BBA - Molecular and Cell Biology of Lipids (v.1771, #2)

LDL particle subspecies are distinct in their capacity to mediate free cholesterol efflux via the SR-BI/Cla-1 receptor by Morgan Tréguier; Martine Moreau; Andrei Sposito; M. John Chapman; Thierry Huby (129-138).
The human scavenger receptor SR-BI/Cla-1 promotes efflux of free cholesterol from cells to both high-density and low-density lipoproteins (HDL, LDL). SR-BI/Cla-1-mediated cholesterol efflux to HDL is dependent on particle size, lipid content and apolipoprotein conformation; in contrast, the capacity of LDL subspecies to accept cellular cholesterol via this receptor is indeterminate. Cholesterol efflux assays were performed with CHO cells stably transfected with Cla-1 cDNA. Expression of Cla-1 in CHO cells induced elevation in total cholesterol efflux to plasma, LDL and HDL. Such Cla-1-specific efflux was abrogated by addition of anti-Cla-1 antibody. LDL were fractionated into five subspecies either on the basis of hydrated density or size. Among LDL subfractions, small dense LDL (sdLDL) were 1.5-to 3-fold less active acceptors for Cla-1-mediated cellular cholesterol efflux. Equally, sdLDL markedly reduced Cla-1-specific cholesterol efflux to large buoyant LDL in a dose-dependent manner. Conversely, sdLDL did not influence efflux to HDL2. These findings provide evidence that LDL particles are heterogeneous in their capacity to promote Cla-1-mediated cholesterol efflux. Relative to HDL2, large buoyant LDL may constitute physiologically-relevant acceptors for cholesterol efflux via Cla-1.
Keywords: SR-BI; Cholesterol efflux; LDL subfractions;

Modulation of apoptotic signalling by 9-hydroxystearic acid in osteosarcoma cells by N. Calonghi; E. Pagnotta; C. Parolin; C. Molinari; C. Boga; F. Dal Piaz; G.L. Brusa; M.A. Santucci; L. Masotti (139-146).
9-hydroxystearic acid (9-HSA) belongs to the class of endogenous lipid peroxidation by-products that greatly diminish in tumors, causing as a consequence the loss of one of the control mechanisms on cell division. We have previously shown that 9-HSA controls cell growth and differentiation by inhibiting histone deacetylase 1 (HDAC1) activity. In this paper our attention has not only been focused on HDAC1 inhibition but also on the hyperacetylation of other substrates such as p53, that is involved in inducing cell cycle arrest and/or apoptosis, and whose activity and stability are known to be regulated by posttranslational modifications, particularly by acetylation at the C-terminus region. 9-HSA administration to U2OS, an osteosarcoma cell line p53 wt, induces a growth arrest of the cells in G2/M and apoptosis via a mitochondrial pathway. In particular hyperacetylation of p53 induced by the HDAC1 inhibitory activity of 9-HSA has been demonstrated to increase Bax synthesis both at the transcriptional and the translational level. The subsequent translocation of Bax to the mitochondria is associated to a significant increase in caspase 9 activity. Our data demonstrate that the effects of 9-HSA on U2OS correlate with posttranslational modifications of p53.
Keywords: 9-hydroxystearic acid; p53; Apoptosis; Histone deacetylase inhibitors; Mass spectrometry;

Palmitate and oleate have distinct effects on the inflammatory phenotype of human endothelial cells by Jolita Ciapaite; Jan van Bezu; Gerco van Eikenhorst; Stephan J.L. Bakker; Tom Teerlink; Michaela Diamant; Robert J. Heine; Klaas Krab; Hans V. Westerhoff; Casper G. Schalkwijk (147-154).
Free fatty acids may create a state of continuous and progressive damaging to the vascular wall manifested by endothelial dysfunction. In this study we determine the mechanisms by which fatty acids palmitate (C16:0) and oleate (C18:1) affect intracellular long chain acyl-CoA (LCAC) content, energy metabolism, cell survival and proliferation and activation of NF-κB in cultured endothelial cells. A 48-h exposure of human umbilical vein endothelial cells (HUVEC) to 0.5 mM palmitate or 0.5 mM oleate increased total long chain acyl-CoA (LCAC) content 1.7 and 2 fold, respectively and decreased ATPtotal/ADPtotal ratio by 26 ± 5% (mean ± SEM) and 15 ± 2%, respectively, which was prevented by the acyl-CoA synthetase inhibitor triacsin C. Furthermore, palmitate inhibited cell proliferation by 34 ± 5%, while oleate stimulated it by 12 ± 2%. α-Tocopherol fully and triacsin C partially abolished the effect of palmitate on cell proliferation. Palmitate and oleate increased caspase-3 activity 3.2 and 1.4 fold, respectively. Palmitate-induced caspase-3 activation was prevented by triacsin C and slightly reduced by α-tocopherol and by the de novo ceramide synthesis inhibitor fumonisin B1. Both fatty acids induced antioxidant-sensitive nuclear translocation of NF-κB after 72 h, but not after 48 h. In conclusion, we showed that fatty acids influence different aspects of HUVEC function resulting in amongst other activation of apoptotic and inflammatory pathways. Our results indicate that the effects depend on the fatty acid type and may be related to accumulation of LCAC.
Keywords: Palmitate; Oleate; Endothelium; Energy metabolism; Inflammatory phenotype;

Differential behavior of sPLA2-V and sPLA2-X in human neutrophils by I. Solodkin-Szaingurten; R. Levy; N. Hadad (155-163).
Neutrophils and differentiated PLB-985 cells contain various types of PLA2s including the 85 kDa cytosolic PLA2 (cPLA2), Ca2+-independent PLA2 (iPLA2) and secreted PLA2s (sPLA2s). The present study focuses on the behavior of sPLA2s in neutrophils and PLB cells and their relationship to cPLA2α. The results of the present research show that the two types of sPLA2 present in neutrophils, sPLA2-V and sPLA2-X, which are located in the azurophil granules, are differentially affected by physiological stimuli. While sPLA2-V is secreted to the extacellular milieu, sPLA2-X is detected on the plasma membranes after stimulation. Stimulation of neutrophils with formyl-Met–Leu–Phe (fMLP), opsonized zymosan (OZ) or A23187 resulted in a different kinetics of sPLA2 secretion as detected by its activity in the neutrophil supernatants. Neutrophil priming by inflammatory cytokines or LPS enhanced sPLA2 activity detected in the supernatant after stimulation by fMLP. This increased activity was due to increased secretion of sPLA2-V to the supernatant and not to release of sPLA2-X. sPLA2 in granulocyte-like PLB cells exhibit identical characteristics to neutrophil sPLA2, with similar activity and optimal pH of 7.5. Granulocyte-like cPLA2α-deficient PLB cells serve as a good model to study whether sPLA2 activity is regulated by cPLA2α. Secretion and activity of sPLA2 were found to be similar in granulocyte-like PLB cells expressing or lacking cPLA2α, indicating that they are not under cPLA2α regulation.
Keywords: Neutrophils; PLB-985 cells; Granulocyte-like PLB cell; Secreted PLA2;

c-Jun N-terminal protein kinase signalling pathway mediates lovastatin-induced rat brain neuroblast apoptosis by Maria Isabel Cerezo-Guisado; Alberto Álvarez-Barrientos; Ricardo Argent; Luis Jesús García-Marín; Maria Julia Bragado; Maria Jesús Lorenzo (164-176).
We have previously shown that lovastatin, an HMG-CoA reductase inhibitor, induces apoptosis in rat brain neuroblasts. c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) are implicated in regulation of neuronal apoptosis. In this work, we investigated the role of JNK and p38 MAPK in neuroblast apoptosis induced by lovastatin. We found that lovastatin induced the activation of JNK, but not p38 MAPK. It also induced c-Jun phosphorylation with a subsequent increase in activator protein-1 (AP-1) binding, AP-1-mediated gene expression and BimEL protein levels. The effects of lovastatin were prevented by mevalonate. Pre-treatment with iJNK-I (a selective JNK inhibitor) prevented the effect of lovastatin on both neuroblast apoptosis and the activation of the JNK cascade. Furthermore, we found that the activation of the JNK signalling pathway triggered by lovastatin is accompanied by caspase-3 activation which is also inhibited by iJNK-I pre-treatment. Finally, a specific inhibitor of p38 MAPK, SB203580, had no effect on lovastatin-induced neuroblast apoptosis. Taken together, our data suggest that the activation of the JNK/c-Jun/BimEL signalling pathway plays a crucial role in lovastatin-induced neuroblast apoptosis. Our findings may also contribute to elucidate the intracellular mechanisms involved in the central nervous system side effects associated with statin therapy.
Keywords: Apoptosis; Statins; Nervous system; JNK; p38 MAPK; Bim;

Effects of rosiglitazone and high fat diet on lipase/esterase expression in adipose tissue by Wen-Jun Shen; Shailja Patel; Zaixin Yu; Dyron Jue; Fredric B. Kraemer (177-184).
A number of intracellular lipase/esterase have been reported in adipose tissue either by functional assays of activity or through proteomic analysis. In the current work, we have studied the relative expression level of 12 members of the lipase/esterase family that are found in white adipose tissue. We found that the relative mRNA levels of ATGL and HSL are the most abundant, being 2–3 fold greater than TGH or ADPN; whereas other intracellular neutral lipase/esterases were expressed at substantially lower levels. High fat feeding did not alter the mRNA expression levels of most lipase/esterases, but did reduce CGI-58 and WBSCR21. Likewise, rosiglitazone treatment did not alter the mRNA expression levels of most lipase/esterases, but did increase ATGL, TGH, CGI-58 and WBSCR21, while reducing ADPN. WAT from HSL−/− mice showed no compensatory increase in any lipase/esterases, rather mRNA levels of most lipase/esterases were reduced. In contrast, BAT from HSL−/− mice showed an increase in ATGL expression, as well as a decrease in ES-1, APEH and WBSCR21. Analysis of the immunoreactive protein levels of some of the lipases confirmed the results seen with mRNA. In conclusion, these data highlight the complexity of the regulation of the expression of intracellular neutral lipase/esterases involved in lipolysis.
Keywords: Lipase; Esterase; Rosiglitazone; High fat diet;

Localization of lipids in the aortic wall with imaging TOF-SIMS by Per Malmberg; Katrin Börner; Yun Chen; Peter Friberg; Birgit Hagenhoff; Jan-Erik Månsson; Håkan Nygren (185-195).
Time-of-flight secondary-ion-mass-spectrometry (TOF-SIMS) was utilized to address the issue of localization of lipids and inorganic ions in healthy rat aorta and human atherosclerotic plaque. Pieces of rat aorta were high pressure frozen, freeze-fractured and freeze dried. The samples were analyzed by imaging TOF-SIMS equipped with a Bi1–7 +-source. Reference lipid samples were analyzed and compared to data obtained by analysis of the rat aorta samples. Fatty acids, cholesterol, oxysterol and diacylglycerols were detected and localized. A heterogeneous lipid distribution could be shown in the aorta, where the lamellae of the aorta, distinguished by imaging of CN, appeared enriched in cholesterol, oxysterol and diacylglycerols, while the smooth muscle tissue, identified by imaging of PO3, appeared enriched in phosphocholine. Palmitic/palmitoleic acid and stearic/oleic acid appeared to be heterogeneously distributed over the aorta with high concentration areas located especially in the tunica media region of the aorta. Human atherosclerotic plaque showed an irregular cholesterol distribution mainly located in spots in the intima region with elongated diacylglycerol regions located mainly in the media region.
Keywords: Lipid; Aortic wall; TOF-SIMS;

Effects of bile diversion in rats on intestinal sphingomyelinases and ceramidase by R.D. Duan; H.J. Verkade; Y. Cheng; R. Havinga; Å. Nilsson (196-201).
Alkaline sphingomyelinase (Alk-SMase) and neutral ceramidase (N-CDase) in the intestinal microvillar membrane are responsible for dietary sphingomyelin digestion. The activities of the enzymes require the presence of bile salt, and the enzymes can be released into the gut lumen in active forms by bile salts and trypsin. It is unclear to what extent that the intestinal presence of bile salts is critical for the intraluminal activity of these enzymes. We compared the activities of Alk-SMase, N-CDase, and other types of SMases in control and permanently bile diverted rats. In the intestinal tract of control rats, the activity of Alk-SMase was profoundly higher than those of acid and neutral SMases. Bile diversion reduced Alk-SMase activity by 85% in the small intestinal content, and by 68% in the faeces, but did not significantly change the activity in the intestinal mucosa. Western blot showed a marked reduction of the enzyme in the intestinal lumen but not mucosa. N-CDase activities both in the intestinal mucosa and content were reduced by bile diversion. Bile diversion also decreased aminopeptidase N activity in the content and increased that in the mucosa, but had no effects on that of alkaline phosphatase. In conclusion, the presence of bile salts is important for maintaining high intraluminal levels of Alk-SMase and N-CDase, two key enzymes for hydrolysis of sphingomyelin in the gut. We speculate that the sphingomyelin hydrolysis in cholestatic conditions is impaired not only by reduced hydrolytic activity but also by deficient dissociation of the enzymes from the membrane.
Keywords: Sphingomyelinase; Ceramidase; Bile diversion; Rat; Small intestine; Colon;

In order to elucidate the biological role of sulfatide it is important to define the cellular and subcellular distribution of its various molecular species (e.g. fatty acid chain length and hydroxylation). We determined sulfatide species distribution in the rat cerebellum using time-of-flight secondary ion mass spectrometry (TOF-SIMS). TOF-SIMS detects ions up to m/z 10000 and enables simultaneous imaging of the lateral distribution of substances on an exposed surface, in this case a section through cerebellum. In addition to TOF-SIMS we analyzed sulfatide distribution in rat cerebellum using a sulfatide monoclonal antibody and confocal laser scanning microscopy. In the white matter, TOF-SIMS showed a uniform distribution of sulfatide with short chain fatty acids and a patchy distribution of sulfatide with C24 fatty acids. These patches had a low cholesterol signal. The granular layer showed a more uniform distribution of the sulfatide species, with the highest signal of C24. The molecular layer and Purkinje cells were devoid of sulfatide signals. Immunofluorescence showed the highest intensity in the white matter, lower intensity in the granular layer and absence of fluorescence in the molecular layer and Purkinje cells. The results are discussed in relation to previously published data and possible functional roles.
Keywords: Sulfatide; TOF-SIMS; Cerebellum; Fatty acid; Glycosphingolipid;

LSDP5 is a PAT protein specifically expressed in fatty acid oxidizing tissues by Knut Tomas Dalen; Tuva Dahl; Elin Holter; Borghild Arntsen; Constantine Londos; Carole Sztalryd; Hilde I. Nebb (210-227).
The PAT family (originally named for Perilipin, ADFP and TIP47) now includes four members: Perilipins, ADFP, TIP47 and S3-12. Significant primary sequence homology and the ability to associate with lipid storage droplets (LSDs) are well conserved within this family and across species. In this study, we have characterized a novel PAT protein, lipid storage droplet protein 5 (LSDP5) of 463 residues. A detailed sequence analysis of all murine PAT proteins reveals that LSDP5, TIP47 and ADFP share the highest order of sequence similarity, whereas perilipin and S3-12 have more divergent carboxyl- and amino-termini, respectively. Ectopically-expressed YFP-LSDP5 or flag-LSDP5 fusion proteins associate with LSDs. In accord with recent published data for perilipin, forced expression of LSDP5 in CHO cells inhibits lipolysis of intracellular LSDs. The LSDP5 gene is primarily transcribed in cells that actively oxidize fatty acids, such as heart, red muscle and liver. Expression of LSDP5 is stimulated by ligand activation of peroxisomal proliferator-activated receptor alpha (PPARα), and significantly reduced in liver and heart in the absence of this transcription factor. PPARα is generally required for regulation of fatty acid metabolism during fasting, but fasting induces LSDP5 mRNA in liver even in the absence of PPARα.
Keywords: LSDP5; Adipophilin; ADFP; S3-12; Perilipin; TIP47; PAT; TAG; Fatty acid; PPAR;

Purification and biochemical characterization of the LIP2 lipase from Yarrowia lipolytica by Ahmed Aloulou; Jorge A. Rodriguez; Delphine Puccinelli; Nicolas Mouz; Julien Leclaire; Yves Leblond; Frédéric Carrière (228-237).
The LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) was obtained from two genetically modified strains with multi-copies of the lip2 gene and further purified using gel filtration and cation exchange chromatography. Four YLLIP2 isoforms were identified and subjected to N-terminal amino-acid sequencing and mass spectrometry analysis. These isoforms differed in their glycosylation patterns and their molecular masses ranged from 36,874 to 38,481 Da, whereas the polypeptide mass was 33,385 Da. YLLIP2 substrate specificity was investigated using short (tributyrin), medium (trioctanoin) and long (olive oil) chain triglyceride substrates at various pH and bile salt concentrations, and compared with those of human gastric and pancreatic lipases. YLLIP2 was not inhibited by bile salts at micellar concentrations with any of the substrates tested, and maximum specific activities were found to be 10,760 ± 115 U/mg on tributyrin, 16,920 ± 480 U/mg on trioctanoin and 12,260 ± 700 U/mg on olive oil at pH 6.0. YLLIP2 was found to be fairly stable and still active on long chain triglycerides (1590 ± 430 U/mg) at pH 4.0, in the presence of bile salts. It is therefore a good candidate for use in enzyme replacement therapy as a means of treating pancreatic exocrine insufficiency.
Keywords: Microbial lipase; Lipolysis; pH-dependency; Bile salts; Yeast; Triglycerides;