAs were observed. In the absence of TDGA, high amounts of [14C]-labeled mitochondrial oxidation products were formed from oxidation of 24:5n-3, 18- and 20-carbon PUFAs. With 18:1n-9, high amounts of mitochondrial oxidation products were formed in the absence of TDGA, and TDGA strongly suppressed the oxidation of this fatty acid. Data of this study indicated that a shift in the partitioning from mitochondrial to peroxisomal oxidation differed for each individual fatty acid and is a specific property of 24:5n-3, 18- and 20-carbon n-3 and n-6 PUFAs.[14C]22:6n-3 was detected with [3-14C]24:5n-3, but not with [1-14C]24:5n-3 as the substrate, while [14C]16:0 was detected with [1-14C]24:5n-3, but not with [3-14C]24:5n-3 as the substrate. Furthermore, the amounts of 14CO2 were similar when cells were incubated with [3-14C]24:5n-3 versus [1-14C]24:5n-3. These findings indicated that the proportion of 24:5n-3 oxidized in mitochondria was high, and that 24:5n-3 and 24:6n-3 were mostly β-oxidized only one cycle in peroxisomes.
Keywords: Fatty acid; Oxidation; Ketone body; Hepatocyte;

Expression and characterization of Ca2+-independent lipase from Bacillus pumilus B26 by Hyung Kwoun Kim; Hwa Jung Choi; Myung Hee Kim; Cheon Bae Sohn; Tae Kwang Oh (205-212).
A lipase-producing Bacillus pumilus strain (B26) was isolated from a soil sample collected in Korea. The cloned gene showed that the lipase B26 composed of a 34-amino-acid signal sequence and a 181-amino-acid mature part corresponding to a molecular mass (M r) of 19,225. Based on the M r and the protein sequence, the lipase B26 belongs to the lipase family I.4. The optimum temperature and pH of the purified enzyme were 35 °C and 8.5, respectively. The lipase B26 showed a ‘Ca2+-independent thermostability and catalytic activity’. These are novel properties observed for the first time in lipase B26 among all bacterial lipases and correspond with the suggestion that this enzyme had no Ca2+-binding motif around the catalytic His156 residue. This enzyme seems to be a true lipase based on the experimental results that it could hydrolyze various long-chain triglycerides (C14–C18) and triolein (C18:1) and that it showed a typical interfacial activation mechanism toward both tripropionin and p-nitrophenyl butyrate.
Keywords: Bacillus pumilus; Lipase; Ca2+-independent thermostability; Ca2+-independent catalytic activity;

Different interactions of egg-yolk phosphatidylcholine and sphingomyelin with detergent bile salts by Catharina P Nibbering; Peter M Frederik; Gerard P van Berge-Henegouwen; Henk A van Veen; Jan van Marle; Karel J van Erpecum (213-220).
To examine physical–chemical aspects of bile salt–phospholipid interactions that could contribute to preferential phosphatidylcholine (PC) secretion into bile, we have compared transitions between vesicles and micelles in model systems containing taurocholate (TC) and either egg-yolk PC (EYPC), egg-yolk sphingomyelin (EYSM), buttermilk SM (BMSM) or dipalmitoyl PC (DPPC). Phase transitions from micelles to vesicles were observed at 4-fold dilution of serially diluted EYPC/TC systems, but not earlier than at 16-fold dilution of SM/TC or DPPC/TC systems, indicating lower concentrations of the detergent required for micellization in the case of SM or DPPC. Cryo-transmission electron microscopy of phase transitions initiated by addition of TC to phospholipid vesicles revealed extremely long SM-containing intermediate structures, but shorter EYPC-containing intermediate structures. Again, larger amounts of bile salt were required to induce phase transitions in the case of EYPC compared to SM. Sizes of TC–phospholipid micelles increased progressively upon increasing phospholipid contents in the rank order: DPPC–TC<EYSM–TC<BMSM–TC<EYPC–TC, consistent with higher micellization concentrations in the case of EYPC. Micelles were also separated from vesicular phases in two-phase model systems composed with TC, both EYPC and EYSM and 0, 10, 20 or 30 mol% cholesterol, by ultracentrifugation and ultrafiltration of the supernatant. At increasing cholesterol contents, EYPC preferentially distributed into the micellar phase. In contrast, no preferential micellar EYPC distribution occurred in the absence of the sterol. These results indicate different structural arrangements of EYPC–TC micelles compared to SM–TC micelles and lower detergent concentrations required for micellization in the case of SM-containing vesicles.
Keywords: Canalicular membrane; Cholesterol; Phosphatidylcholine; Micelle; Sphingomyelin; Vesicle;

Effects of hydrophobic and hydrophilic bile salt mixtures on cholesterol crystallization in model biles by Niels G Venneman; Sebastiaan J Huisman; Antonio Moschetta; Gerard P vanBerge-Henegouwen; Karel J van Erpecum (221-228).
The hydrophilic bile salt ursodeoxycholate is frequently used to dissolve cholesterol gallstones. We have now quantitated crystallization as a function of bile salt hydrophobicity, phospholipid content, cholesterol saturation and total lipid concentration (TLCo). Methods: Crystallization in supersaturated model biles with low phospholipid contents (left two-phase-micelles and crystal-containing-zone) was assessed during 21 days by microscopy and chemical measurement of crystal mass. For model biles with higher phospholipid contents (central three-phase-micelles, vesicles and crystal-containing-zone), lipid distribution into various phases was determined by combined ultracentrifugation–filtration–dialysis methodology (Biochim. Biophys. Acta 1532 (2001) 15–27). Results: In the left two-phase zone, crystal numbers and masses were highest in case of more hydrophilic bile salt composition (TUDC 100%>TC/TUDC 70%/30%>TC 100%>TC/TDC 70%/30%>TDC 100%) and decreased with increasing phospholipid contents, lower TLCo and lower cholesterol saturation index (CSI). In contrast, in the presence of vesicles (three-phase zone), crystallization decreased at increasing bile salt hydrophilicity, with concomitant increased vesicular cholesterol solubilization. Conclusions: Presence of vesicular phases is a prerequisite for inhibition of cholesterol crystallization by tauroursodeoxycholate.
Keywords: Bile salt hydrophobicity; Bile salt; Cholesterol; Crystallization; Phosphatidylcholine; Ursodeoxycholate;

Cholesterol 7α-hydroxylase (cyp7a) mediates cholesterol elimination in the liver by catalyzing the first and rate-limiting step in the conversion of cholesterol into bile acids. Peroxisome proliferator-activated receptor α (PPARα; NR1C1) and liver X receptor α (LXRα; NR1H3) are two nuclear receptors that stimulate the murine Cyp7a1 gene. Here we report that co-expression of PPARα and LXRα in hepatoma cells abolishes the stimulation of Cyp7a1 gene promoter in response to their respective agonists. PPARα and LXRα form an atypical heterodimer that binds to two directly adjacent hexameric sequences localized within overlapping PPARα and LXRα response elements (termed Site I), antagonizing the interaction of PPARα:retinoid X receptor α (RXRα) or RXRα:LXRα with the Cyp7a1 gene promoter. Mutations within either hexameric sequences that specifically abolished LXRα:PPARα heterodimer binding to the murine Cyp7a1 Site I also relieved promoter inhibition. The LXRα:PPARα heterodimer may be important in coordinating the expression of genes that encode proteins involved in metabolism of fats and cholesterol.
Keywords: Bile acid; Fatty acid; Nuclear receptor; Oxysterol; Transcription factor;

Characterization of a new endogenous vitamin A metabolite by Carsten K Schmidt; Jutta Volland; Gerd Hamscher; Heinz Nau (237-251).
Here, we describe the discovery of a new major endogenous vitamin A metabolite with particularly high hepatic concentrations. This metabolite was isolated from mouse livers and was characterized as 9-cis-4-oxo-13,14-dihydro-retinoic acid (RA) based on mass spectral, ultraviolet, and nuclear magnetic resonance analyses. It was also detected in one human liver. To gain further insight into endogenous retinoid metabolism, mice were fed over a period of 14 days ad libitum with diets enriched with different amounts of retinyl palmitate [15,000, 45,000 or 150,000 international units (IU)/kg diet]. Higher retinyl palmitate amounts in the diet resulted surprisingly in a dose-dependent decrease in all-trans-RA levels in serum, kidney, and brain, whereas levels of 9-cis-4-oxo-13,14-dihydro-RA, retinol, and retinyl esters were dose-dependently elevated in serum, kidney, and liver. 13-cis-RA levels could be detected in serum, liver, and kidney, but were unaffected by the dietary vitamin A status. 9-cis-RA levels were below the detection limit of 0.2 ng/ml serum or 0.4 ng/g tissue. This study indicates that the oxidation at C4 of the cyclohexenyl ring, isomerization of the C9/C10 double bond, and reduction of the C13/C14 double bond are major endogenous metabolic pathways of vitamin A.
Keywords: Vitamin A metabolism; Retinoic acid; Retinoid; Mouse; Human; 9-cis-4-oxo-13,14-dihydro-retinoic acid;