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BBA - Molecular and Cell Biology of Lipids (v.1811, #4)

Editorial Board (pp. i).

Inhibition of the isoprenoid biosynthesis pathway; detection of intermediates by UPLC–MS/MS by Linda Henneman; Arno G. van Cruchten; Willem Kulik; Hans R. Waterham (pp. 227-233).

The isoprenoid biosynthesis pathway provides the cell with a variety of compounds which are involved in multiple cellular processes. Inhibition of this pathway with statins and bisphosphonates is widely applied in the treatment of hypercholesterolemia and metabolic bone disease, respectively. In addition, since isoprenylation of proteins is an important therapeutic target in cancer research there is interest in interfering with isoprenoid biosynthesis, for which new inhibitors to block farnesylation and geranylgeranylation of small GTPases are being developed. We recently developed a sensitive method using UPLC–MS/MS that allows the direct detection and quantification of all intermediates of the mevalonate pathway from MVA to GGPP which can be used to verify the specificity of inhibitors of the isoprenoid biosynthesis pathway. We here investigated the specificity of several inhibitors of the isoprenoid biosynthesis pathway in HepG2 cells, fibroblasts and lymphoblasts. The nitrogen-containing bisphosphonates pamidronate and zoledronate specifically inhibit farnesyl pyrophosphate synthase indicated by the accumulation of IPP/DMAPP. However, zaragozic acid A, a squalene synthase inhibitor, causes an increase of MVA in addition to the expected increase of FPP. Analysis of isoprenoid intermediate profiles after incubation with 6-fluoromevalonate showed a very nonspecific result with an increase in MVA, MVAP, MVAPP and IPP/DMAPP. These results show that inhibitors of a particular enzyme of the isoprenoid biosynthesis pathway can have additional effects on other enzymes of the pathway either direct or indirect through accumulation of isoprenoid intermediates. Our method can be used to test new inhibitors and their effect on overall isoprenoid biosynthesis.► New UPLC–MS/MS method for detection of isoprenoid intermediates. ► Useful tool to investigate the specificity of inhibitors of isoprenoid biosynthesis. ► ZAA indirectly inhibits mevalonate kinase in addition to squalene synthase inhibition. ► FMVA affects multiple enzymes of the isoprenoid biosynthesis pathway.

Keywords: Abbreviations; DMAPP; dimethylallyl pyrophosphate; DMEM; Dulbecco's Modified Eagles Medium; FCS; fetal calf serum; FMVA; 6-fluoromevalonate; FPP; farnesyl pyrophosphate; FPPS; farnesyl pyrophosphate synthase; FTI; farnesyltransferase inhibitor; GGPP; geranylgeranyl pyrophosphate; GGTI; geranylgeranyltransferase inhibitor; GPP; geranyl pyrophosphate; HMG-CoA; 3-hydroxy-3-methylglutaryl-CoA; IPP; isopentenyl pyrophosphate; IPPI; isopentenyl pyrophosphate isomerase; IS; internal standard; MK; mevalonate kinase; MPD; mevalonate pyrophosphate decarboxylase; MRM; multiple reaction monitoring; MVA; mevalonate; MVAL; mevalonolactone; MVAP; 5-phosphomevalonate; MVAPP; 5-pyrophosphomevalonate; PAM; pamidronate; PMK; phosphomevalonate kinase; UPLC–MS/MS; ultra performance liquid chromatography–tandem mass spectrometry; ZAA; zaragozic acid A; ZOL; zoledronateIsoprenoid biosynthesis; Mevalonate pathway; Mass spectrometry; Farnesyl pyrophosphate; Geranylgeranyl pyrophosphate

Watching intracellular lipolysis in mycobacteria using time lapse fluorescence microscopy by Rabeb Dhouib; Adrien Ducret; Pierre Hubert; Carriere Frédéric Carrière; Sam Dukan; Stéphane Canaan (pp. 234-241).

The fact that Mycobacterium tuberculosis mobilizes lipid bodies (LB) located in the cytosol during infection process has been proposed for decades. However, the mechanisms and dynamics of mobilization of these lipid droplets within mycobacteria are still not completely characterized. Evidence in favour of this characterization was obtained here using a combined fluorescent microscopy and computational image processing approach. The decrease in lipid storage levels observed under nutrient depletion conditions was correlated with a significant increase in the size of the bacteria. LB fragmentation/condensation cycles were monitored in real time. The exact contribution of lipases in this process was confirmed using the lipase inhibitor tetrahydrolipstatin, which was found to prevent LB degradation and to limit the bacterial cell growth. The method presented here provides a powerful tool for monitoring in vivo lipolysis in mycobacteria and for obtaining new insights on the growth of cells and their entry into the dormant or reactivation phase. It should be particularly useful for studying the effects of chemical inhibitors and activators on cells as well as investigating other metabolic pathways.► Observation of the intracellular lipolysis in real time in M. smegmatis. ► Proof of involvement of intracellular lipases in the process of LIBs degradation. ► Lipid consumption account for at least 25% of the energy required in cell growth. ► No lipid consumption in dormant cells suggesting a role in the reactivation process. ► Lipid storage contributes to survival of mycobacteria in a non- replicating state.

Keywords: Lipid bodies; Lipases; Dormancy; Mycobacteria; Growth; Fluorescence

Purification and characterization of a second type of neutral ceramidase from rat brain: A second more hydrophobic form of rat brain ceramidase by Faisal Thayyullathil; Shahanas Chathoth; Abdulkader Hago; Mahendra Patel; Zdzislaw M. Szulc; Yusuf Hannun; Sehamuddin Galadari (pp. 242-252).

Ceramidases (CDase) are enzymes that catalyze the hydrolysis of N-acyl linkage of ceramide (Cer) to generate sphingosine and free fatty acids. In this study we report the purification and characterization of a novel second type of neutral ceramidase from rat brain (RBCDase II). Triton X-100 protein extract from rat brain membrane was purified sequentially using Q-Sepharose, HiLoad16/60 Superdex 200pg, heparin-Sepharose, phenyl-Sepharose HP, and Mono Q columns. After Mono Q, the specific activity of the enzyme increased by ~15,000-fold over that of the rat brain homogenate. This enzyme has pH optima of 7.5, and it has a larger apparent molecular weight (110kDa) than the previously purified (90kDa) and characterized neutral rat brain CDase (RBCDase I). De-glycosylation experiments show that the differences in molecular mass of RBCDase I and II on SDS-PAGE are not due to the heterogeneity with N-glycan. RBCDase II is partially stimulated by Ca²⁺ and is inhibited by pyrimidine mono nucleotides such as TMP and UMP. This finding is significant as it demonstrates for the first time an effect by nucleotides on a CDase activity. The enzyme was also inhibited by both oxidized and reduced GSH. The effects of metal ions were examined, and we found that the enzyme is very sensitive to Hg²⁺ and Fe³⁺, while it is not affected by Mn²⁺. EDTA was somewhat inhibitory at a 20mM concentration.► Purified a second more hydrophobic type of nCDase (RBCDase II) from rat brain. ► Characterized many biochemical properties of the RBCDase II. ► The purified RBCDase II is different from those reported data of RBCDase I in several aspects.

Keywords: Abbreviations; RBCDase I; rat brain ceramidase 1; RBCDase II; rat brain ceramidase II; Cer; ceramide; SPH; sphingosine; S-1-P; sphingosine-1-phosphate; C; ₁₂; -NBD- Cer; 4-nitrobenzo-2- oxa-l,3-diazole ceramide; AMP; adenosine monophosphate; ADP; adenosine diphosphate; ATP; adenosine triphosphate; GPM; guanosine monophosphate; GDP; guanosine diphosphate; GTP; guanosine triphosphate; TMP; thymidine monophosphate; TDP; thymidine diphosphate; TTP; thymidine triphosphate; UMP; uridine monophosphate; UDP; uridine diphosphate; UTP; uridine triphosphate; SDS-PAGE; sodium dodicyl suphate polyacrylamide gel electrophoresis; PA; phosphatic acid; PC; phosphatidylcholine; PG; phosphatidylglycerol; PE; phosphatidylethanolamine; PI; phosphatidylinositol; PS; phosphatidylserineCeramidase; Ceramide; Sphingosine; Chromatography; Glycosidase F

Microbial Baeyer–Villiger oxidation of steroidal ketones using Beauveria bassiana: Presence of an 11α-hydroxyl group essential to generation ofD-homo lactones by Swizdor Alina Świzdor; Kolek Teresa Kołek; Anna Panek; Bialonska Agata Białońska (pp. 253-262).

This paper demonstrates for the first time transformation of a series of 17-oxo steroidal substrates (epiandrosterone, dehydroepiandrosterone, androstenedione) by the most frequently used whole cell biocatalyst, Beauveria bassiana, to 11α-hydroxy-17a-oxa-d-homo-androst-17-one products, in the following sequence of reactions: 11α-hydroxylation and subsequent Baeyer–Villiger oxidation to a ring-D lactone. 11α-Hydroxyprogesterone, the product of the first stage of the progesterone metabolism, was further converted along two routes: hydroxylation to 6β,11α-dihydroxyprogesterone or 17β-acetyl chain degradation leading to 11α-hydroxytestosterone, the main metabolite of the substrate. Part of 11α-hydroxytestosterone underwent a rare reduction to 11α-hydroxy-5β-dihydrotestosterone. The experiments have demonstrated that the Baeyer–Villiger monooxygenase produced by the strain catalyzes solely oxidation of C-20 or C-17 ketones with 11α-hydroxyl group. 17-Oxo steroids, beside the 11α-hydroxylation and Baeyer–Villiger oxidation, also underwent reduction to 17β-alcohols; activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) has significant impact on the amount of the formed ring-D δ-lactone.► 17-Oxo steroids are metabolized to 11α-hydroxy-d-lactones, in a one-step process. ► BVMOs oxidize solely substrates with 11α-hydroxyl group. ► Activity of 17β-HSD has significant impact on the amount of the formedd-lactone.

Keywords: Steroid biotransformation; Steroidal lactone; Microbial Baeyer–Villiger oxidation; Beauveria bassiana; 17β-hydroxysteroid dehydrogenase

1α,25-Dihydroxyvitamin D₃ exerts tissue-specific effects on estrogen and androgen metabolism by Johan Lundqvist; Maria Norlin; Kjell Wikvall (pp. 263-270).

It is well-known that 1α,25-dihydroxyvitamin D₃ and analogs exert anti-proliferative and pro-differentiating effects and these compounds have therefore been proposed to be of potential use as anti-cancer agents. Due to its effects on aromatase gene expression and enzyme activity, 1α,25-dihydroxyvitamin D₃ has been proposed as an interesting substance in breast cancer treatment and prevention. In the present study, we have examined the effects of 1α,25-dihydroxyvitamin D₃ on estrogen and androgen metabolism in adrenocortical NCI-H295R cells, breast cancer MCF-7 cells and prostate cancer LNCaP cells. The NCI-H295R cell line has been proposed as a screening tool to study endocrine disruptors. We therefore studied whether this cell line reacted to 1α,25-dihydroxyvitamin D₃ treatment in the same way as cells from important endocrine target tissues. 1α,25-Dihydroxyvitamin D₃ exerted cell line-specific effects on estrogen and androgen metabolism. In breast cancer MCF-7 cells, aromatase gene expression and estradiol production were decreased, while production of androgens was markedly increased. In NCI-H295R cells, 1α,25-dihydroxyvitamin D₃ stimulated aromatase expression and decreased dihydrotestosterone production. In prostate cancer LNCaP cells, aromatase expression increased after the same treatment, as did production of testosterone and dihydrotestosterone. In summary, our data show that 1α,25-dihydroxyvitamin D₃ exerts tissue-specific effects on estrogen and androgen production and metabolism. This is important knowledge about 1α,25-dihydroxyvitamin D₃ as an interesting substance for further research in the field of breast cancer prevention and treatment. Furthermore, the observed cell line-specific effects are of importance in the discussion about NCI-H295R cells as a model for effects on estrogen and androgen metabolism.►1α,25-Dihydroxyvitamin D₃ alters sex hormone metabolism in a cell line-specific way. ►1α,25-Dihydroxyvitamin D₃ increases androgen production in breast cancer MCF-7 cells. ►1α,25-Dihydroxyvitamin D₃ increases aromatase expression in LNCaP and H295R cells. ►Adds data supporting that vitamin D acts as a selective aromatase modulator ►New data important in the discussion of H295R as a model for sex hormone metabolism

Keywords: Vitamin D; Calcitriol; Androgens; Estrogens; Breast cancer; AromataseAbbreviations; 17β-HSD; 17β-hydroxysteroid dehydrogenase; ELISA; enzyme-linked immunosorbent assay; SAM; selective aromatase modulator; TBP; TATA box binding protein; VDR; vitamin D receptor

Synthesis of enantiopure 2-carba-cyclic phosphatidic acid and effects of its chirality on biological functions by Emi Nozaki; Mari Gotoh; Harumi Hotta; Shuwa Hanazawa; Susumu Kobayashi; Kimiko Murakami-Murofushi (pp. 271-277).

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator, which has a quite unique cyclic phosphate ring at sn-2 and sn-3 positions of the glycerol backbone. We have designed and chemically synthesized several metabolically stabilized derivatives of cPA. 2-Carba-cPA (2ccPA) is one of the synthesized compounds in which the phosphate oxygen was replaced with a methylene group at the sn-2 position, and it showed much more potent biological activities than natural cPA. Here, we developed a new method of 2ccPA enantiomeric synthesis. And we examined the effects of 2ccPA enantiomers on autotaxin (ATX) activity, cancer cell invasion and nociceptive reflex. As well as racemic-2ccPA, both enantiomers showed inhibitory effects on ATX activity, cancer cell invasion and nociceptive reflex. As their effects were not significantly different from each other, the chirality of 2ccPA may not be critical for these biological functions of 2ccPA.► Synthesis of enantiopure 2ccPA. ► Effects of chirality of 2ccPA on ATX activity and cancer cell invasion. ► Effects of chirality of 2ccPA on somato-somatic C-reflexexs in anesthetized rats.

Keywords: Abbreviations; cPA; Cyclic phosphatidic acid; LPA; Lysophosphatidic acid; ATX; Autotaxin; 2ccPA; 2-carba-cyclic phosphatidic acidEnantiopure 2-carba-cyclic phosphatidic acid; Stereoselectivity; Chirality; Autotaxin; Cancer cell invasion; Nociceptive reflex

The role of phosphatidylethanolamine methyltransferase in a mouse model of intrahepatic cholestasis by Zhaoyu Li; Luis B. Agellon; Dennis E. Vance (pp. 278-283).

Intrahepatic cholestasis eventually leads to liver failure. We report here a condition that decreases liver damage in intrahepatic cholestasis based on a mouse model that lacks multiple drug resistant protein 2 (ABCB4). We found that lack of phosphatidylethanolamine N-methyltransferase (PEMT) decreased liver damage in Abcb4^−/− mice caused by exposure of the liver to excess bile acids. The protective effect was not related to hepatic ratio of phosphatidylcholine to phosphatidylethanolamine or the level of cholesterol. The decreased concentration of bile acids in liver was related to impaired re-absorption of bile acids in intestine and increased disposal of bile acids in feces in Abcb4^−/− /Pemt^−/− mice as compared to Abcb4^−/− mice. PEMT deficiency affected intestinal Na⁺ absorption resulting in an impaired Na⁺ concentration gradient along the length of the small intestine and abnormal absorption of bile acids mediated by apical sodium-dependent bile acid transporter (ASBT). The findings of this study suggest that inhibition of PEMT and/or reduction of intestinal sodium concentration may be helpful in attenuating liver damage and prolonging hepatic function in intrahepatic cholestasis.► Lack of phospholipid methylation decreases liver damage in Abcb4^−/− mice. ► Protective effect not due to phosphatidylcholine/phosphatidylethanolamine ratio. ► Lack of methylation impairs ileal Na+ absorption. ► Mice that lack phospholipid methylation have impaired bile acid re-absorption.

Keywords: Abbreviations; PFIC; progressive familiar intrahepatic cholestasis type 3; ABCB4; multiple drug resistant protein 2; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PEMT; PE; N-; methyltransferase; ALT; alanine aminotransferases; TCA; taurocholic acid; ASBT; apical sodium-dependent bile acid transporterMdr2; ABCB4; PFIC3; Cholestasis; Sodium

Participation of mammary gland in long-chain polyunsaturated fatty acid synthesis during pregnancy and lactation in rats by Maricela Rodriguez-Cruz; Sanchez Raúl Sánchez; Sanchez Apolos M. Sánchez; Shannon L. Kelleher; Sanchez-Munoz Fausto Sánchez-Muñoz; Jorge Maldonado; Lopez-Alarcon Mardia López-Alarcón (pp. 284-293).

Metabolic adaptations are triggered in the maternal organism to synthesize milk with an adequate concentration of long-chain polyunsaturated fatty acids (LC-PUFAs) required to the newborn. They may be a high uptake of dietary linoleic acid and its conversion to LC-PUFAs by desaturases of fatty acids (FADS) 1 and 2 in the mammary gland (MG). It is unknown if they also occur from onset of pregnancy. The aim of this study was to explore the participation of the MG as a mechanism involved in LC-PUFAs synthesis to support their demand during pregnancy and lactation in rats. The expression of desaturases in MG was significantly ( P<0.05) higher (12.3-fold for FADS1 and 41.2-fold for FADS2) during the late pregnancy and throughout lactation (31.7-fold for FADS1 and 67.1-fold higher for FADS2) than in nonpregnant rats. SREBF-1c showed a similar pattern of increase during pregnancy but remained higher only during the early lactation (11.7-fold, P<0.005). Transcript of ELOVL6 and FASN increased throughout pregnancy and lactation, respectively. ELOVL5 mRNA increased in MG only during lactation (2.8 to 5.3-fold, P<0.005). Accordingly, a higher content of LC-PUFAs was found in lactating MG than in nonpregnant rats. Results suggest that MG participates from late pregnancy and throughout lactation by expressing desaturases and elongases as a mechanism involved in LC-PUFAs synthesis, probably by SREBF-1c. Because desaturases and ELOVL5 were expressed in cultured lactocytes and such expression was downregulated by linoleic and arachidonic acid, these cells may be a useful model for understanding the regulatory mechanisms for LC-PUFAs synthesis in MG.►Mammary gland (MG) expresses elongases for LC-PUFAs synthesis. ►MG expresses desaturases and elongases since late pregnancy throughout lactation. ►Transcription factor SREBF-1c probably upregulate desaturases and elongases in MG. ►MG participate in the LC-PUFAs synthesis from late pregnancy throughout lactation. ►Cultured lactocytes may be useful to understand the LC-PUFAs synthesis in MG.

Keywords: Abbreviations; LC-PUFAs; Long-chain polyunsaturated fatty acids; EFAs; essential fatty acids; AA; arachidonic acid; EPA; eicosapentaenoic acid; DHA; docosahexaenoic acid; LA; linoleic acid; ALA; α-linolenic acid; FADS1; Fatty acid desaturase 1; FADS2; Fatty acid desaturase 2; Elovl; elongation of very long chain fatty acids Elovl; SREBF-1c; sterol-regulatory element binding transcription factor 1c; FASN; fatty acid synthase; MFA; monounsaturated fatty acids; FBS; fetal bovine serum; PRL; prolactin; DEPC; diethylpyrocarbonate; VLDL; very low-density lipoproteins; CHREBP; carbohydrate-responsive element-binding proteinFatty acid desaturase 1; Fatty acid desaturase 2; Elongase 5; Sterol-regulatory element binding factor; Pregnancy; Lactation

Overexpression of apolipoprotein O does not impact on plasma HDL levels or functionality in human apolipoprotein A-I transgenic mice by Niels Nijstad; Jan Freark de Boer; William R. Lagor; Markus Toelle; David Usher; Wijtske Annema; Markus van der Giet; Daniel J. Rader; Uwe J.F. Tietge (pp. 294-299).

Apolipoprotein (apo) O is a newly discovered apolipoprotein preferentially contained within HDL; however, currently, no data are available on the (patho)physiological effects of apoO. Therefore, the present study assessed the impact of apoO overexpression on (i) plasma lipids and lipoproteins as well as on (ii) HDL functionality. Human apoO was overexpressed by means of recombinant adenovirus (AdhapoO) in human apoA-I transgenic mice, a humanized mouse model of HDL metabolism. AdhapoO substantially increased apoO in plasma and within HDL. However, plasma triglycerides, phospholipids, total cholesterol and HDL cholesterol did not change. HDL size distribution, lipid composition and the apoA-I and the apoO distribution over the different HDL fractions separated by FPLC remained unaltered. Furthermore, enrichment of HDL with apoO did not impact on HDL functionality assessed in four independent ways, namely (i) stimulation of cholesterol efflux from macrophage foam cells, (ii) protection against LDL oxidation, (iii) anti-inflammatory activity on endothelial cells, and (iv) induction of vasodilation in isolated aortic rings ex vivo as a measure of stimulating vascular NO production. These results demonstrate that although overexpression of apoO results in a substantial enrichment of HDL particles with this novel apolipoprotein, apoO does not impact the plasma lipoprotein profile or HDL functionality.►apoO is a novel apolipoprotein preferentially associated with HDL. ►Overexpression of apoO does not impact plasma HDL levels or size. ►Overexpression of apoO does not change key atheroprotective functions of HDL.

Keywords: Apolipoprotein; High density lipoprotein; HDL function; Cholesterol; Liver

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