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BBA - Molecular and Cell Biology of Lipids (v.1761, #1)

Editorial Board (pp. ii).

Reviewer Acknowledgement (pp. 1-3).

Pancreatic lipase and pancreatic lipase-related protein 2, but not pancreatic lipase-related protein 1, hydrolyze retinyl palmitate in physiological conditions by Emmanuelle Reboul; Am�lie Berton; Myriam Moussa; Corinne Kreuzer; Isabelle Crenon; Patrick Borel (pp. 4-10).

The major sources of vitamin A in the human diet are retinyl esters (mainly retinyl palmitate) and provitamin A carotenoids. It has been shown that classical pancreatic lipase (PL) is involved in the luminal hydrolysis of retinyl palmitate (RP), but it is not known whether pancreatic lipase-related proteins 1 (PLRP1) and 2 (PLRP2), two other lipases recovered in the human pancreatic juice, are also involved. The aim of this study was to assess whether RP acts a substrate for these lipase-related proteins. Pure horse PL, horse PLRP2 and dog PLRP1 were incubated with RP solubilized in its physiological vehicles, i.e., triglyceride-rich lipid droplets, mixed micelles and vesicles. High performance liquid chromatography (HPLC) was used to assess RP hydrolysis by the free retinol released in the incubation medium. Incubation of RP-containing emulsions with horse PL and colipase resulted in RP hydrolysis (0.051±0.01 μmol/min/mg). This hydrolysis was abolished when colipase was not added to the medium. PLRP2 and PLRP1 were unable to hydrolyze RP solubilized in emulsions, regardless of whether colipase was added to the medium. PL hydrolyzed RP solubilized in mixed micelles as well (0.074±0.014 μmol/min/mg). Again, this hydrolysis was abolished in the absence of colipase. PLRP2 hydrolyzed RP solubilized in micelles but less efficiently than PL (0.023±0.005 μmol/min/mg). Colipase had no effect on this hydrolysis. PLRP1 was unable to hydrolyze RP solubilized in micelles, regardless of whether colipase was present or absent. Both PL and PLRP2 hydrolyzed RP solubilized in a vesicle rich-solution, and a synergic phenomenon between the two lipases was enlighten. Taken together, these results show that (1) PL hydrolyzes RP whether RP is solubilized in emulsions or in mixed micelles, (2) PLRP2 hydrolyzes RP only when RP is solubilized in mixed micelles, and (3) PLRP1 is unable to hydrolyze RP regardless of whether RP is solubilized in emulsions or in mixed micelles.

Keywords: Vitamin A; Intestinal absorption; PLRP2; PLRP1; Pancreatic lipase-related protein; Micelles; Emulsion; Colipase

Regulation of Akt by arachidonic acid and phosphoinositide 3-kinase in angiotensin II-stimulated vascular smooth muscle cells by Maria L. Wildroudt; Ernest J. Freeman (pp. 11-16).

Angiotensin (Ang) II stimulates cytosolic phospholipase A2(cPLA₂)-dependent release of arachidonic acid (ArAc) in vascular smooth muscle cells (VSMC). ArAc release and production of reactive oxygen species (ROS) lead to the activation of downstream kinases resulting in VSMC growth. To determine the role of Akt in this pathway, we used VSMC to link Ang II-induced ArAc release and ROS production to the activation of Akt and VSMC growth. We observed that Ang II, ArAc, or H₂O₂ increased Akt activation. The Akt inhibitor SH6 blocked Ang II-, ArAc-, or H₂O₂-induced Akt activation, as did inhibition of phosphoinositide 3-kinase (PI₃K). Inhibition of cPLA₂ blocked Ang II effects, while the ROS scavenger NaC decreased Ang II- and ArAc-induced Akt activation. Inhibition of Akt blocked the³H-thymidine incorporation induced by all three agonists. Thus, Ang II-induced ArAc release and ROS production leads to the PI₃K-dependant activation of Akt and VSMC growth.

Keywords: Angiotensin II; Arachidonic acid; Akt; Phosphoinositide 3-kinase; Reactive oxygen species; Vascular smooth muscle cell

Acetate generation in rat liver mitochondria; acetyl-CoA hydrolase activity is demonstrated by 3-ketoacyl-CoA thiolase by Hiromi Yamashita; Ai Itsuki; Masumi Kimoto; Miki Hiemori; Hideaki Tsuji (pp. 17-23).

Acetate has been found as an endogenous metabolite of β-oxidation of fatty acids in liver. In order to investigate the regulation of acetate generation in liver mitochondria, we attempted to purify a mitochondrial acetyl-CoA hydrolase in rat liver. This acetyl-CoA-hydrolyzing activity in isolated mitochondria was induced by the treatment of rats with di(2-ehtylhexyl)phthalate (DEHP), a peroxisome proliferator which induces expression of several peroxisomal and mitochondrial enzymes involved in β-oxidation of fatty acids. The purified enzyme was 43-kDa in molecular mass by SDS/PAGE. Internal amino acid sequencing of this enzyme revealed that it was identical with mitochondrial 3-ketoacyl-CoA thiolase, suggesting that this enzyme has two kinds of activities, 3-ketoacyl-CoA thiolase and acetyl-CoA hydrolase activities. Kinetic studies clearly indicated that this enzyme had the both activities and each activity was inhibited by the substrates of the other activity, that is, 3-ketoacyl-CoA thiolase activity was inhibited by acetyl-CoA, on the other hand, acetyl-CoA hydrolase activity was inhibited by acetoacetyl-CoA in a competitive manner. These findings suggested that acetate generation in liver mitochondria is a side reaction of this known enzyme, 3-ketoacyl-CoA thiolase, and this enzyme may regulate its activities depending on each substrate level.

Keywords: Abbreviations; DEHP; di(2-ethylhexyl)phthalate; PAGE; polyacrylamide gel electrophoresis; BSA; bovine serum albumin; DTNB; 5,5′-dithiobis (2-nitrobenzoate); CoA; coenzyme AAcetyl-CoA hydrolase; 3-ketoacyl-CoA thiolase; Rat liver mitochondria; Acetate metabolism; Peroxisome proliferator; β-oxidation of fatty acidPACS; Enzymes: acetyl-CoA hydrolase (EC 3.1.2.1); 3-ketoacyl-CoA thiolase (EC 2.3.1.16)

Opposite effect of caveolin-1 in the metabolism of high-density and low-density lipoproteins by To Quyen Truong; Dominique Aubin; Philippe Bourgeois; Louise Falstrault; Louise Brissette (pp. 24-36).

Receptors of the scavenger class B family were reported to be localized in caveolae, the cell surface microdomains rich in free cholesterol and glycosphyngolipids, which are characterized by the presence of caveolin-1. Parenchymal hepatic and hepatoma HepG2 cells express very low levels of caveolin-1. In the present study, stable transformants of HepG2 cells expressing caveolin-1 were generated to address the effect of caveolin-1 on receptor activity. Compared to normal cells, these cells show higher¹²⁵I-bovine serum albumin (BSA) uptake and cholesterol efflux, two indicators of functional caveolae. By immunoprecipitation, cell fractionation and confocal analyses, we found that caveolin-1 is well colocalized with the cluster of differentiation-36 (CD36) and the low-density lipoprotein (LDL) receptor (LDLr) but to a lesser extent with the scavenger receptor class B type I (SR-BI) in HepG2 cells expressing caveolin-1. However, caveolin-1 expression favors the dimerization of SR-BI. Two clones of cells expressing caveolin-1 were investigated for their lipoprotein metabolism activity. Compared to normal cells, these cells show a 71–144% increase in¹²⁵I-LDL degradation. The analysis of the cholesteryl esters (CE)-selective uptake (CE association minus protein association) revealed that the expression of caveolin-1 in HepG2 cells decreases by 59%–73% LDL-CE selective uptake and increases high-density lipoprotein (HDL)-CE selective uptake by 44%–66%. We conclude that the expression of caveolin-1 in HepG2 cells moves the balance of LDL degradation/CE selective uptake towards degradation and favors HDL-CE selective uptake. Thus, in the normal hepatic parenchymal situation where caveolin-1 is poorly expressed, LDL-CE selective uptake is the preferred pathway.

Keywords: Caveolin-1; LDL; HDL; SR-BI; Cholesteryl ester selective uptake

Effects of interleukin-15 on lipid oxidation by Vanessa Almendro; S�lvia Busquets; Elisabet Ametller; Neus Carb�; Maite Figueras; Gemma Fuster; Josep M. Argil�s; Francisco J. L�pez-Soriano (pp. 37-42).

Interleukin 15 (IL-15) has previously been shown to have important effects on lipid metabolism in adipose tissue, particularly influencing the rate of the de novo fatty acid synthesis. The results presented here show that chronic administration to rats (100 μg/kg body weight) has important effects on the metabolic fate of an exogenous [¹⁴C]-triolein load, decreasing the incorporation of lipid into adipose tissue and significantly increasing the total¹⁴CO₂ formation from [¹⁴C]-triolein. Skeletal muscle and possibly liver seem to be the main organs involved in the action of IL-15 on lipid oxidation, since the presence of the cytokine in incubated EDL muscle with [¹⁴C]-palmitic acid increased¹⁴CO₂ formation by 39%. Concerning the mechanism, the results suggest that the transport of fatty acids into mitochondria could be involved in the action of IL-15 since the cytokine clearly increases the presence of L-CPT-I and CPT-II in liver tissue. In addition, IL-15 treatment resulted in a significant increment in the gene expression of PPARδ, a transcription factor clearly related with lipid catabolism in many tissues. Altogether, the results presented here suggest that IL-15 alters exogenous lipid partitioning, limiting adipose tissue uptake and favouring oxidation.

Keywords: IL-15; Lipid metabolism; Skeletal muscle; Liver; PPAR

Sphingosine 1-phosphate inhibits cell migration in C2C12 myoblasts by Laura Becciolini; Elisabetta Meacci; Chiara Donati; Francesca Cencetti; Elena Rapizzi; Paola Bruni (pp. 43-51).

This study shows that sphingosine 1-phosphate (S1P) exerts an anti-migratory action in C2C12 myoblasts by reducing directional cell motility and fully abrogating the chemotactic response to insulin-like growth factor-1. The anti-migratory response to S1P required ligation to S1P₂, being attenuated in myoblasts where the receptor was down-regulated by specific antisense oligodeoxyribonucleotides or small interfering RNA (siRNA) and conversely potentiated in S1P₂-overexpressing myoblasts. The investigation of RhoA and Rac GTPases, critically implicated in cell motility regulation, demonstrated that RhoA was rapidly activated by S1P, while Rac1 was unaffected within the first 5 min but stimulated thereafter. RhoA, but not Rac activation, was identified as a S1P₂-dependent pathway in experiments in which receptor expression was attenuated by siRNA treatment or up-regulated by S1P₂-encoding plasmid transfection. Finally, by expression of the dominant negative mutant of RhoA, the GTPase was found implicated in the anti-migratory action of S1P, whereas modulation of Rac1 functionality unaffected the antichemotactic effect of S1P, ruling out a role for this protein in the biological response. Since S1P was previously shown to inhibit myoblast proliferation and stimulate myogenesis, the here identified novel biological activity is in favour of a complex physiological role of the sphingolipid in the process of muscle repair.

Keywords: Abbreviations; IGF-1; insulin-like growth factor 1; S1P; sphingosine 1-phosphate; S1PR; sphingosine 1-phosphate receptors; PBS; phosphate-buffered saline, FCS fetal calf serum; BSA; bovine serum albumin; ODN; oligodeoxyribonucleotides; siRNA; small interfering RNA; ECL; enhanced chemiluminescenceSphingosine 1-phosphate; Cell migration; S1P; ₂; receptor; RhoA; C2C12 myoblast

Sphingolipid biosynthesis in pathogenic fungi: Identification and characterization of the 3-ketosphinganine reductase activity of Candida albicans and Aspergillus fumigatus by Michelle Fornarotto; Li Xiao; Yan Hou; Keith A. Koch; Edcon Chang; Robert M. O'Malley; Todd A. Black; Michael B. Cable; Scott S. Walker (pp. 52-63).

An early step in sphingolipid biosynthesis, the reduction of 3-ketosphinganine, is catalyzed in the yeast Saccharomyces cerevisiae by Tsc10p ( TSC10 ( YBR265W)). We have identified orthologs of TSC10 in two clinically important fungal pathogens, Candida albicans and Aspergillus fumigatus. The translated sequences of the putative C. albicans ortholog, KSR1 (orf6.5112), and the putative A. fumigatus ortholog, ksrA, show significant homology to the yeast protein. All three proteins contain the signature motifs of NAD(P)H-dependent oxidoreductases in the short-chain dehydrogenase/reductase family and a conserved putative substrate-binding domain. Despite being essential in S. cerevisiae, we demonstrate that the C. albicans ortholog, KSR1, is not required for cell viability. However, ksr1 null mutants produce lower levels of inositolphosphorylceramides, are significantly more sensitive than the wildtype to an inhibitor of a subsequent step in sphingolipid biosynthesis, and are defective for the transition from yeast to filamentous growth, a key virulence determinant. Recombinant, purified Ksr1p and KsrA can carry out the reduction of 3-ketosphinganine in an NADPH-dependent manner. Molecular modeling of Ksr1p with bound substrates suggests that a significant portion of the aliphatic chain of 3-ketosphinganine protrudes from the enzyme. Guided by this molecular model, we developed shorter, water-soluble derivatives of 3-ketosphinganine that are substrates for 3-ketosphinganine reductase.

Keywords: Yeast; Fungal; Sphingolipid; Mutant; Filamentation; Oxidoreductase

Apolipoprotein A-I lysine modification: Effects on helical content, lipid binding and cholesterol acceptor activity by Gregory Brubaker; Dao-Quan Peng; Benjamin Somerlot; Davood J. Abdollahian; Jonathan D. Smith (pp. 64-72).

We examined the role of the positively charged lysine residues in apoAI by chemical modification. Lysine modification by reductive methylation did not alter apoAI's net charge, secondary or tertiary structure as observed by circular dichroism and trytophan fluorescence, respectively, or have much impact on lipid binding or ABCA1-dependent cholesterol acceptor activity. Acetylation of lysine residues lowered the isoelectric point of apoAI, altered its secondary and tertiary structure, and led to a 40% decrease in cholesterol acceptor activity, while maintaining 93% of its lipid binding activity. Exhaustive lysine acetoacetylation lowered apoAI's isoelectric point, profoundly disrupted its secondary and tertiary structure, and led to 90% and 82% reductions in cholesterol acceptor and lipid binding activities, respectively. The dose-dependent acetoacetylation of an increasing proportion of apoAI lysine residues demonstrated that cholesterol acceptor activity was more sensitive to this modification than lipid binding activity, suggesting that apoAI lysine positive charges play an important role in ABCA1 mediated lipid efflux beyond the role needed to maintain α-helical content and lipid binding activity.

Keywords: Abbreviations; apoAI; apolipoprotein A-I; OPA; o-phthaldialdehyde; DGGB; DMEM supplemented with 50 mM glucose, 2 mM glutamine, and 0.2% BSA; PLC; phospholipase C; LDL; low density lipoprotein; WMF; wavelength of maximum fluorescenceReverse cholesterol transport; Lipid binding; Class A amphipathic helix; Circular dichroism; Alpha helical content

ATP-induced apoptosis of thymocytes is mediated by activation of P2X7 receptor and involves de novo ceramide synthesis and mitochondria by S. L�pine; H. Le Stunff; B. Lakatos; J.C. Sulpice; F. Giraud (pp. 73-82).

Thymocytes were reported to undergo apoptosis in the presence of extracellular ATP through the activation of the purinergic receptors P2X1R, P2X7R or both. We investigated the identity of the P2XR and the signaling pathways involved in ATP-mediated apoptosis. Apoptosis elicited by ATP was prevented by inhibition of P2X7R, or in thymocytes bearing a mutated P2X7R, and reproduced with a P2X7R agonist, but not with a P2X1R agonist. Stimulation of thymocytes with either ATP or a P2X7R agonist was found to stimulate a late de novo ceramide synthesis and mitochondrial alterations. Inhibition of either processes attenuated apoptosis. Interestingly, stimulation with either ATP or a P2X1R agonist induced an early ceramide accumulation and a weak caspases-3/7 activation that did not lead to apoptosis. In conclusion, de novo ceramide generation and mitochondrial alterations, both resulting from P2X7R activation, were implicated in ATP-induced thymocyte apoptosis.

Keywords: Abbreviations; BA; bongkrekic acid; BzATP; 3′-O(4-benzoylbenzoyl) ATP; C12-NBD-SM; N[12(7-nitro-2-1,3-benzoxidiazol-4-yl)amino]dodecanoyl]-sphingosine-1-phosphocholine; Cer; ceramide; CS; cycloserine; DAG; diacylglycerol; GSH; glutathione. DiOC; ₆; (3), 3,3′-dihexylocarbocyanine iodide; D-MAPP; (1S,2R)-; d; -; erythro; -2-(N-myristoylamino)-1-phenyl-1-propanol; FCS; fetal calf serum; FITC; fluorescein isothiocyanate; GlucCer; glucosylceramide; LC; long chain; NOE; N-oleoyl ethanolamine; oATP; oxidized ATP; P2XR; P2X receptor; PI; propidium iodide; PS; phosphatidylserine; PTP; permeability transition pore; SM; sphingomyelin; SMase; sphingomyelinase; Sp; sphingosine; SPT; serine palmitoyl transferase; SR33557; ((2-isopropyl-1-4-[3-; N; -methyl-N(3,4-dimetoxy-β-phenetyl)amino]-propyloxy) benzenesulfonyl)) indolizine; VLC; very long chain; z-VAD-fmk; N-benzoyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; αβmATP; αβ methylene ATP; β-Cl; β-chloro alanineCeramide; Purinergic receptor; Apoptosis; Thymocyte

Degradation of perilipin is mediated through ubiquitination-proteasome pathway by Guoheng Xu; Carole Sztalryd; Constantine Londos (pp. 83-90).

Perilipin protein coats the surface of intracellular lipid droplets and plays fundamental roles in lipid droplet formation and triacylglycerol hydrolysis. Perilipin is transcriptionally regulated through peroxisome proliferator-activated receptor and post-translationally stabilized by stored intracellular neutral lipids. In this study, we show that perilipin protein accumulates in transfected Chinese hamster ovary cells cultured in the presence of fatty acids but in turn is destabilized when lipid precursors for triacylglycerol synthesis are removed from culture serum. Adding fatty acids in the culture medium prevents the degradation of perilipin. Moreover, specific proteasome inhibitors, MG132, lactacystin, and ALLN, block the degradation, whereas inhibitors of other proteases are ineffective. Pulse-chase experiments confirm that perilipin is degraded through proteasome, a process that is inhibited by MG132 or ALLN and blunted by the addition of oleic acid. We have detected the co-immunoprecipitation of perilipin and ubiquitin, thus confirming that perilipin is conjugated to poly-ubiquitin and targeted for proteasomal degradation. Treatment with MG132 increases the expression of perilipin associated with lipid droplets as well as modestly throughout the cytosol. We conclude that the degradation of perilipin is mediated through an ubiquitination-proteasome pathway, which suggests another mode for the post-translational regulation of perilipin.

Keywords: Abbreviations; ADRP; adipose differentiation-related protein; ALLN; N-acetyl-L-leucyl-L-leucyl-L-norleucinal; BSA; bovine serum albumin; CHO; Chinese Hampster ovary; DMEM; Dulbecco's modified Eagles medium; FCS; fetal calf serum; G418; Geneticin; MG132; Z-leu-leu-leu-al; OA; oleic acid; PAT; Perilipin/ADRP/TIP47 domain; PBS; phosphate-buffered saline; TAG; triacylglycerolPerilipin; Ubiquitination; Proteasome; Degradation; Lipid droplet; Fatty acid; Triacylglycerol; Adipose differentiation-related protein

Immunolocalization and high affinity interactions of acyl-CoAs with proteins: An original study with anti-acyl-CoA antibodies by Paraskevi Diakou; C�cile Faurie; Juliette Puyaubert; Agn�s Hemar; Lilly Maneta-Peyret (pp. 91-99).

Anti-acyl-Coenzyme A (acyl-CoA) antibodies were used to detect fatty acyl-CoAs in cultured rat hippocampal neurons, in which important lipid metabolism and transport occur. Hippocampus was chosen because of his involvement in many cerebral functions and diseases. Immunofluorescence experiments showed an intense labelling within neurites and cell bodies. Labelling seems to be associated with vesicles and membrane domains. We have shown by immunoblot experiments that the labelling corresponded to acyl-CoAs which were in strong interaction with proteins, without being covalently bound to them. Immunoprecipitation experiments, followed by proteomic analysis, showed that anti-acyl-CoA antibodies were also able to immunoprecipitate multiprotein complexes, principally related to vesicle trafficking and/or to membrane rafts.

Keywords: Fatty acyl-CoA; Neuron; Anti-acyl-CoA antibody; Immunofluorescence; Raft

Triacylglycerol accumulation is not primarily affected in myotubes established from type 2 diabetic subjects by Michael Gaster; Henning Beck-Nielsen (pp. 100-110).

In the present study, we investigated triacylglycerol (TAG) accumulation, glucose and fatty acid (FA) uptake, and glycogen synthesis (GS) in human myotubes from healthy, lean, and obese subjects with and without type 2 diabetes (T2D), exposed to increasing palmitate (PA) and oleate (OA) concentrations with/without high glucose and/or high insulin concentrations for 4 days. We showed that these myotubes expressed an increased TAG accumulation ( P<0.001) without differences between groups. Chronically high insulin, but not high glucose concentrations, increases TAG accumulation by 25% ( P<0.001). Inhibition of oxidative phosphorylation by antimycin A and oligomyin was followed by a reduced lipid oxidation ( P<0.05) and increased TAG accumulation ( P<0.05), but only in the presence of FAs. Both chronic PA and OA exposure reduced the insulin-mediated PA and OA uptake (fold change) ( P<0.001), but could not induce insulin resistance at the level of glucose uptake, whereas high insulin concentrations induced insulin resistance ( P<0.001). Chronic, high PA, but not OA, induced insulin resistance at the GS level in control subjects ( P<0.05). The TAG content correlated negatively with insulin-stimulated FA uptake ( P<0.001), but did not correlate with insulin-stimulated glucose uptake for PA or OA ( P>0.05). These results indicate that (1) TAG accumulation is not primarily affected in skeletal muscle tissue of obese and T2D; (2) induced inhibition of oxidative phosphorylation is followed by TAG accumulation; (3) increasing FA and insulin availability, and reduced oxidative phosphorylation, and to a lesser extent glucose, are determinants for differences in intramyocellular TAG accumulation; (4) quantitative TAG content may not be the best marker for insulin resistance. Thus, increased TAG content in skeletal muscle of obese and T2D subjects is adaptive.

Keywords: Abbreviations; CPT-1; Carnitine palmitoyltransferase-1; FFA; Free fatty acids; OA; oleate; PA; palmitate; TAG; Triacylglycerol; T2D; Type 2 diabetic/type 2 diabetesGlucose metabolism; Insulin resistance; Lipid metabolism; Oleic acid; Palmitic acid; Skeletal muscle; T2D

Deletion and alanine mutation analyses for the formation of active homo- or hetero-dimer complexes of mouse choline kinase-α and -β by Huanan Liao; Chieko Aoyama; Kozo Ishidate; Hirobumi Teraoka (pp. 111-120).

Choline kinase (CK) is the first-step regulatory enzyme for the biosynthesis of phosphatidylcholine in all mammalian cells. It exists as at least three isoforms (α1, α2 and β) that are encoded by two separate genes termed ck-α and ck-β. The active enzyme has been proposed to consist of either their homo- or hetero-dimeric forms. Here, we report on the identification of several essential domains and amino acid residues involved in their active dimer formation. Full-length cDNAs or their truncated or alanine-mutated versions for mouse CK-α1 and CK-β tagged with either HA or Myc at their N-termini were expressed in COS-7 cells. Each dimer formation was analyzed by immuno-precipitation followed by Western blotting. Kinetic analysis for CK reaction was performed with different expression products. Both the N-terminal domain-1 and C-terminal portions (E424–K430 for CK-α1 and Q379–K385 for CK-β) were shown to be critical for the formation of active homo- or hetero-dimer complex. Interestingly, D320 in the CK-motif of CK-α1 was found to be essential for α1/α1 homo-dimerization but not for α1/β hetero-dimerization. A mutation of the corresponding D276 of CK-β to A276 did not show any effect on either its homo- or hetero-dimerization but it caused a strong inhibition of CK activity in either case.

Keywords: Choline kinase; Phosphocholine; Isoform; Homo-dimer; Hetero-dimer

High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS) by Gerhard Liebisch; Marion Binder; Rainer Schifferer; Thomas Langmann; Berta Schulz; Gerd Schmitz (pp. 121-128).

Analysis of free cholesterol (FC) is not well suited for electrospray ionization (ESI); however, cholesteryl ester (CE) form ammonium adducts in positive ion mode and generate a fragment ion of m/ z 369 upon collision-induced fragmentation. In order to allow parallel analysis of FC and CE using ESI tandem mass spectrometry (ESI-MS/MS), we developed an acetyl chloride derivatization method to convert FC to cholesteryl acetate (CE 2:0). Derivatization conditions were chosen to provide a quantitative conversion of FC to CE 2:0 without transesterification of naturally occurring CE species. FC and CE were analyzed by direct flow injection analysis using a fragment of m/ z 369 in a combination of selected reaction monitoring (SRM) and precursor ion scan for FC and CE, respectively. Quantification was achieved using deuterated D₇-FC and CE 17:0/CE 22:0 as internal standards as well as calibration lines generated by addition of FC and naturally occurring CE species to the respective sample matrix. The developed assay showed a precision and detection limit sufficient for routine analysis. A run time of 1.3 min and automated data analysis allow high throughput analysis. Loading of human skin fibroblast and monocyte derived macrophages with stable isotope labeled FC showed a potential application of this method in metabolism studies. Together with existing mass spectrometry methodologies for lipid analysis, the present methodology will provide a useful tool for clinical and biochemical studies and expands the lipid spectrum that can be analyzed from one lipid sample on a single instrumental platform.

Keywords: Abbreviations; CE; cholesteryl ester; CV; coefficient of variation; E-LDL; enzymatically modified LDL; FC; free cholesterol; IS; internal standard; MCSF; monocyte colony stimulating factor; SRM; selected reaction monitoring; TLC; thin layer chromatographyCholesterol; Cholesteryl ester; Lipid; Stable isotope labeling; Lipidomics; Sterol; Mass spectrometry; Electrospray ionization

Phosphorylation regulates mitochondrial glycerol-3-phosphate-1 acyltransferase activity in T-lymphocytes by Lauren W. Collison; Christopher A. Jolly (pp. 129-139).

Recently, we have shown that stimulation and recombinant ACBP increase mitochondrial glycerol-3-phosphate acyltransferase (mtGPAT) activity in rat splenic T-lymphocytes and that this effect is blunted in aged T-lymphocytes. In addition to decreased mtGPAT activity, aged T-lymphocytes also have altered membrane lipid composition and decreased proliferation in response to antigen. Therefore, we wanted to determine the mechanism by which mtGPAT activity is regulated in aged T-lymphocytes. We show that aged T-lymphocyte mtGPAT activity is not increased by ex vivo stimulation or in vitro phosphorylation with casein kinase II and protein kinase C theta as is seen in young T-lymphocytes. However, other factors that might impact mtGPAT activity such as reduced mtGPAT protein levels, gene expression or alterations in the soluble acyl-CoA pool were not affected by age or stimulation. The age effect was also not compensated for by increased acyl-CoA binding protein expression in aged T-lymphocytes. Currently, two mitochondrial GPAT (mtGPAT) isoforms (mtGPAT1 and mtGPAT2) have been identified. We found that T-lymphocytes express mtGPAT1, but not mtGPAT2, suggesting that at least mtGPAT1 is sensitive to phosphorylation in vitro. Support for direct phosphorylation of mtGPAT1 in young T-lymphocytes is shown by mtGPAT1 immunoprecipitation where a phosphoprotein band was detected migrating at the same molecular weight (85 kDa) as mtGPAT1. This is significant because we also show that T-lymphocytes from mtGPAT1 KO mice have reduced proliferation ex vivo as is seen in aged T-lymphocytes. These data provide evidence for a novel mechanism by which T-lymphocyte proliferation may be regulated and, for the first time, give a potential mechanistic explanation for the correlation between reduced proliferation and membrane lipid changes seen in aged T-lymphocytes.

Keywords: Abbreviations; GPAT; glycerol-3-phosphate acyltransferase; LPA; lysophosphatidic acid; PA; phosphatidic acid; mtGPAT; mitochondrial glycerol-3-phosphate acyltransferase; AGPAT; acylglycerolphosphate acyltransferase; CK2; casein kinase 2; PKCθ; protein kinase C theta; ACBP; acyl-CoA binding protein; rACBP; recombinant ACBPAging; T-lymphocyte; Phosphatidic acid; Acyltransferase; Protein kinase C

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BBA - Molecular and Cell Biology of Lipids (v.1761, #1)