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BBA - Molecular and Cell Biology of Lipids (v.1801, #7)
Bile acids and their nuclear receptor FXR: Relevance for hepatobiliary and gastrointestinal disease by Raffaella M. Gadaleta; Saskia W.C. van Mil; Bas Oldenburg; Peter D. Siersema; Leo W.J. Klomp; Karel J. van Erpecum (pp. 683-692).
The nuclear receptor Farnesoid X Receptor (FXR) critically regulates nascent bile formation and bile acid enterohepatic circulation. Bile acids and FXR play a pivotal role in regulating hepatic inflammation and regeneration as well as in regulating extent of inflammatory responses, barrier function and prevention of bacterial translocation in the intestinal tract. Recent evidence suggests, that the bile acid–FXR interaction is involved in the pathophysiology of a wide range of diseases of the liver, biliary and gastrointestinal tract, such as cholestatic and inflammatory liver diseases and hepatocellular carcinoma, inflammatory bowel disease and inflammation-associated cancer of the colon and esophagus. In this review we discuss current knowledge of the role the bile acid–FXR interaction has in (patho)physiology of the liver, biliary and gastrointestinal tract, and proposed underlying mechanisms, based on in vitro data and experimental animal models. Given the availability of highly potent synthetic FXR agonists, we focus particularly on potential relevance for human disease.
Keywords: Bile acid; Carcinogenesis; Enterohepatic circulation; Farnesoid X Receptor; Inflammation
13C-labelling studies indicate compartmentalized synthesis of triacylglycerols in C6 rat glioma cells by MariaRosa Quintero; Cabanas Miquel E. Cabañas; Arus Carles Arús (pp. 693-701).
NMR-visible mobile lipid (ML) signals have been detected in1H-NMR spectra of tissues in vivo, ex vivo and in vitro, and have been shown to change in apparent intensity in association with pathology (necrosis in brain tumours) and normal processes (cell differentiation, cell growth arrest and apoptosis). Although it is widely accepted that ML signals originate mainly from fatty-acyl chains in triacylglycerols (TAG) contained in cytosolic lipid droplets (LD), the dynamics of TAG in LD is not yet fully understood. In order to better understand the synthesis of cellular TAG and its relationship to ML dynamics we carried out a set of labelling experiments with C6 rat glioma cells in culture. TAG and phospholipid metabolism was monitored by incubating C6 cells with [1-13C]-glucose at two time points during cell growth curve –24h incubation starting at log-phase; 48h incubation starting at saturation density– and by acquiring the 2D-HMQC NMR spectra of the respective total lipid extracts. The resulting TAG, diacylglycerol (DAG) and phospholipid labelling patterns can only be explained if TAG synthesis takes place in two different subcellular compartments. One compartment would be the endoplasmic reticulum, which is known to be involved in TAG metabolism, while the other compartment could be the plasma membrane and/or the LD. This possible role of LD is further supported by the recent description of diacylglycerolacyltranferase-activity associated with LD. Accordingly, we postulate the existence of a carbon-shuttling mechanism between plasma membrane phospholipids and endoplasmic reticulum by way of LD content. The results we have obtained with C6 cells may also apply to other cellular systems and should be taken into account when interpreting ML dynamics detected by NMR in vivo.
Keywords: Abbreviations; ATCC; American Tissue Culture Collection; CCT; CTP:phosphocholine cytidylyl transferase; Chol; cholesterol; CholEst; cholesteryl esters; DAG; diacylglycerol; DMEM; Dulbecco's Modified Eagle's Medium; FCS; foetal calf serum; GPC; glycerophosphocholine; Hepes; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HBSS; Hank's Balanced Salt Solution; HMQC; Heteronuclear Multiple Quantum Coherence transfer; LD; lipid droplets; ML; NMR-visible Mobile lipids; PBS; phosphate buffered saline; ppm; parts per million; PtdCho; 3-sn-alpha-phosphatidylcholine; PtdEt; 1, 2-diacyl-sn-glycero-3-phosphoethanolamine; TAG; triacylglycerol; VLDL; very-low-density lipoproteinsNMR-visible lipid droplet; Triacylglycerol synthesis; 13; C labelling
Cholesterol increases adhesion of monocytes to endothelium by moving adhesion molecules out of caveolae by Chenglai Fu; Jinlong He; Chenghong Li; John Y.-J. Shyy; Yi Zhu (pp. 702-710).
Caveolae and its structural protein caveolin-1 (Cav-1) are abundant in vascular endothelial cells (ECs). We examined whether caveolae are involved in monocyte adhesion to ECs responding to a synergy of hypercholesterolemia and inflammation. Treating human umbilical vein ECs with cholesterol enhanced endotoxin lipopolysaccharide (LPS)-induced monocyte adhesion. Use of isolated caveolae-enriched membranes revealed that cell adhesion molecules (CAMs), including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), co-localized with Cav-1 in caveolae. LPS upregulated CAMs expression and increased the co-localization. Cholesterol exposure decreased the level of CAMs in the caveolae. Co-immunoprecipitation and confocal microscopy revealed that ICAM-1 interacted with Cav-1. Electron microscopy showed that ICAM-1 was mainly located in caveolae. Cholesterol exposure decreased this interaction and drove ICAM-1 out of caveolae. Knockdown of Cav-1 reduced the synergistic effects of cholesterol and inflammation. In vivo, ICAM-1 and Cav-1 co-localization was lower in the aortic endothelium of ApoE−/− mice than in that of wild-type controls. Cav-1 negatively regulates monocyte adhesion by the co-localization of CAMs in caveolae, which is disturbed by cholesterol. Thus, our study suggests a molecular basis underlying the synergistic effects of hypercholesterolemia and inflammation in atherogenesis.
Keywords: Endothelial; Caveolae; ICAM-1; Cholesterol; Atherosclerosis
Generation of lysophosphatidylinositol by DDHD domain containing 1 (DDHD1): Possible involvement of phospholipase D/phosphatidic acid in the activation of DDHD1 by Atsushi Yamashita; Tsukasa Kumazawa; Hiroki Koga; Naotaka Suzuki; Saori Oka; Takayuki Sugiura (pp. 711-720).
GPR55 is a seven-transmembrane G-protein-coupled receptor that has been proposed as a novel type of cannabinoid receptor. Previously, we identified lysophosphatidylinositol (LPI), in particular 2-arachidonoyl-LPI, as an agonist for GPR55. In the present study, we examined whether intracellular phospholipase A1 (DDHD domain containing 1, or DDHD1), previously identified as phosphatidic acid (PA)-preferring PLA1 (PA-PLA1), is involved in the formation of 2-arachidonoyl-LPI. HEK293 cells expressing DDHD1 produced [3H]arachidonic acid-containing LPI after prelabeling with [3H]arachidonic acid and subsequent activation by ionomycin; the formation of [3H]LPI was inhibited by n-butanol and the overexpression of an inactive PLD1 mutant PLD1K898R. DDHD1 was translocated from the cytosol to membranes upon ionomycin treatment. A purified recombinant DDHD1 formed [3H]LPI when incubated with [3H]PI; the Vmax and apparent Km were 190µmol/min/mg protein and 10mol% PI, respectively. DDHD1 binds PA, and the addition of PA to DDHD1 increased the affinity for PI ( Km ; 3mol%) and augmented the PI-PLA1 activity. DDHD1 activated by PA was returned to a basal state by its own PA-hydrolytic activity. These results implicate DDHD1 in the formation of 2-arachidonoyl-LPI and indicate that the process is modulated by PA released by phospholipase D. Similar observations for the production of arachidonic acid-containing LPI in neuroblastoma cells suggest the DDHD1-LPI-GPR55 axis to be involved in functions in the brain.
Keywords: Abbreviations; FFA; free fatty acid; GPCR; G-protein-coupled receptor; LPA; lysophosphatidic acid; LPI; lysophosphatidylinositol; MAFP; methyl arachidonoyl fluorophosphate; PA; phosphatidic acid; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PI; phosphatidylinositol; PLA1; phospholipase A1; PLA2; phospholipase A2; PS; phosphatidylserine; SDS–PAGE; SDS–polyacrylamide gel electrophoresisLysophosphatidylinositol; Lysophospholipid; Phospholipase A1; Cannabinoid receptor; GPR55; Phosphatidic acid
Increase of hepatic fat accumulation by liver specific expression of Hepatitis B virus X protein in zebrafish by Yun-Sheng Shieh; Yin-Shan Chang; Jiann-Ruey Hong; Li-Je Chen; Luen-Kuang Jou; Chia-Chun Hsu; Guor Mour Her (pp. 721-730).
The pathogenesis of fatty liver disease remains largely unknown. Here, we assessed the importance of hepatic fat accumulation on the progression of hepatitis in zebrafish by liver specific expression of Hepatitis B virus X protein (HBx). Transgenic zebrafish lines, GBXs, which selectively express the GBx transgene (GFP-fused HBx gene) in liver, were established. GBX Liver phenotypes were evaluated by histopathology and molecular analysis of fatty acid (FA) metabolism-related genes expression. Most GBXs (66–81%) displayed obvious emaciation starting at 4months old. Over 99% of the emaciated GBXs developed hepatic steatosis or steatohepatitis, which in turn led to liver hypoplasia. The liver histology of GBXs displayed steatosis, lobular inflammation, and balloon degeneration, similar to non-alcoholic steatohepatitis (NASH). Oil red O stain detected the accumulation of fatty droplets in GBXs. RT-PCR and Q-rt-PCR analysis revealed that GBx induced hepatic steatosis had significant increases in the expression of lipogenic genes, C/EBP-α, SREBP1, ChREBP and PPAR-γ, which then activate key enzymes of the de novo FA synthesis, ACC1, FAS, SCD1, AGAPT, PAP and DGAT2. In addition, the steatohepatitic GBX liver progressed to liver degeneration and exhibited significant differential gene expression in apoptosis and stress. The GBX models exhibited both the genetic and functional factors involved in lipid accumulation and steatosis-associated liver injury. In addition, GBXs with transmissible NASH-like phenotypes provide a promising model for studying liver disease.
Keywords: Abbreviations; HBV; Hepatitis B virus; HBx; HBV X protein; HCC; hepatocellular carcinoma; GBXs; zebrafish transgenic GBX lines; GBx; GFP-fused HBx; C/EBP-α; CCAAT enhancer binding protein-alpha; PPAR-γ; peroxisome proliferator-activated receptor gamma; SREBP1; sterol regulatory element binding protein1; ChREBP; carbohydrate-response element-binding protein; ACC; Acetyl-CoA carboxylase 1; FAS; Fatty acid synthase; SCD; stearoyl-CoA desaturase 1; AGAPT; acyl-CoA: 1-acylglycerol-sn-3-phosphate acyltransferase; PAP; phosphatidic acid phosphatase; DGAT2; diacylglycerol O-acyltransferase 2Hepatitis B virus X protein; Fatty liver disease; Non-alcoholic steatohepatitis; Liver degeneration; Transgenic zebrafish; GFP
Decrease in expression or activity of cytosolic phospholipase A2α increases cyclooxygenase-1 action: A cross-talk between key enzymes in arachidonic acid pathway in prostate cancer cells by Marzieh Niknami; Soma Vignarajan; Mu Yao; Sheng Hua; Paul K. Witting; Yoshihiro Kita; Takao Shimizu; Paul Sved; Manish I. Patel; Qihan Dong (pp. 731-737).
The eicosanoid pathway is activated in many types of cancers including prostate. Eicosanoids are synthesized from intracellular arachidonic acid (AA), which is released from membrane glycerophospholipids mainly by the action of cytosolic phospholipase A2α (cPLA2α). Thus, targeting cPLA2α has been proposed as a treatment option. The aim of this study was to determine the effect of cPLA2α inhibition on cyclooxygenase (COX) expression and PGE2 production. Inhibition of cPLA2α expression by siRNA or activity by Efipladib in prostate cancer cell lines (PC3 and LNCaP) led to an increase in COX-1 protein and PGE2 levels in a dose-dependent manner from 24 to 72h. The COX-2 response was less evident. Efipladib treatment increased COX-1 promoter transcriptional activity without changing the rate of COX-1 protein degradation. Treatment with Efipladib also led to a decrease in most LOX products (HETEs) as measured by LC/MS/MS. Replenishing 5- and 12-HETEs abolished Efipladib-induced COX-1 and PGE2 levels. Decreasing 5- and 12-HETE production, as a result of treating cells with inhibitors MK886 and Baicalein, respectively, mimicked the effect of Efipladib on COX-1 and PGE2 levels. Hence, the mechanism underlying the cPLA2α inhibition-induced COX-1 is likely due to a decrease in LOX products, which may exert a negative feedback on COX-1 gene expression in prostate cancer cells. Considering that PGE2 is a potent promoter of cancer cell proliferation and survival, understanding the mechanism coupling cPLA2α with COX-1 is of potential clinical significance.
Keywords: Abbreviations; cPLA; 2; α; cytosolic phospholipase A; 2; α; AA; arachidonic acid; COX; cyclooxygenase; LOX; lipoxygenase; HETE; hydroxyeicosatetraenoic acid; PGE2; prostaglandin E2; PC; prostate cancer; DMSO; dimethylsulfoxidecPLA; 2; -α; Cyclooxygenase; Lipoxygenase; Prostate cancer
Naturally occurring human plasminogen, like genetically related apolipoprotein(a), contains oxidized phosphatidylcholine adducts by Celina Edelstein; Ditta Pfaffinger; Ming Yang; John S. Hill; Angelo M. Scanu (pp. 738-745).
Human apolipoprotein(a) (apo(a)), synthesized in the liver, contains oxidized phosphatidylcholine (oxPtdPC) adducts probably generated at the hepatic site. Since plasminogen (Plg), also synthesized in the liver, is genetically related and structurally homologous to apo(a), we wanted to determine whether it contains oxPtdPCs and their location. We used Plg isolated from fresh or frozen normal human plasma and several commercial preparations. Some were freed of non-covalently bound lipids by organic solvent extraction. By immunoblot analyses, all products reacted against T15, a natural IgM monoclonal antibody specific for phosphorylcholine -containing oxidized phospholipids (ox-PLs). This immunoreactivity was retained in urokinase type plasminogen activator -generated plasmin and was abrogated in Plg previously digested with lipoprotein-associated phospholipase A2 (Lp-PLA2), a reaction that generated predominantly C16:0 lysophosphatidylcholine species as determined by mass spectrometry. Lyso derivatives were also generated upon the cleavage by Lp-PLA2 of a model ox-PL chemically linked to a lysine-containing pentapeptide. From inorganic phosphorous analyses, we found 2mol of oxPtdPC/mole of Plg distributed between the kringles 1–4 and mini-Plg domain. OxPtdPCs were also present in the Plg isolated from the serum-free medium of cultured human HepG2 cells. In conclusion, our results provide strong evidence that naturally occurring Plg contains oxPtdPC probably linked by a Schiff base and also suggest that the linkage occurs at the hepatic site. Given the emerging evidence for the cardiovascular pathogenicity of oxPtdPCs, we speculate that they may impart athero-thrombogenic properties to Plg under inflammatory conditions.
Keywords: Abbreviations; apo(a); apolipoprotein(a); oxPtdPC; oxidized phosphatidylcholine; Lp-PLA; 2; lipoprotein-associated phospholipase A; 2; Plg; plasminogen; Glu-Plg; plasminogen native form; PL; phospholipid; PC; phosphorylcholine; PLC; phospholipase C; uPA; urokinase type plasminogen activator; POVPC; 1-palmitoyl-2-(5′-oxo)valeroyl-; sn; -glycero-3-phosphorylcholinePlasminogen; Phosphatidylcholine; Lysophosphatidylcholine; Oxidation; Monoclonal antibody T15