BBA - Molecular and Cell Biology of Lipids (v.1801, #2)

The mammalian pineal gland is a prominent secretory organ with a high metabolic activity. Melatonin (N-acetyl-5-methoxytryptamine), the main secretory product of the pineal gland, efficiently scavenges both the hydroxyl and peroxyl radicals counteracting lipid peroxidation in biological membranes. Approximately 25% of the total fatty acids present in the rat pineal lipids are represented by arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3). These very long chain polyunsaturated fatty acids play important roles in the pineal gland. In addition to the production of melatonin, the mammalian pineal gland is able of convert these polyunsaturated fatty acids into bioactive lipid mediators. Lipoxygenation is the principal lipoxygenase (LOX) activity observed in the rat pineal gland. Lipoxygenation in the pineal gland is exceptional because no other brain regions express significant LOX activities under normal physiological conditions. The rat pineal gland expresses both 12- and 15-lipoxygenase (LOX) activities, producing 12- and 15-hydroperoxyeicosatetraenoic acid (12- and 15-HpETE) from arachidonic acid and 14- and 17-hydroxydocosahexaenoic acid (14- and 17-HdoHE) from docosahexaenoic acid, respectively. The rat pineal also produces hepoxilins via LOX pathways. The hepoxilins are bioactive epoxy-hydroxy products of the arachidonic acid metabolism via the 12S-lipoxygenase (12S-LOX) pathway. The two key pineal biochemical functions, lipoxygenation and melatonin synthesis, may be synergistically regulated by the status of n-3 essential fatty acids.
Keywords: Pineal gland; Arachidonic acid; Docosahexaenoic acid; Lipoxygenation; Hepoxilin; Melatonin;

Apolipoprotein M (apoM) is a novel apolipoprotein belonging to the lipocalin protein superfamily. In humans, the apoM gene is mainly expressed in the liver and kidney, where it is highly regulated by transcription factors, hormones, and cytokines. Recent studies have shown that apoM expression is reduced in some patients with diabetes and that it has a paradoxical effect on atherosclerosis. Here, we review the progress made in understanding the structure, distribution, and regulation of apoM and its potential role in lipoprotein metabolism, diabetes, and atherosclerosis.
Keywords: ApoM; HDL; Cholesterol; Transcription factor; Hormones; Cytokines; Diabetes; Atherosclerosis;

NDRG2 induced by oxidized LDL in macrophages antagonizes growth factor productions via selectively inhibiting ERK activation by Shumei Liu; Pengyuan Yang; Hui Kang; Ling Lu; Yuefan Zhang; Jingwei Pan; Yao-Cheng Rui (106-113).
During atherogenesis, macrophage foam cells produce prodigious growth factors, cytokines, and chemokines, which play the central roles in inflammatory process in atherosclerotic plaque formation. In the present study, we identified a new protein marker, N-Myc downstream-regulated protein 2 (NDRG2), which is significantly up-regulated in oxidized low density lipoprotein (oxLDL) treated macrophages and in human atherosclerotic plaques. Over-expression and siRNA knockdown studies showed that NDRG2 is a negative regulator of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) productions in macrophages. Furthermore, we investigated the effects of NDRG2 on MAPK signal activation. Our results showed ERK1/2 activation, but not P38 or JNK1/2 activation, is responsible for regulation of NDRG2 on VEGF and PDGF productions. Consistent with the PDGF levels, the vascular smooth muscle cell (VSMC) proliferation was also regulated by the conditional medium of the oxLDL treated macrophages with NDRG2 knockdown or over-expression. Neutralizing anti-PDGF antibody can significantly inhibit the enhanced VSMC proliferation by macrophage medium with NDRG2 knockdown. Our present results demonstrate that NDRG2 participates in oxLDL-induced macrophage activation and modulates ERK1/2-dependent PDGF and VEGF production, which has potential application in atherogenesis.
Keywords: N-Myc downstream-regulated protein 2; Oxidized low density lipoprotein; Macrophage; Smooth muscle cell; Platelet-derived growth factor; Vascular endothelial growth factor; MAPK signal;

There is accumulating evidence that LDL oxidation is essential for atherogenesis and antioxidants that prevent oxidation may either decelerate or reduce atherogenesis. Current study focused on the effect and mechanism of 3′,4′-dihydroxy-5,6,7,8-tetramethoxyflavone (DTF), a major metabolite of nobiletin (NOB, a citrus polymethoxylated flavone) on atherogenesis. We found DTF had stronger inhibitory activity than α-tocopherol on inhibiting Cu2+-mediated LDL oxidation measured by thiobarbituric acid-reactive substances assay (TBARS), conjugated diene formation and electrophoretic mobility. Monocyte-to-macrophage differentiation plays a vital role in early atherogenesis. DTF (10–20 μM) dose-dependently attenuated differentiation along with the reduced gene expression of scavenger receptors, CD36 and SR-A, in both PMA- and oxidized low-density lipoprotein (oxLDL)-stimulated THP-1 monocytes. Furthermore, DTF treatment of monocytes and macrophages led to reduction of fluorescent DiI-acLDL and DiI-oxLDL uptake. In conclusion, at least three mechanisms are at work in parallel: DTF reduces LDL oxidation, attenuates monocyte differentiation into macrophage and blunts uptake of modified LDL by macrophage. The effect is different from that of NOB, from which DTF is derived. This study thus significantly enhanced our understanding on how DTF may be beneficial against atherogenesis.
Keywords: Low-density lipoprotein (LDL); Atherogenesis; Scavenger receptor A (SR-A); CD36;

Atlantic salmon (Salmo salar) muscle precursor cells differentiate into osteoblasts in vitro: Polyunsaturated fatty acids and hyperthermia influence gene expression and differentiation by Elisabeth Ytteborg; Anne Vegusdal; P. Eckhard Witten; Gerd Marit Berge; Harald Takle; Tone-Kari Østbye; Bente Ruyter (127-137).
The formation and mineralisation of bone are two critical processes in fast-growing Atlantic salmon (Salmo salar). The mechanisms of these processes, however, have not been described in detail. Thus, in vitro systems that allow the study of factors that influence bone formation in farmed Atlantic salmon are highly warranted. We describe here a method by which unspecialised primary cells from salmon white muscle can differentiate to osteoblasts in vitro. We have subsequently used the differentiated cells as a model system to study the effects of two factors that influence bone formation in Atlantic salmon under commercial farming conditions, namely polyunsaturated fatty acids, PUFAs, and temperature. Muscle precursor cells changed their morphology from triangular or spindle-shaped cells to polygonal or cubical cells after 3 weeks in osteogenic medium. In addition, gene expression studies showed that marker genes for osteoblastogenesis; alp, col1a1, osteocalcin, bmp2 and bmp4 increased after 3 weeks of incubation in osteogenic media showing that these cells have differentiated to osteoblasts at this stage. Adding CLA or DHA to the osteoblast media resulted in a reduced PGE2 production and increased expression of osteocalcin. Further, temperature studies showed that differentiating osteoblasts are highly sensitive to increased incubation temperature at early stages of differentiation. Our studies show that unspecialised precursor cells isolated from salmon muscle tissue can be caused to differentiate to osteoblasts in vitro. Furthermore, this model system appears to be suitable for the study of osteoblast biology in vitro.
Keywords: Atlantic salmon; Differentiation; Hyperthermia; In vitro; Osteoblast; PUFA;

Effect of gallbladder hypomotility on cholesterol crystallization and growth in CCK-deficient mice by Helen H. Wang; Piero Portincasa; Min Liu; Patrick Tso; Linda C. Samuelson; David Q.-H. Wang (138-146).
We investigated the effect of gallbladder hypomotility on cholesterol crystallization and growth during the early stage of gallstone formation in CCK knockout mice. Contrary to wild-type mice, fasting gallbladder volumes were enlarged and the response of gallbladder emptying to a high-fat meal was impaired in knockout mice on chow or the lithogenic diet. In the lithogenic state, large amounts of mucin gel and liquid crystals as well as arc-like and tubular crystals formed first, followed by rapid formation of classic parallelogram-shaped cholesterol monohydrate crystals in knockout mice. Furthermore, three patterns of crystal growth habits were observed: proportional enlargement, spiral dislocation growth, and twin crystal growth, all enlarging solid cholesterol crystals. At day 15 on the lithogenic diet, 75% of knockout mice formed gallstones. However, wild-type mice formed very little mucin gel, liquid, and solid crystals, and gallstones were not observed. We conclude that lack of CCK induces gallbladder hypomotility that prolongs the residence time of excess cholesterol in the gallbladder, leading to rapid crystallization and precipitation of solid cholesterol crystals. Moreover, during the early stage of gallstone formation, there are two pathways of liquid and polymorph anhydrous crystals evolving to monohydrate crystals and three modes for cholesterol crystal growth.
Keywords: Bile; Bile flow; Bile salt; Cholesterol monohydrate crystal; Cholesterol nucleation; Mucin;

In the present study we have applied a novel form of Total Internal Reflection Fluorescence Microscopy (LG-TIRFM) in combination with fluorescently labeled cholera toxin to the study of lipid rafts dynamics in living cells. We demonstrate the usefulness of such approach by showing the dynamic formation/disaggregation of islands of cholera toxin on the surface of cells. Using multicolor LG-TIRFM with co-localization studies we show for the first time that two receptors previously identified as constituents of lipid rafts are found on different and independent “raft domains” on the cell plasma membrane. Furthermore, LG-TIRFM studies revealed limited association and dissociation of both domains overtime on different areas of the plasma membrane. The implications of different “raft domains” on cell physiology are discussed.
Keywords: TIRFM; Lipid raft; p75 receptor; Decay accelerating factor; Cholera toxin; Imaging;

Interactions of oxidosqualene cyclase (Erg7p) with 3-keto reductase (Erg27p) and other enzymes of sterol biosynthesis in yeast by S. Taramino; M. Valachovic; S. Oliaro-Bosso; F. Viola; B. Teske; M. Bard; G. Balliano (156-162).
In Saccharomyces cerevisiae and Candida albicans, two enzymes of the ergosterol biosynthetic pathway, oxidosqualene cyclase (Erg7p) and 3-keto reductase (Erg27p) interact such that loss of the 3-keto reductase also results in a concomitant loss of activity of the upstream oxidosqualene cyclase. This interaction wherein Erg27p has a stabilizing effect on Erg7p was examined to determine whether Erg7p reciprocally has a protective effect on Erg27p. To this aim, three yeast strains each lacking the ERG7 gene were tested for 3-ketoreductase activity by incubating either cells or cell homogenates with unlabeled and radiolabeled 3-ketosteroids. In these experiments, the ketone substrates were effectively reduced to the corresponding alcohols, providing definitive evidence that oxidosqualene cyclase is not required for the 3-ketoreductase activity. This suggests that, in S. cerevisiae, the protective relationship between the 3-keto reductase (Erg27p) and oxidosqualene cyclase (Erg7p) is not reciprocal. However, the absence of the Erg7p, appears to affect other enzymes of sterol biosynthesis downstream of lanosterol formation. Following incubation with radiolabeled and non-radiolabeled 3-ketosteroids we detected differences in hydroxysteroid accumulation and ergosterol production between wild-type and ERG7 mutant strains. We suggest that oxidosqualene cyclase affects Erg25p (C-4 sterol oxidase) and/or Erg26p (C-3 sterol dehydrogenase/C-4 decarboxylase), two enzymes that, in conjunction with Erg27p, are involved in C-4 sterol demethylation.
Keywords: Saccharomyces cerevisiae; Ergosterol; Erg7; Erg27; 3-keto reductase; Oxidosqualene cyclase; Protein-protein interaction;

Remodeling of phosphatidylglycerol in Synechocystis PCC6803 by Hajnalka Laczko-Dobos; Petr Fryčák; Bettina Ughy; Ildiko Domonkos; Hajime Wada; Laszlo Prokai; Zoltan Gombos (163-170).
The phosphatidylglycerol deficient ΔpgsA mutant of Synechocystis PCC6803 provided a unique experimental system for investigating in vivo retailoring of exogenously added dioleoylphosphatidylglycerol in phosphatidylglycerol-depleted cells. Gas chromatographic analysis of fatty acid composition suggested that diacyl-phosphatidylglycerols were synthesized from the artificial synthetic precursor. The formation of new, retailored lipid species was confirmed by negative-ion electrospray ionization–Fourier-transform ion cyclotron resonance and ion trap tandem mass spectrometry. Various isomeric diacyl-phosphatidylglycerols were identified indicating transesterification of the exogenously added dioleoylphosphatidyl-glycerol at the sn-1 or sn-2 positions. Polyunsaturated fatty acids were incorporated selectively into the sn-1 position. Our experiments with Synechocystis PCC6803/ΔpgsA mutant cells demonstrated lipid remodeling in a prokaryotic photosynthetic bacterium. Our data suggest that the remodeling of diacylphosphatidylglycerol likely involves reactions catalyzed by phospholipase A1 and A2 or acyl-hydrolase, lysophosphatidylglycerol acyltransferase and acyl-lipid desaturases.
Keywords: Phosphatidylglycerol; Remodeling; Membrane lipid; Photosynthetic organism; Mass spectrometry; Synechocystis PCC6803;

Ischemia–reperfusion alters cardiac lipoprotein lipase by Thomas Pulinilkunnil; Prasanth Puthanveetil; Min Suk Kim; Fang Wang; Veronika Schmitt; Brian Rodrigues (171-175).
Ischemia–reperfusion (I/R) is associated with changes in energy metabolism in the heart. However, the majority of studies have focused on examining rates and extent of fatty acid (FA) oxidation, with limited emphasis on FA delivery. We examined the influence of acute myocardial I/R on coronary lipoprotein lipase (LPL), the key enzyme responsible for triglyceride-lipoprotein hydrolysis and FA delivery to the heart. In a whole animal and an ex vivo model of I/R, we demonstrate increases in luminal LPL activity, an effect that involved signaling through nitric oxide. Given the damaging effect of excess FA utilization by the ischemic heart, strategies to restrict LPL at the vascular lumen would be an attractive therapeutic option in limiting I/R related cardiac injury.
Keywords: Ischemia; Reperfusion; Myocytes; NOS; LPL;

Lipoprotein-associated phospholipase A2 decreases oxidized lipoprotein cellular association by human macrophages and hepatocytes by Ming Yang; Eugene M. Chu; Muriel J. Caslake; Celina Edelstein; Angelo M. Scanu; John S. Hill (176-182).
We investigated whether the presence of endogenous or exogenous lipoprotein-associated phospholipase A2 (Lp-PLA2) can modify the cellular association of oxidized low density lipoprotein (oxLDL) and oxidized lipoprotein(a) (oxLp(a)) by human monocyte-derived macrophages (MDM) and hepatocytes (HepG2). Purified recombinant Lp-PLA2 was used as a source of exogenous enzyme whereas Pefabloc (serine esterase inhibitor) was used to inhibit the endogenous Lp-PLA2 activity associated with isolated lipoproteins. Cellular association studies were performed with DiI-labeled oxLDL or oxLp(a) and human monocyte-derived macrophages and HepG2 cells. Active Lp-PLA2 decreased the cellular association of oxLDL and oxLp(a) in macrophages and HepG2 cells by approximately 30–40%, whereas the inactive enzyme did not significantly change oxidized lipoprotein cellular association by either cell type. OxLDL pretreated by Pefabloc increased oxLDL cellular association by MDM and HepG2 cells compared to untreated oxLDL. Therefore, unlike some lipases, Lp-PLA2 did not appear to have any catalytic independent function in oxLDL cellular association. To assess whether the reduced cellular association mediated by Lp-PLA2 was due to the hydrolysis of oxidized phosphatidylcholine (oxPC), we measured the concentration of lysophosphatidylcholine (lysoPC) in lipoprotein fractions after Lp-PLA2 treatment. LysoPC was increased by 20% (0.4 μM) and 87% (0.7 μM) by active Lp-PLA2 compared to inactive Lp-PLA2 for oxLDL and Lp(a), respectively. LysoPC at higher concentration dose-dependently increased the cellular association of oxLDL and oxLp(a) in MDM and HepG2 cells. We conclude that Lp-PLA2 mediates a decrease in oxidized lipoprotein cellular association in human macrophages and HepG2 cells by reducing the concentration of oxPC within these lipoproteins.
Keywords: Lipoprotein-associated phospholipase A2; Platelet-activating factor acetylhydrolase; Macrophage; HepG2; Oxidized LDL; Lipoprotein(a);

Primary hypercholesterolaemia impairs glucose homeostasis and insulin secretion in low-density lipoprotein receptor knockout mice independently of high-fat diet and obesity by Maria Lúcia Bonfleur; Emerielle Cristine Vanzela; Rosane Aparecida Ribeiro; Gabriel de Gabriel Dorighello; Carolina Prado de França Carvalho; Carla Beatriz Collares-Buzato; Everardo Magalhães Carneiro; Antonio Carlos Boschero; Helena Coutinho Franco de Oliveira (183-190).
We investigated whether primary hypercholesterolaemia per se affects glucose homeostasis and insulin secretion in low-density lipoprotein receptor knockout mice (LDLR−/−). Glucose plasma levels were increased and insulin decreased in LDLR−/− compared to the wild-type mice. LDLR−/− mice presented impaired glucose tolerance, but normal whole body insulin sensitivity. The dose–response curve of glucose-stimulated insulin secretion was shifted to the right in LDLR−/− islets. Significant reductions in insulin secretion in response to l-leucine or 2-ketoisocaproic acid were also observed in LDLR−/−. Islet morphometric parameters, total insulin and DNA content were similar in both groups. Glucose uptake and oxidation were reduced in LDLR−/− islets. Removal of cholesterol from LDLR−/− islets corrected glucose-stimulated insulin secretion. These results indicate that enhanced membrane cholesterol content due to hypercholesterolaemia leads to a lower insulin secretion and glucose intolerance without affecting body insulin sensitivity. This represents an additional risk factor for diabetes and atherosclerosis in primary hypercholesterolaemia.
Keywords: Cholesterol; Hypercholesterolaemia; LDL receptor; Pancreatic islet; Insulin secretion; Glucose homeostasis;

Facilitation of fatty acid uptake by CD36 in insulin-producing cells reduces fatty-acid-induced insulin secretion and glucose regulation of fatty acid oxidation by Tina Wallin; Zuheng Ma; Hirotaka Ogata; Ingrid Hals Jørgensen; Mariella Iezzi; Haiyan Wang; Claes B. Wollheim; Anneli Björklund (191-197).
Facilitation of fatty acid uptake in beta cells could potentially affect beta cell metabolism and secretory function; however such effects have not been clearly documented. CD36 facilitates uptake of fatty acids (FA) in muscle and adipose tissue and is likely to exert a similar effect in beta cells. We investigated the impact of over-expressing CD36 on fatty acid uptake and beta cell function by a Tet-on system in INS-1 cells. Doxycycline dose-dependently increased the CD36 protein with localization mainly in the cell membrane. Over-expression increased both specific uptake and efflux of oleate whereas intracellular glycerides were only marginally increased and incorporation of 14C-oleate or -palmitate into di- or triglycerides not affected. The normal potentiation of glucose-induced insulin secretion by acute addition of FA (50–100 μmol/l oleate and palmitate) was lost and the normal inhibitory effect of high glucose both on oleate oxidation and on the activity of carnitine palmitoyltransferase I was reduced. Over-expression did not induce apoptosis.We conclude that induction of the CD36 transporter increases uptake of FA, the consequences of which are blunting of the functional interplay between glucose and FA on insulin secretion and oxidative metabolism.
Keywords: Insulin secretion; CD36; INS-1; Palmitate; Oleate; Carnitine palmitoyltransferase I;

Scavenger receptor, Class B, Type I provides an alternative means for β-VLDL uptake independent of the LDL receptor in tissue culture by Clemens Röhrl; Stefanie Fruhwürth; Sabine Maria Schreier; Alfred Lohninger; Andrea Dolischka; Manfred Hüttinger; Nina Zemann; Marcela Hermann; Witta Strobl; Herbert Stangl (198-204).
Recent evidence suggests that scavenger receptor, class B, type I (SR-BI) plays a physiological role in VLDL metabolism. SR-BI was reported to mediate β-VLDL uptake; however, cellular details of this process are not well characterized. In the present study we show that SR-BI delivers cholesterol derived from β-VLDL to LDL receptor negative SR-BI over-expressing Chinese Hamster Ovarian cells (ldlA7-SRBI). Cell association of β-VLDL was ∼ 3 times higher after SR-BI over-expression, which was competed by β-VLDL, but only to a lesser extent by HDL and LDL. Almost all of the associated β-VLDL was located intracellularly, and therefore could not be released by a 50-fold excess of unlabeled β-VLDL. β-VLDL was degraded at a rate of 6 ng β-VLDL/mg cell protein and hour. In contrast to ldlA7 cells, β-VLDL association was competed by LDL in cells with a functional LDL receptor like CHO and HepG2 cells, indicating a strong impact of the LDL receptor in β-VLDL uptake. β-VLDL degradation was similar to ldlA7-SRBI cells. When β-VLDL uptake was followed using fluorescence microscopy, β-VLDL showed a different uptake pattern in SR-BI over-expressing cells, ldlA7-SRBI, compared to LDL receptor containing cells, CHO and HepG2.
Keywords: SR-BI; β-VLDL; LDL receptor; Chylomicrons; Cholesterol; Chinese Hamster Ovarian Cells; HepG2;