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BBA - Molecular and Cell Biology of Lipids (v.1761, #2)
Intestinal levels of anandamide and oleoylethanolamide in food-deprived rats are regulated through their precursors
by Gitte Petersen; Camilla Sørensen; Patricia C. Schmid; Andreas Artmann; Mads Tang-Christensen; Steen H. Hansen; Philip Just Larsen; Harald H.O. Schmid; Harald S. Hansen (pp. 143-150).
The anorectic lipid oleoylethanolamide and the orexigenic lipid anandamide both belong to the group of N-acylethanolamines that are generated by the enzyme N-acylphosphatidylethanolamine-hydrolyzing phospholipase D. The levels of the two bioactive lipids were investigated in rat intestines after 24 h of starvation as well as after 1 and 4 h of re-feeding. Total levels of precursor phospholipids and N-acylethanolamines were decreased upon food-deprivation whereas the level of the anandamide precursor molecule was significantly increased. The level of 2-arachidonoyl-glycerol was unchanged as was the activity of N-acyltransferase, N-acylphosphatidylethanolamine-hydrolyzing phospholipase D, and fatty acid amide hydrolase upon starvation and re-feeding. It is concluded that remodeling of the amide-linked fatty acids of N-acylphosphatidylethanolamine is responsible for the opposite effects on levels of anandamide and oleoylethanolamide in intestines of food-deprived rats and not an alternative biochemical route for anandamide synthesis. Furthermore, linoleoylethanolamide, which accounted for more than 50 mol% of the endogenous pool of N-acylethanolamines, was found not to have the same inhibitory effect on food intake, as did oleoylethanolamide following oral administration.
Keywords: Abbreviations; AEA; N; -arachidonoylethanolamine or anandamide; 2-AG; 2-arachidonoyl-glycerol; FAAH; fatty acid amide hydrolase; GC-MS; gas chromatography-mass spectrometry; i.p.; intraperitoneal; LEA; linoleoylethanolamide; NAE; N; -Acylethanolamine; NAPE; N; -acylated phosphatidylethanolamine; NAPE-PLD; NAPE-hydrolyzing phospholipase D; NAT; N; -acyltransferase; OEA; N; -oleoylethanolamine; PPAR; peroxisome proliferator-activated receptor N; -Acyltransferase; Anandamide; 2-arachidonoyl-glycerol; Oleoylethanolamide; Linoleoylethanolamide
Signalling diacylglycerol pyrophosphate, a new phosphatidic acid metabolite
by Bas van Schooten; Christa Testerink; Teun Munnik (pp. 151-159).
Diacylglycerol pyrophosphate (DGPP) is a novel phospholipid that has been found in plants and yeast but not in higher animals. It is produced through phosphorylation of phosphatidic acid (PA) by the novel enzyme PA kinase (PAK). In plants, DGPP is virtually absent in non-stimulated cells but its concentration increases within minutes in response to various stimuli, including osmotic stress and pathogen attack, implying a role in stress signalling. DGPP is broken down by the enzyme DGPP phosphatase (DPP). DPP-encoding genes have been cloned from Arabidopsis thaliana and Saccharomyces cerevisiae ( DPP1). In S. cerevisiae, the expression of DPP1 is regulated coordinately with the majority of genes encoding enzymes involved in phospholipid biosynthesis.
Keywords: Diacylglycerol pyrophosphate; Phosphatidic acid kinase; Diacylglycerol pyrophosphate phosphatase; Phosphatidic acid; Plant stress signalling; Yeast phospholipid biosynthesis
The 2-series prostaglandins suppress VLDL secretion in an inflammatory condition-dependent manner in primary rat hepatocytes
by Silvia Pérez; Patricia Aspichueta; Begoña Ochoa; Yolanda Chico (pp. 160-171).
In the liver, prostaglandins (PG) generated mainly by activated non-parenchymal cells can modulate the parenchymal cell function during homeostasis and inflammation. Whether prostaglandins regulate the hepatocyte VLDL assembling/secretor phenotype in both conditions remains unresolved. We sought to determine whether and how PGE2, PGD2, and PGF2α (5 and 50 μM) have a role in VLDL secretion regulation in resting and interleukin-6 (IL-6) stimulated rat hepatocytes. Prostaglandins led to comparable, concentration-dependent reductions in the secretion of VLDL apoB and lipids by resting, 24 h-cultured cells. Moreover, each apoB copy recruited less of each lipid class, correlating with reduced particle size, lipogenesis and cholesterogenesis, and impaired cellular triacylglycerol recycling. Triacylglycerol output reduction occurred early, as the transient PGD2- and PGF2α-promoted apoB mRNA decreases. IL-6 markedly increased the apoB mRNA expression and the secretion of its protein in triacylglycerol-poor VLDL. The latter was uniquely blunted by PGE2, which unaffected basal or IL-6-activated apoB gene expression. Collectively, our findings show inflammation condition-based roles for 2-series-prostaglandins in VLDL secretion modulation. Whereas in non-stimulated hepatocytes, they all inhibited VLDL-apoB output, interfered with lipid provision for lipoprotein assembly and may be regarded as pro-steatotic, the anti-inflammatory PGE2 antagonized the IL-6-promoted VLDL secretion contributing in restoring liver homeostasis.
Keywords: Triacylglycerol secretion; Cholesteryl ester metabolism; Hepatocyte gene expression; VLDL secretion; Acute-phase reaction control
Adipocyte-type fatty acid-binding protein as inter-compartmental shuttle for peroxisome proliferator activated receptor γ agonists in cultured cell
by Anne Adida; Friedrich Spener (pp. 172-181).
We and others showed earlier that liver-type, epidermal-type and adipocyte-type (A-) fatty acid-binding proteins (FABPs) mediate peroxisome proliferator activated receptor (PPAR) dependent gene expression by channelling their ligand (fatty acid or drug) to the nuclear receptors via direct protein/protein interaction. To clarify mechanistic details of this signaling path, we address here A-FABP import into the nucleus and its interaction with PPARγ. Making use of COS cells transfected with wild-type or mutant A-FABPs, we exclude posttranslational modification of A-FABP as import signal and provide evidence for both, ligand-dependent and ligand-independent nuclear translocation. With the aid of in vitro pull down assay we demonstrate that specific interaction of A-FABP with PPARγ isoforms does not require ligand. Moreover, A-FABP binds not only to the ligand-binding domain including hinge domain (domains DEF), but also to the DNA-binding domain including AB domains (domains ABC) of PPARγ.
Keywords: Abbreviations; A-FABP; adipocyte-type fatty acid-binding protein; B-FABP; brain-type FABP; DBD; DNA binding domain; DMSO; dimethylsulfoxide; CRABP; cellular retinoic acid-binding protein; E-FABP; epidermal-type FABP; GFP; green fluorescent protein; HSL; hormone sensitive lipase; iLBP; intracellular lipid binding protein; LBD; ligand binding domain; L-FABP; liver-type FABP; mt; mutant; NLS; nuclear localization signal; PPAR; peroxisome proliferator activated receptor; RAR; retinoic acid receptor; RXR; retinoic acid X receptor; wt; wild-typeFatty acid-binding protein; Peroxisome proliferator activated receptor; Fatty acid signaling; Ligand dependence; Subcellular localization; Protein/protein interaction
A mouse monoclonal antibody specific for mouse apoB48 and apoB100 produced by immunizing “apoB39-only� mice with mouse apoB48
by Anh T. Nguyen; Sylvie Braschi; Michèle Geoffrion; Loren G. Fong; Rosanne M. Crooke; Mark J. Graham; Stephen G. Young; Ross Milne (pp. 182-185).
We have generated and characterized a murine monoclonal antibody (mAb) that binds to both mouse apolipoprotein (apo) B48 and apoB100. We immunized “apoB39-only� mice (mice that synthesize a truncated form of apoB, apoB39, but no apoB48 or apoB100) with lipoproteins containing mouse apoB48 and then used splenocytes from the immunized mice to create hybridomas. We identified a hybridoma, 2G11, that secretes a mAb that binds to mouse apoB48 and apoB100 but not to apoB39. Antibody 2G11 also binds apoB48 and apoB100 from rats and hamsters but not from humans. The mAb recognizes mouse apoB equally in very low and low density lipoproteins and was used to quantify apoB in wild-type, apoE-deficient and low-density lipoprotein receptor-deficient mice and in mice treated with an antisense drug that lowers plasma apoB levels. The antibody will be an important reagent for studying mouse models of atherosclerosis. The study also underscores the utility of genetically modified mice for generating mouse mAbs against mouse proteins.
Keywords: Monoclonal antibody; Mouse model; Apolipoprotein B
Binding, internalization and transport of apolipoprotein A-I by vascular endothelial cells
by Lucia Rohrer; Clara Cavelier; Séverine Fuchs; Marc Alexander Schlüter; Wolfgang Völker; Arnold von Eckardstein (pp. 186-194).
High density lipoproteins (HDL) and their main protein constituent, apolipoprotein A-I (apoA-I), exert potentially anti-atherogenic properties within the arterial wall. However, it is unknown how they are transported from the blood stream into the vascular wall. Here we investigated the interaction of apoA-I with endothelial cells. At 4 °C endothelial cells bound125I-apoA-I with high affinity, Kd=2.1 μg/ml and in a saturable manner ( Bmax of 35 ng/mg cell protein). At 37 °C, the cell association of apoA-I revealed similar affinity as at 4 °C ( Kd=2.2 μg/ml) but the maximum specific cell association was much enhanced ( Bmax=360 ng/mg cell protein). Binding and cell association was competed by excess unlabeled apoA-I and HDL but not by albumin. Biotinylation experiments and electron microscopy studies showed that endothelial cells internalize labeled apoA-I. Only minor amounts of the internalized apoA-I were degraded. Cultivated in a Transwell system, the cells transported a fraction of125I-apoA-I from the apical to the basolateral compartment in a competable and temperature-sensitive manner. Furthermore, after specific transport the originally prebeta-mobile and lipid-free apoA-I was recovered as particles which have electrophoretic alpha-mobility. We conclude that endothelial cells transcytose and lipidate lipid-free apoA-I.
Keywords: Abbreviations; apo; apolipoprotein; apoA-I; apolipoprotein A-I; BAEC; bovine aortic endothelial cells; BSA; bovine serum albumin; HDL; high density lipoprotein; HUVEC; human umbilical vein endothelial cells; LDL; low-density lipoproteinHDL; Apolipoprotein A-I; ApoA-I; Endothelial cell; Transcytosis; Vascular wall
Involvement of the Na+/H+ exchanger in membrane phosphatidylserine exposure during human platelet activation
by Robert Bucki; Jennifer J. Pastore; Françoise Giraud; Paul A. Janmey; Jean-Claude Sulpice (pp. 195-204).
Platelet membrane phosphatidylserine (PS) exposure that regulates the production of thrombin represents an important link between platelet activation and the coagulation cascade. Here, we have evaluated the involvement of the Na+/H+ exchanger (NHE) in this process in human platelets. PS exposure induced in human platelets by thrombin, TRAP, collagen or TRAP+ collagen was abolished in a Na+-free medium. Inhibition of the Na+/H+ exchanger (NHE) by 5-(N-Ethyl-N-Isopropyl) Amiloride (EIPA) reduced significantly PS exposure, whereas monensin or nigericin, which mimic or cause activation of NHE, respectively, reproduced the agonist effect. These data suggest a role for Na+ influx through NHE activation in the mechanism of PS exposure. This newly identified pathway does not discount a role for Ca2+, whose cytosolic concentration varies together with that of Na+ after agonist stimulation. Ca2+ deprivation from the incubation medium only attenuated PS exposure induced by thrombin, measured from the uptake of FM1-43 (a marker of phospholipid scrambling independent of external Ca2+). Surprisingly, removal of external Ca2+ partially reduced FM1-43 uptake induced by A23187, known as a Ca2+ ionophore. The residual effect can be attributed to an increase in [Na+]i mediated by the ionophore due to a lack of its specificity. Finally, phosphatidylinositol 4,5-bisphosphate (PIP2), previously reported as a target for Ca2+ in the induction of phospholipid scrambling, was involved in PS exposure through a regulation of NHE activity. All these results would indicate that the mechanism that results in PS exposure uses redundant pathways inextricably linked to the physio-pathological requirements of this process.
Keywords: Abbreviations; BSA; bovine serum albumin; EIPA; 5-(N-Ethyl-N-Isopropyl) amiloride; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PS; phosphatidylserine; LUVs; large unilamellar vesicles; IP; 3; inositol triphosphate; PIP; 2; phosphatidylinositol 4, 5-bisphosphate; NHE; Na; +; /H; +; exchanger; TRAP; thrombin receptor agonist fragment; PLSCR1; phospholipid scramblase 1Platelet; Thrombin; Phosphatidylserine; Na; +; /H; +; exchanger (NHE) phosphatidylinositol 4,5-bisphosphate (PIP; 2; )
Development of the first potent and specific inhibitors of endocannabinoid biosynthesis
by Tiziana Bisogno; Maria Grazia Cascio; Bijali Saha; Anu Mahadevan; Paolo Urbani; Alberto Minassi; Giovanni Appendino; Carmela Saturnino; Billy Martin; Raj Razdan; Vincenzo Di Marzo (pp. 205-212).
Enzymes for the biosynthesis and degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG) have been cloned and are the sn-1-selective-diacylglycerol lipases α and β (DAGLα and β) and the monoacylglycerol lipase (MAGL), respectively. Here, we used membranes from COS cells over-expressing recombinant human DAGLα to screen new synthetic substances as DAGLα inhibitors, and cytosolic fractions from wild-type COS cells to look for MAGL inhibitors. DAGLα and MAGL activities were assessed by using sn-1-[14C]-oleoyl-2-arachidonoyl-glycerol and 2-[3H]-arachidonoylglycerol as substrates, respectively. We screened known compounds as well as new phosphonate derivatives of oleic acid and fluoro-phosphinoyl esters of different length. Apart from the general lipase inhibitor tetrahydrolipstatin (orlistat®) (IC50∼60 nM), the most potent inhibitors of DAGLα were O-3640 [octadec-9-enoic acid-1-(fluoro-methyl-phosphoryloxymethyl)-propylester] (IC50=500 nM), and O-3841 [octadec-9-enoic acid 1-methoxymethyl-2-(fluoro-methyl-phosphinoyloxy)-ethyl ester] (IC50=160 nM). Apart from being almost inactive on MAGL, these two compounds showed high selectivity over rat liver triacylglycerol lipase, rat N-acylphosphatidyl-ethanolamine-selective phospholipase D (involved in anandamide biosynthesis), rat fatty acid amide hydrolase and human recombinant cannabinoid CB1 and CB2 receptors. Methylarachidonoyl-fluorophosphonate and the novel compound UP-101 [O-ethyl- O- p-nitro-phenyl oleylphosphonate] inhibited both DAGLα and MAGL with similar potencies (IC50=0.8–0.1 and 3.7–3.2 μM, respectively). Thus, we report the first potent and specific inhibitors of the biosynthesis of 2-AG that may be used as pharmacological tools to investigate the biological role of this endocannabinoid.
Keywords: Abbreviations; AA-5-HT; arachidonoyl-5-hydroxytryptamine; 2-AG; 2-Arachidonoyl-glycerol; AMT; anandamide membrane transporter; CB; 1; cannabinoid receptor type 1; CB; 2; cannabinoid receptor type 2; [; 3; H]CP-55,940; [; 3; H]-(−)-; cis; -3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-; trans; -4-(3-hydroxy-propyl)-cyclohexanol; DAGLα; sn; -1-selective-diacylglycerol lipase α; DAGLβ; sn; -1-selective-diacylglycerol lipase β; DAGs; diacylglycerols; DAST; diethylamino-sulfur trifluoride; DCC; N,N′; -Dicycloexylcarbodiimide; DMAP; 4-dimethylaminopyridine; DSE; depolarization-induced suppression of excitatory neurotransmission; DSI; depolarization-induced suppression of inhibitory neurotransmission; EDCI; N-(3-dimethylaminopropyl)-; N′; -ethylcarbodiimide hydrochloride; FAAH; fatty acid amide hydrolase; MAFP; methylarachidonoyl-fluorophosphonate; MAGL; monoacylglycerol lipase; NAPE-PLD; N; -acylphosphatidyl-ethanolamine-selective phospholipase D; O-3453; octadec-9-enoic acid-2-(methoxy-fluoro-phosphinoyl)-1-octadec-9-enoyloxymethyl-ethyl ester; O-3640; octadec-9-enoic acid-1-(fluoro-methyl-phosphoryloxymethyl)-propylester; O-3696; octadec-9-enoic acid-1-(fluoro-methoxy-phosphinoylmethyl)-hexyl ester; O-3841; octadec-9-enoic acid 1-methoxymethyl-2-(fluoro-methyl-phosphinoyloxy)-ethyl ester; OMDM-1; 4-hydroxybenzil-N-oleoyl-ethanolamide; PA; phosphatidic acid; PI; phosphoinositides; PI-PLC; PI-selective phospholipase C; RHC80267; 1,6-bis-(cyclohexyloximino-carbonylamino)-hexane; solketal; 2,2-dimethyl-[1,3]dioxolan-4-yl-methanol; TEP; triethylphosphite; THL; tetrahydrolipstatin (orlistat®); UP99; O,O; -diethyl oleylphosphonate; UP100; O; -Ethyl oleylphosphonate; UP-101; O-ethyl-; O; -; p; -nitro-phenyl oleylphosphonate; UP104; O; -ethyl-; O; -oleyl oleylphosphonate; VDM-11; 2-methyl-4-hydroxybenzyl-; N; -arachidonoyl-ethanolamineInhibitor; Diacylglycerol; Cannabinoid; 2-Arachidonoylglycerol; Lipase
Apolipoprotein CI causes hypertriglyceridemia independent of the very-low-density lipoprotein receptor and apolipoprotein CIII in mice
by Caroline C. van der Hoogt; Jimmy F.P. Berbée; Sonia M.S. Espirito Santo; Gery Gerritsen; Yvonne D. Krom; André van der Zee; Louis M. Havekes; Ko Willems van Dijk; Patrick C.N. Rensen (pp. 213-220).
We have recently shown that the predominant hypertriglyceridemia in human apolipoprotein C1 ( APOC1) transgenic mice is mainly explained by apoCI-mediated inhibition of the lipoprotein lipase (LPL)-dependent triglyceride (TG)-hydrolysis pathway. Since the very-low-density lipoprotein receptor (VLDLr) and apoCIII are potent modifiers of LPL activity, our current aim was to study whether the lipolysis-inhibiting action of apoCI would be dependent on the presence of the VLDLr and apoCIII in vivo. Hereto, we employed liver-specific expression of human apoCI by using a novel recombinant adenovirus (Ad APOC1). In wild-type mice, moderate apoCI expression leading to plasma human apoCI levels of 12–33 mg/dl dose-dependently and specifically increased plasma TG (up to 6.6-fold, P<0.001), yielding the same hypertriglyceridemic phenotype as observed in human APOC1 transgenic mice. Ad APOC1 still increased plasma TG in vldlr −/− mice (4.1-fold, P<0.001) and in apoc3 −/− mice (6.8-fold, P<0.001) that were also deficient for the low-density lipoprotein receptor (LDLr) and LDLr-related protein (LRP) or apoE, respectively. Thus, irrespective of receptor-mediated remnant clearance by the liver, liver-specific expression of human apoCI causes hypertriglyceridemia in the absence of the VLDLr and apoCIII. We conclude that apoCI is a powerful and direct inhibitor of LPL activity independent of the VLDLr and apoCIII.
Keywords: ApoE; LDL receptor; LRP; Triglyceride; VLDL receptor; Adenoviral gene transfer
Evidence for the activation of 1α-hydroxyvitamin D2 by 25-hydroxyvitamin D-24-hydroxylase: Delineation of pathways involving 1α,24-dihydroxyvitamin D2 and 1α,25-dihydroxyvitamin D2
by Sonoko Masuda; Stephen A. Strugnell; Joyce C. Knutson; René St-Arnaud; Glenville Jones (pp. 221-234).
While current dogma argues that vitamin D prodrugs require side-chain activation by liver enzymes, recent data suggest that hydroxylation may also occur extrahepatically. We used keratinocytes and recombinant human enzyme to test if the 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) is capable of target cell activation and inactivation of a model prodrug, 1α-hydroxyvitamin D2 (1α(OH)D2) in vitro. Mammalian cells stably transfected with CYP24A1 (V79-CYP24A1) converted 1α(OH)D2 to a series of metabolites similar to those observed in murine keratinocytes and the human cell line HPK1A- ras, confirming the central role of CYP24A1 in metabolism. Products of 1α(OH)D2 included the active metabolites 1α,24-dihydroxyvitamin D2 (1α,24(OH)2D2) and 1α,25-dihydroxyvitamin D2 (1α,25(OH)2D2); the formation of both indicating the existence of distinct activation pathways. A novel water-soluble metabolite, identified as 26-carboxy-1α,24(OH)2D2, was the presumed terminal degradation product of 1α(OH)D2 synthesized by CYP24A1 via successive 24-hydroxylation, 26-hydroxylation and further oxidation at C-26. This acid was absent in keratinocytes from Cyp24a1 null mice. Slower clearance rates of 1α(OH)D2 and 1α,24(OH)2D2 relative to 1α,25(OH)2D2 and 1α,25(OH)2D3 were noted, arguing for a role of 24-hydroxylated metabolites in the altered biological activity profile of 1α(OH)D2. Our findings suggest that CYP24A1 can activate and inactivate vitamin D prodrugs in skin and other target cells in vitro, offering the potential for treatment of hyperproliferative disorders such as psoriasis by topical administration of these prodrugs.
Keywords: Abbreviations; CYP; cytochrome P450; 1α,25(OH); 2; D; 3; 1α,25-dihydroxyvitamin D; 3; 25(OH)D; 3; 25-hydroxyvitamin D; 3; 1α(OH)D; 3; 1α-hydroxyvitamin D; 3; 1α(OH)D; 2; 1α-hydroxyvitamin D; 2; 1α,24(OH); 2; D; 2; 1α,24-dihydroxyvitamin D; 2; 1α,25(OH); 2; D; 2; 1α,25-dihydroxyvitamin D; 2; 1α,24,25(OH); 3; D; 2; 1α,24,25-trihydroxyvitamin D; 2; 1α,24,26(OH); 3; D; 2; 1α,24,26-trihydroxyvitamin D; 2; PTH; parathyroid hormone; VDR; vitamin D receptor; HPLC; High Performance Liquid Chromatography; LC-MS; Liquid Chromatographic-Mass Spectrometry; NMR; Nuclear magnetic resonance; DMEM; Dulbecco's modified Eagle's medium; DPPD; N′N; -diphenyl-; p; -phenylenediamine; GAA; Glacial acetic acidCYP24A1; 1α-hydroxyvitamin D; 2; 1α,24-dihydroxyvitamin D; 2; 1α,25-dihydroxyvitamin D; 2; 1α,25-dihydroxyvitamin D; 3; Vitamin D catabolism
Biosynthesis of novel carotenoid families based on unnatural carbon backbones: A model for diversification of natural product pathways
by Alexander V. Tobias; Frances H. Arnold (pp. 235-246).
We show that the C40 carotenoid desaturase CrtI from Pantoea ananatis ( Erwinia uredovora) is capable of desaturating unnaturally long C45 and C50 carotenoid backbones in recombinant E. coli. Desaturation step number in these pathways is not very specific, and at least ten new C45 and C50 carotenoids were synthesized. We also present evidence for a novel asymmetric C40 backbone formed by the condensation of farnesyl diphosphate (C15PP) with farnesylgeranyl diphosphate (C25PP), and the subsequent desaturation of this backbone by CrtI in an atypical manner. Under some conditions, the C40, C45, and C50 carotenoid backbones synthesized in E. coli were monohydroxylated; their desaturation by CrtI in vitro led to yet more novel carotenoids. Challenging CrtI with larger-than-natural substrates in vivo has allowed us to show that this enzyme regulates desaturation step number by sensing the end groups of its substrate. Analysis of the mechanisms by which chemical diversity is generated and propagated through the nascent pathways provides insight into how natural product diversification occurs in nature.
Keywords: Molecular evolution; Carotenoid biosynthetic pathway
Calyculin and okadaic acid promote perilipin phosphorylation and increase lipolysis in primary rat adipocytes
by Jinhan He; Hongfeng Jiang; John T. Tansey; Chaoshu Tang; Shenshen Pu; Guoheng Xu (pp. 247-255).
Lipolysis is primarily regulated by protein kinase A (PKA), which phosphorylates perilipin and hormone-sensitive lipase (HSL), and causes translocation of HSL from cytosol to lipid droplets in adipocytes. Perilipin coats lipid droplet surface and assumes to prevent lipase access to triacylglycerols, thus inhibiting basal lipolysis; phosphorylated perilipin facilitates lipolysis on PKA activation. Here, we induced lipolysis in primary rat adipocytes by inhibiting protein serine/threonine phosphatase with specific inhibitors, okadaic acid and calyculin. The incubation with calyculin promotes incorporation of32Pi into perilipins, thus, confirming that perilipin is hyperphosphorylated. The lipolysis response to calyculin is gradually accompanied by increased accumulation of phosphorylated perilipin A in a concentration- and time-responsive manner. When perilipin phosphorylation is abrogated by the addition of N-ethylmaleimide, lipolysis ceases. Different from a considerable translocation of HSL upon PKA activation with isoproterenol, calyculin does not alter HSL redistribution in primary or differentiated adipocytes, as confirmed by both immunostaining and immunoblotting. Thus, we suggest that inhibition of the phosphatase by calyculin activates lipolysis via promoting perilipin phosphorylation rather than eliciting HSL translocation in adipocytes. Further, we show that when the endogenous phosphatase is inhibited by calyculin, simultaneous PKA activation with isoproterenol converts most of the perilipin to the hyperphosphorylated species, and induces enhanced lipolysis. Apparently, as PKA phosphorylates perilipin and stimulates lipolysis, the phosphatase acts to dephosphorylate perilipin and attenuate lipolysis. This suggests a two-step strategy governed by a kinase and a phosphatase to modulate the steady state of perilipin phosphorylation and hence the lipolysis response to hormonal stimulation.
Keywords: Abbreviations; AGTL; Adipose triglyceride lipase; fDMEM; Phenol red-free and serum-free Dulbecco's modified Eagle's medium; HSL; Hormone-sensitive lipase; PCV; Packed cell volume; PKA; cAMP-dependent protein kinase A; PPase; Protein serine/threonine phosphatase; PP1; protein phosphatase-1; PP2A; Protein phosphatase-2A; SDS-PAGE; SDS polyacrylamide gel electrophoresisLipolysis; Perilipin; Okadaic acid; Calyculin; Phosphorylation; Dephosphorylation; Protein phosphatase; Hormone-sensitive lipase; Adipose triglyceride lipase; Lipid droplet; Isoproterenol
Down-regulation of apolipoprotein M expression is mediated by phosphatidylinositol 3-kinase in HepG2 cells
by Ning Xu; Bo Ahrén; Jingting Jiang; Peter Nilsson-Ehle (pp. 256-260).
Apolipoprotein M (apoM) is a novel apolipoprotein present mostly in high-density lipoprotein (HDL) in human plasma. In the present study, we demonstrate that insulin, insulin-like growth factor I (IGF-I), and IGF-I potential peptide (IGF-IPP) significantly inhibits apoM expression, in a dose- and a time-dependent manner, in the human hepatoma cell line, HepG2 cells. Insulin-induced down-regulation of apoM was blocked by AG1024 (a specific insulin receptor inhibitor) and LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor), which indicates that it is mediated via the activation of PI3K pathway. In contrast, PD98059 (a MAP kinase inhibitor) did not influence insulin-induced down-regulation of apoM expression, and activation of neither PPAR-α agonist (GW7647) nor PPAR-γ agonist (GW1929) influences apoM expression in HepG2 cells, which indicates that regulation of apoM expression is not related to the activation of PPAR-α and PPAR-γ in hepatic cells, whereas, both PPAR-α and PPAR-γ agonists could inhibit apoB expression. Moreover, in the present study, we demonstrated that PPAR β/δ agonist (GW501516) could inhibit both apoM and apoB expression in the HepG2 cells. In conclusion, this study shows that apoM expression is regulated by PI3-kinase in HepG2-cells.
Keywords: Apolipoprotein M; Insulin; Phosphatidylinositol 3-kinase and HepG2 cell line; PPARs
Contribution of lipid mediators to the regulation of phosphatidylcholine synthesis by angiotensin
by Theresa E. Kitos; Adrienne Drobnies; Michael N.P. Ng; Yeshao Wen; Rosemary B. Cornell (pp. 261-271).
Angiotensin stimulates a cellular mitogenic response via the AT1 receptor. We have examined the effect of angiotensin on the rate of phosphatidylcholine (PC) synthesis and have begun to dissect the pathway linking the AT1 receptor to the rate-limiting enzyme in PC synthesis, CTP: phosphocholine cytidylyltransferase (CCT), using CHO cells engineered to express the AT1a receptor. Since CCT can be directly activated by lipid mediators, we probed for their involvement in the PC synthesis response to angiotensin. Angiotensin stimulated CCT activity and PC synthesis two- to threefold after a 30-min delay. The kinetics of this stimulation most closely paralleled an increase in diacylglycerol (DAG) derived from myristic acid-enriched phospholipids. The production of arachidonic acid, phosphatidic acid, or reactive oxygen species either peaked much earlier or not at all. Moreover, manipulation of the intracellular supply of oxygen free radicals, arachidonic acid, HETEs, or phosphatidic acid (using inhibitors and/or exogenous addition) did not generate parallel effects on the rate of PC synthesis. Restricting the production of DAG by inhibition of PLCβ with U73122 reduced both basal and angiotensin-stimulated PC synthesis. The U73122 inhibition of PC synthesis was accompanied by a similar inhibition of ERK1/2 phosphorylation. Addition of exogenous DAG stimulated basal and angiotensin-dependent PC synthesis, and partially reversed the effect of the PLC inhibitor on PC synthesis. These results do not provide support for lipid mediators as direct stimulators of CCT and PC synthesis downstream of angiotensin, but give rise to the idea that angiotensin effects might be mediated via ERK1/2.
Keywords: Abbreviations; CCT; CTP:phosphocholine cytidylyltransferase; PC; phosphatidylcholine; PA; phosphatidic acid; PE; phosphatidylethanolamine; PIP; 2; phosphatidylinositol bisphosphate; PA; phosphatidic acid; AA; arachidonic acid; OA; oleic acid; DAG; diacylglycerol; HETE; hydroxyeicosatetraenoic acid; PtdBut; phosphatidylbutanol; PLC; phospholipase C; iPLA; 2; calcium-independent phospholipase A; 2; cPLS; 2; calcium-dependent phospholipase A; 2; PLD; phospholipase D; DTT; dithiothreitol; FBS; fetal bovine serum; MAFP; methyl arachidonyl fluorophosphonate; BEL; bromoenol lactone; ETYA; eicosatetraynoic acid; ROS; reactive oxygen species; CDC; cinnamyl-3,4-dihydroxy-α-cyanocinnamate; CM-H; 2; DCFDA; 5-(and 6-)chloromethyl-2,′,7′-dichlorodihydrofluorescin diacetate, acetyl esterCTP:phosphocholine cytidylyltransferase; Phospholipase A; 2; Phospholipase C; Fatty acid; HETE; Diacylglycerol
Angiotensin stimulates phosphatidylcholine synthesis via a pathway involving diacylglycerol, protein kinase C, ERK1/2, and CTP:phosphocholine cytidylyltransferase
by Theresa E. Kitos; Chrystal M.Y. Choi; Rosemary B. Cornell (pp. 272-279).
We are probing the regulation of phosphatidylcholine (PC) synthesis by angiotensin II. In the accompanying paper, we showed that manipulation of the lipid second messengers, arachidonic acid or hydroxyeicosatetraenoic acid, produced downstream of the angiotensin AT1a receptor did not affect the PC synthesis rates in a manner consistent with direct activation of the rate limiting enzyme in the pathway, CTP:phosphocholine cytidylyltransferase (CCT). However, suppression of diacylglycerol (DAG) production with an inhibitor of phospholipase C-β reduced angiotensin-dependent PC synthesis as well as ERK1/2 phosphorylation. Here, we show that the stimulation of PC synthesis and activation of CCT by angiotensin requires a signaling pathway that involves protein kinase C and ERK1/2. The inhibitors bis-indolylmaleimide I and PD98059 blocked ERK1/2 phosphorylation and completely eliminated angiotensin stimulation of the CCT-catalyzed reaction and PC synthesis. Exogenous addition of DAG using a lipid vesicle delivery system exactly mimicked the kinetics of angiotensin-promoted PC synthesis, suggesting that this mode of DAG delivery can effectively substitute for the DAG generated downstream of the activated AT1a receptor. Moreover, exogenous DAG activated ERK1/2, and the activation of PC synthesis by DAG was blocked by inhibition of protein kinase C and MEK. These data suggest that angiotensin-dependent DAG and the exogenously supplied DAG stimulate PC synthesis, not solely by direct action on CCT, but via a signaling pathway involving protein kinase C and ERK1/2. Angiotensin did not alter the net phosphorylation state of CCT as probed by immunoprecipitation of32P-labeled CCT. Angiotensin stimulation of ERK1/2 likely mediates effects on CCT via a process other than CCT dephosphorylation.
Keywords: Abbreviations; CCT; CTP:phosphocholine cytidylyltransferase; PC; phosphatidylcholine; AA; arachidonic acid; PA; phosphatidic acid; PIP; 2; phosphatidylinositol (4,5) bisphosphate; DAG; diacylglycerol; HETE; hydroxyeicosatetraenoic acid; PLC(D); phospholipase C(D), Bisi, Bis-indolylmaleimide; BSA; bovine serum albumin; ROS; reactive oxygen species; DTT; dithiothreitolCTP:phosphocholine cytidylyltransferase; phospholipase C; Protein kinase C; ERK; Diacylglycerol; Angiotensin II
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