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BBA - Molecular and Cell Biology of Lipids (v.1734, #1)

Editorial Board (pp. ii).

Increases in NMR-visible lipid and glycerophosphocholine during phenylbutyrate-induced apoptosis in human prostate cancer cells by Matthew Milkevitch; Hyunsuk Shim; Ulrich Pilatus; Stephen Pickup; Janna P. Wehrle; Dvorit Samid; Harish Poptani; Jerry D. Glickson; Edward J. Delikatny (pp. 1-12).

DU145 human prostatic carcinoma cells were treated with the differentiating agents phenylacetate (PA) and phenylbutyrate (PB) and examined in perfused cultures by diffusion-weighted¹H and³¹P nuclear magnetic resonance spectroscopy (NMR). PA and PB (10 mM) induced significant (>3-fold) time-dependent increases in the level of NMR-visible lipids and total choline in¹H spectra, and glycerophosphocholine levels in the³¹P spectra, with the increases being greater for PB. These effects were accompanied by significant increases in cytoplasmic lipid droplets and intracellular lipid volume fraction as observed by morphometric analysis of Oil Red O-stained cells. PB treatment caused cell cycle arrest in the G₁ phase and induction of apoptosis. In contrast, PA-treated DU145 cells showed an accumulation of cells in G₂/M and no evidence of apoptosis. These results demonstrate that significant differences exist in the mechanism of PA and PB activity, although both compounds cause similar, but graded alterations in lipid metabolism. The simultaneous accumulation of mobile lipid and glycerophosphocholine suggests that PB and PA induce phospholipid catabolism via a phospholipase-mediated pathway. The mobile lipid accumulation following the induction of either apoptosis and cytostasis by related differentiating agents indicate that the presence of NMR-visible lipids may not be a specific event causally resulting from the induction of apoptosis.

Keywords: Abbreviations; CHESS; chemical shift selective (water suppression); EMEM; Eagle's Minimum Essential Medium; FACS; fluorescence activated cell sorter; FBS; fetal bovine serum; G; gauss; GPC; sn; -glycero-3-phosphocholine; NMR; nuclear magnetic resonance; NTP; nucleoside triphosphate; PA; phenylacetate; PB; phenylbutyrate; PBS; phosphate buffered saline; PPAR; peroxisome proliferator-activated receptor; rf; radiofrequency; tCho; total choline; TE; echo time; TM; mixing time; TUNEL; terminal deoxynucleotidyl transferase dUTP nick end labelingPhenylacetate; Phenylbutyrate; NMR; Differentiation; Apoptosis; NMR-visible lipid; Glycerophosphocholine

Environmental and dietary influences on highly unsaturated fatty acid biosynthesis and expression of fatty acyl desaturase and elongase genes in liver of Atlantic salmon ( Salmo salar) by Xiaozhong Zheng; Bente E. Torstensen; Douglas R. Tocher; James R. Dick; R. James Henderson; J. Gordon Bell (pp. 13-24).

Highly unsaturated fatty acid (HUFA) synthesis in Atlantic salmon ( Salmo salar) was known to be influenced by both nutritional and environmental factors. Here we aimed to test the hypothesis that both these effectors involved similar molecular mechanisms. Thus, HUFA biosynthetic activity and the expression of fatty acyl desaturase and elongase genes were determined at various points during an entire 2 year production cycle in salmon fed diets containing either 100% fish oil or diets in which a high proportion (75% and 100%) of fish oil was replaced by C₁₈ polyunsaturated fatty acid-rich vegetable oil. The results showed that HUFA biosynthesis in Atlantic salmon varied during the growth cycle with peak activity around seawater transfer and subsequent low activities in seawater. Consistent with this, the gene expression of Δ6 desaturase, the rate-limiting step in the HUFA biosynthetic pathway, was highest around the point of seawater transfer and lowest during the seawater phase. In addition, the expression of both Δ6 and Δ5 desaturase genes was generally higher in fish fed the vegetable oil-substituted diets compared to fish fed fish oil, particularly in the seawater phase. Again, generally consistent with this, the activity of the HUFA biosynthetic pathway was invariably higher in fish fed diets in which fish oil was substituted by vegetable oil compared to fish fed only fish oil. In conclusion, these studies showed that both nutritional and environmental modulation of HUFA biosynthesis in Atlantic salmon involved the regulation of fatty acid desaturase gene expression.

Keywords: Abbreviations; FO; fish oil; HUFA; highly unsaturated fatty acids (carbon chain length ≥C; ₂₀; with ≥3 double bonds); VO; vegetable oilPolyunsaturated fatty acid; Gene; Desaturase; Elongase; Nutrition; Salmon

Role of MEK-ERK pathway in sphingosylphosphorylcholine-induced cell death in human adipose tissue-derived mesenchymal stem cells by Eun Su Jeon; Yong Jung Kang; Hae Young Song; Jae Suk Woo; Jin Sup Jung; Yong Keun Kim; Jae Ho Kim (pp. 25-33).

Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in a variety of cellular responses. In the present study, we demonstrated that treatment of human adipose tissue-derived mesenchymal stem cells (hATSCs) withd- erythro-SPC resulted in apoptosis-like cell death, as demonstrated by decreased cell viability, DNA strand breaks, the increase of sub-G1 fraction, cytochrome c release into cytosol, and activation of caspase-3. In contrast, the exposure of hATSCs tol- threo-SPC did not induce the cell death, suggesting that the SPC-induced cell death was selective for thed- erythro-stereoisomer of SPC. Thed- erythro-SPC-induced cell death was prevented by DEVD-CHO, a caspase-3 specific inhibitor, and Z-VAD-FMK, a general caspase inhibitor, suggesting that the SPC-induced cell death of hATSCs occurs through the cytochrome c- and caspase-3-dependent pathways. In addition,d- erythro-SPC treatment stimulated the activation of mitogen-activated protein kinases, such as ERK and c- Jun NH₂-terminal protein kinase (JNK), and thed- erythro-SPC-induced cell death was completely prevented by pretreatment with the MEK inhibitor, U0126, but not by pretreatment with the JNK inhibitor, SP600125, and the p38 MAPK inhibitor, SB202190, suggesting a specific involvement of ERK in thed- erythro-SPC-induced cell death. Pretreatment with U0126 attenuated thed- erythro-SPC-induced release of cytochrome c. From these results, we suggest that ERK is involved in the SPC-induced cell death of hATSC through stimulation of the cytochrome c/caspase-3-dependent pathway.

Keywords: Abbreviations; MSCs; mesenchymal stem cells; SPC; sphingosylphosphorylcholine; MAPKs; mitogen-activated protein kinases; ERK; extracellular signal-regulated kinase; JNK; c-; Jun; NH2-terminal protein kinase; GPCRs; G protein-coupled receptors; hATSCs; human adipose tissue-derived mesenchymal stem cells; PBS; phosphate-buffered saline; MTT; 3-(4,5-dimethyl-2-thiozol)-2,5-diphenyl-2; H; -tetrazolium bromide; DMSO; dimethylsulfoxide; TUNEL; terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelingMesenchymal stem cell; Adipose tissue; SPC; Cell death; ERK

Isolation of very low density lipoprotein phospholipids enriched in ethanolamine phospholipids from rats injected with Triton WR 1339 by Jyrki J. �gren; Juha-Pekka Kurvinen; Arnis Kuksis (pp. 34-43).

Phospholipids carried by very low density lipoprotein (VLDL) are hydrolysed in circulation by lipoprotein and hepatic lipases and lecithin-cholesterol acyltransferase. We have previously demonstrated [6] [J.J. Ågren, A. Ravandi, A. Kuksis, G. Steiner, Structural and compositional changes in very low density lipoprotein triacylglycerols during basal lipolysis, Eur. J. Biochem. 269 (2002) 6223–6232] that the infusion of Triton WR 1339 (TWR), which inhibits these lipases, leads in 2 h to five-fold increase in VLDL triacylglycerol concentration along with major differences in the composition of their molecular species. The present study demonstrates that the accumulation of triacylglycerols is accompanied by major changes in the content of the VLDL phospholipids, of which the most significant is the enrichment of phosphatidylethanolamine (PtdEtn). This finding coincides with the enrichment in PtdEtn demonstrated in the VLDL of a hepatocytic Golgi fraction but it had not been demonstrated that the Golgi VLDL, along with its unusual phospholipid composition, can be directly transferred to plasma. Aside from providing an easy access to nascent plasma VLDL, the TWR infusion demonstrates that lipoprotein and hepatic lipases are also responsible for the degradation of plasma VLDL PtdEtn, as independently demonstrated for plasma phosphatidylcholine. Our results indicate also, with the exception of lysophosphatidylcholine, that preferential basal hydrolysis no dot lead to major differences in molecular species composition between circulating and newly secreted VLDL phospholipids. The comparison of the molecular species composition of VLDL and liver phospholipids suggests a selective secretion of PtdEtn and sphingomyelin molecular species during VLDL secretion.

Keywords: Very low density lipoprotein; Phospholipid; Phosphatidylethanolamine; Phosphatidylcholine; Sphingomyelin; Molecular species

Endocytosis is not required for the selective lipid uptake mediated by murine SR-BI by Thomas J.F. Nieland; Marcelo Ehrlich; Monty Krieger; Tomas Kirchhausen (pp. 44-51).

The scavenger receptor class B, type I (SR-BI) mediates the cellular selective uptake of cholesteryl esters and other lipids from high-density lipoproteins (HDL) and low-density lipoproteins (LDL). This process, unlike classical receptor-mediated endocytosis, does not result in lipoprotein degradation. Instead, the lipid depleted particles are released into the medium. Here we show that selective lipid uptake mediated by murine SR-BI can be uncoupled from the endocytosis of HDL or LDL particles. We found that blocking selective lipid uptake by incubating cells with the small chemical inhibitors BLT-1 or BLT-4 did not affect endocytosis of HDL. Similarly, blocking endocytosis by hyperosmotic sucrose or K⁺ depletion did not prevent selective lipid uptake from HDL or LDL. These findings suggest that mSR-BI-mediated selective uptake occurs at the cell surface upon the association of lipoproteins with mSR-BI and does not require endocytosis of HDL or LDL particles.

Keywords: High-density lipoprotein; Low-density lipoprotein; Scavenger receptor; Scavenger receptor class B, type I; Endocytosis; BLT

Agonist-induced activation releases peroxisome proliferator-activated receptor β/δ from its inhibition by palmitate-induced nuclear factor-κB in skeletal muscle cells by Mireia Jov�; Juan C. Laguna; Manuel V�zquez-Carrera (pp. 52-61).

The mechanisms by which elevated levels of free fatty acids cause insulin resistance are not well understood, but there is a strong correlation between insulin resistance and intramyocellular lipid accumulation in skeletal muscle. In addition, accumulating evidence suggests a link between inflammation and type 2 diabetes. The aim of this work was to study whether the exposure of skeletal muscle cells to palmitate affected peroxisome proliferator-activated receptor (PPAR) β/δ activity. Here, we report that exposure of C2C12 skeletal muscle cells to 0.75 mM palmitate reduced (74%, P<0.01) the mRNA levels of the PPARβ/δ-target gene pyruvatedehydrogenase kinase 4 (PDK-4), which is involved in fatty acid utilization. This reduction was not observed in the presence of the PPARβ/δ agonist L-165041. This drug prevented palmitate-induced nuclear factor (NF)-κB activation. Increased NF-κB activity after palmitate exposure was associated with enhanced protein–protein interaction between PPARβ/δ and p65. Interestingly, treatment with the PPARβ/δ agonist L-165041 completely abolished this interaction. These results indicate that palmitate may reduce fatty acid utilization in skeletal muscle cells by reducing PPARβ/δ signaling through increased NF-κB activity.

Keywords: Nuclear factor-κB; Peroxisome proliferator-activated receptor β/δ; Palmitate; Pyruvatedehydrogenase kinase-4; L-165041

Acidic residues emulate a phosphorylation switch to enhance the activity of rat hepatic neutral cytosolic cholesterol esterase by E. Milad-Kodsi; T.B. Langston; M.R. Gergis; W.M. Grogan (pp. 62-73).

Site-directed mutagenesis of rat hepatic neutral cytosolic cholesteryl ester hydrolase (rhncCEH) was used to substitute acidic, basic or neutral amino acid residues for Ser506, required for activation by protein kinase A. The substitution of acidic Asp506 resulted in esterase activities with cholesteryl oleate, p-nitrophenylcaprylate (PNPC) and p-nitrophenylacetate (PNPA) equivalent to those of native rhncCEH with Ser506. The substitution of 2 acidic residues (Asp505/506), emulating the 2 negative charges of phosphoserine, resulted in a 10-fold greater cholesterol esterase activity than that of native rhncCEH, similar to the activity of rhncCEH treated with protein kinase A. In contrast to mutants with Ser506, protein kinase A did not increase the specific activities of mutants with Asp505/506. The substitution of basic (Lys506) or neutral (Asn506) residues abolished activity with cholesteryl oleate but not PNPC or PNPA. The substitution of neutral Gln for basic residues Lys496/Arg503 also abolished cholesterol esterase activity but not PNPC- and PNPA-esterase activities. These structure–activity relationships are modeled by homology with a recently reported crystal structure for the homologous human triacylglycerol hydrolase. The results suggest that the cholesterol esterase activity of carboxylesterases is enhanced by interactions between one or more basic residues on helix α16 (residues 485–503) and acidic groups at residues 505–506 in the adjacent surface loop.

Keywords: Abbreviations; α16; α-helix 16 (residues 485–503) of rhncCEH and RLCE; CEL; carboxylester lipase (bile salt stimulated lipase); rhncCEH; rat hepatic neutral cytosolic cholesteryl ester hydrolase; rLCE; rat lung cholesteryl ester hydrolase

Increase in vulnerability to oxidative damage in cholesterol-modified erythrocytes exposed to t-BuOOH by Abel L�pez-Revuelta; Jos� I. S�nchez-Gallego; Angel Hern�ndez-Hern�ndez; Jes�s S�nchez-Yag�e; Marcial Llanillo (pp. 74-85).

During the course of radical oxidation, cholesterol may exert seemingly contradictory effects. In order to gain a better understanding of the relationship between cholesterol levels and membrane susceptibility to oxidative damage induced by reactive oxygen species (ROS), here we analyze the integrity and structural stability of cholesterol-modified (enriched or depleted) and unmodified (control) erythrocytes exposed to tert-butyl hydroperoxide. The oxidant significantly increased ROS production, with almost complete oxidation of hemoglobin and a reduction in GSH content in the different erythrocyte groups at 2 mM concentration. These changes were accompanied by losses of cholesterol and total phospholipids, the main decreases being in phosphatidylethanolamine and phosphatidylcholine. The highest lipid loss was found in the cholesterol-depleted group. Fatty acid analyses revealed changes only in peroxidized cholesterol-modified erythrocytes, with decreases in linoleic and arachidonic acids. Fluorescence anisotropy studies showed an increase in the fluidity of the negatively charged surface of peroxidized control erythrocytes. Increased hemolysis and a positive correlation between cellular osmotic fragility and malondialdehyde contents were found in all peroxidized groups. These findings provide evidence that the modification of cholesterol levels in the erythrocyte membrane has provoking effects on peroxidation, with corresponding increases in oxidative damage in the treated cell, possibly as a consequence of lipid bilayer destabilization.

Keywords: Abbreviations; BCD; β-cylodextrin; Ch; cholesterol; DCF; 2′,7′-dichlorofluorescein; DPH; 1,6-diphenyl-1,3,5-hexatriene; Hb; hemoglobin; MBCD; methyl-β-cyclodextrin; MDA; malonyldialdehyde; MetHb; methemoglobin; PL; phospholipids; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PS+PI; phosphatidylserine+phospatidylinositol; PUFA; polyunsaturated fatty acids; PVP; polyvinylpyrrolidone; ROS; reactive oxygen species; SM; sphingomyelin; t; -BuOOH; tert; -butylhydroperoxide; TBARS; thiobarbituric acid-reactive substances; TMA-DPH; 1[4′-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatrieneCholesterol; Oxidative damage; Lipid oxidation; tert; -Butyl hydroperoxide; Erythrocyte

Erratum to “High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope corrections algorithm�? [Biochimica et Biophysics Acta, 1686 (2004) 108–117] by Gerhard Liebisch; Bernd Lieser; Jan Rahtenberg; Wolfgang Drobnik; Gerd Schmitz (pp. 86-89).

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BBA - Molecular and Cell Biology of Lipids (v.1734, #1)