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BBA - Molecular and Cell Biology of Lipids (v.1811, #10)

Title page/Section Editors (pp. i).

Enzymatic formation of N-acylethanolamines from N-acylethanolamine plasmalogen through N-acylphosphatidylethanolamine-hydrolyzing phospholipase D-dependent and -independent pathways by Kazuhito Tsuboi; Yasuo Okamoto; Natsuki Ikematsu; Manami Inoue; Yoshibumi Shimizu; Toru Uyama; Jun Wang; Dale G. Deutsch; Matthew P. Burns; Nadine M. Ulloa; Akira Tokumura; Natsuo Ueda (pp. 565-577).

Bioactive N-acylethanolamines include anandamide (an endocannabinoid), N-palmitoylethanolamine (an anti-inflammatory), and N-oleoylethanolamine (an anorexic). In the brain, these molecules are formed from N-acylphosphatidylethanolamines (NAPEs) by a specific phospholipase D, called NAPE-PLD, or through NAPE-PLD-independent multi-step pathways, as illustrated in the current study employing NAPE-PLD-deficient mice. Although N-acylethanolamine plasmalogen (1-alkenyl-2-acyl-glycero-3-phospho( N-acyl)ethanolamine, pNAPE) is presumably a major class of N-acylethanolamine phospholipids in the brain, its enzymatic conversion to N-acylethanolamines is poorly understood. In the present study, we focused on the formation of N-acylethanolamines from pNAPEs. While recombinant NAPE-PLD catalyzed direct release of N-palmitoylethanolamine from N-palmitoylethanolamine plasmalogen, the same reaction occurred in the brain homogenate of NAPE-PLD-deficient mice, suggesting that this reaction occurs through both the NAPE-PLD-dependent and -independent pathways. Liquid chromatography-mass spectrometry revealed a remarkable accumulation of 1-alkenyl-2-hydroxy-glycero-3-phospho( N-acyl)ethanolamines (lyso pNAPEs) in the brain of NAPE-PLD-deficient mice. We also found that brain homogenate formed N-palmitoylethanolamine, N-oleoylethanolamine, and anandamide from their corresponding lyso pNAPEs by a Mg²⁺-dependent “lysophospholipase D”. Moreover, the brain levels of alkenyl-type lysophosphatidic acids, the other products from lyso pNAPEs by lysophospholipase D, also increased in NAPE-PLD-deficient mice. Glycerophosphodiesterase GDE1 can hydrolyze glycerophospho- N-acylethanolamines to N-acylethanolamines in the brain. In addition, we discovered that recombinant GDE1 has a weak activity to generate N-palmitoylethanolamine from its corresponding lyso pNAPE, suggesting that this enzyme is at least in part responsible for the lysophospholipase D activity. These results strongly suggest that brain tissue N-acylethanolamines, including anandamide, can be formed from N-acylated plasmalogen through an NAPE-PLD-independent pathway as well as by their direct release via NAPE-PLD.► N-Acylethanolamine (NAE) plasmalogens are precursors of NAEs in the brain. ► The phospholipase D-type enzyme NAPE-PLD converted NAE plasmalogen to NAE. ► Brain homogenates of NAPE-PLD-null mice also formed NAE from NAE plasmalogen. ► The homogenates could hydrolyze N-acylated plasmalogen lysophospholipids to NAEs. ► Thus NAPE-PLD-independent formation of NAEs from NAE plasmalogens was also shown.

Keywords: N; -acylethanolamine; Anandamide; Endocannabinoid; NAPE-PLD; Phospholipase; Plasmalogen

Presence, formation and putative biological activities of N-acyl serotonins, a novel class of fatty-acid derived mediators, in the intestinal tract by Kitty C.M. Verhoeckx; Trudy Voortman; Michiel G.J. Balvers; Henk F.J. Hendriks; Heleen M.Wortelboer; Renger F. Witkamp (pp. 578-586).

Following the discovery of the endocannabinoid arachidonoyl ethanolamide (anandamide) and other N-acyl-ethanolamines, several other compounds have been found in which amino acids or neurotransmitters rather than ethanolamide are linked to fatty acids. Studies have shown that the local availability of fatty acid precursors, which in turn is modulated by dietary intake of lipids, determines the pattern of conjugates formed. Less information is available whether the same might be true for the amines or neurotransmitters involved. We hypothesized that N-arachidonoyl-serotonin (AA-5-HT) and its analogs could be endogenously present in those tissues that have high contents of serotonin. We investigated the endogenous presence of N-acyl serotonins in different parts of the gastro-intestinal tract of pigs and mice. We discovered that AA-5-HT, oleoyl-serotonin, palmitoyl-serotonin, and stearoyl-serotonin were endogenously present, particularly in the jejunum and ileum. Their formation in vitro was stimulated by the addition of serotonin to intestinal tissue incubations. Furthermore, in a mouse study we showed that the pattern of formation is dependent on the relative amount of fatty acids in the diet. The formation of docosahexaenoyl-serotonin and eicosapentaenoyl-serotonin was elevated in mice fed with a diet containing fish oil. Preliminary data showed that several of the serotonin conjugates are able to inhibit glucagon-like peptide-1 secretion and FAAH activity in vitro. Taken together, our data suggest that N-acyl serotonins are a novel class of lipid mediators present in the gut with highly promising biological properties.► Acyl-serotonins are endogenously present in the gastro-intestinal tract. ► Incubation of ileal tissue with serotonin elevated the levels of acyl-serotonins. ► N-acyl-serotonin patterns in the intestine is related to fatty acids in the diet. ► N-acyl-serotonins are able to inhibit GLP-1 secretion and FAAH activity in vitro.

Keywords: Abbreviations; AA-5-HT; arachidonoyl-serotonin; AEA; arachidonoyl ethanolamide; AMC; fluorophore7-amino-4-methylcoumarin; CB1; cannabinoid receptors 1; CID; collision-induced dissociation; DHA; docosahexaenoic acid; DHA-5-HT; docosahexaenoyl-serotonin; EPA-5-HT; eicosapentaenoyl-serotonin; EPA; eicosapentaenoic acid; FAAH; fatty acid amide hydrolase; GLP-1; glucagon-like peptide-1; HOSF; high-oleic acid sunflower oil; LDH; lactate dehydrogenase; LOD; limit of detection; LLOQ; lower limit of quantification; MRM; multiple reactions monitoring mode; NADA; N-arachidonoyl dopamine; NAEs; N-acyl ethanolamines; OA-5-HT; oleoyl-serotonin; OLDA; oleoyl dopamine; PA-5-HT; palmitoyl-serotonin; PPARα; peroxisome proliferator-activated receptor; SA-5-HT; stearoyl-serotonin; SPE; solid phase extraction; TFA; trifluoro acetic acid; THF; Tetrahydrofuran; TRPV1; transient receptor potential channel type V1; QC; quality controlFish oil; N; -acyl serotonin; FAAH; GLP-1; Gastro-intestinal tract

YALI0E32769g ( DGA1) and YALI0E16797g ( LRO1) encode major triacylglycerol synthases of the oleaginous yeast Yarrowia lipolytica by Karin Athenstaedt (pp. 587-596).

The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to produce and store triacylglycerols resembling adipocytes of higher eukaryotes. Here, the identification of two genes YALI0E32769g ( DGA1) and YALI0E16797g ( LRO1) encoding major triacylglycerol synthases of Yarrowia lipolytica is reported. Heterologous expression of either DGA1 or LRO1 in a mutant of the budding yeast Saccharomyces cerevisiae defective in triacylglycerol synthesis restores the formation of this neutral lipid. Whereas Dga1p requires acyl-CoA as a substrate for acylation of diacylglycerol, Lro1p is an acyl-CoA independent triacylglycerol synthase using phospholipids as acyl-donor. Growth of Yarrowia lipolytica strains deleted of DGA1 and/or LRO1 on glucose containing medium significantly decreases triacylglycerol accumulation. Most interestingly, when oleic acid serves as the carbon source the ratio of triacylglycerol accumulation in mutants to wild-type is significantly increased in strains defective in DGA1 but not in lro1Δ. In vitro experiments revealed that under these conditions an additional acyl-CoA dependent triacylglycerol synthase contributes to triacylglycerol synthesis in the respective mutants. Taken together, evidence is provided that Yarrowia lipolytica contains at least four triacylglycerol synthases, namely Lro1p, Dga1p and two additional triacylglycerol synthases whereof one is acyl-CoA dependent and specifically induced upon growth on oleic acid.►Identification of 2 triacylglycerol synthases of Y. lipolytica, Dga1p and Lro1p. ►Dga1p is an acyl-CoA dependent triacylglycerol synthase. ►Lro1p uses phospholipids as acyl donor for triacylglycerol formation. ►Growth on oleic acid induces additional triacylglycerol synthase(s).

Keywords: Abbreviation; TAG; triacylglycerolsTriacylglycerol synthesis; Oleic acid; Yarrowia lipolytica; Yeast

Subcellular localization and regulation of StarD4 protein in macrophages and fibroblasts by Daniel Rodriguez-Agudo; Maria Calderon-Dominguez; Shunlin Ren; Dalila Marques; Kaye Redford; Miguel Angel Medina-Torres; Phillip Hylemon; Gregorio Gil; William M. Pandak (pp. 597-606).

StarD4 is a member of the StarD4 subfamily of START domain proteins with a characteristic lipid binding pocket specific for cholesterol. The objective of this study was to define StarD4 subcellular localization, regulation, and function. Immunobloting showed that StarD4 is highly expressed in the mouse fibroblast cell line 3T3-L1, in human THP-1 macrophages, Kupffer cells (liver macrophages), and hepatocytes. In 3T3-L1 cells and THP-1 macrophages, StarD4 protein appeared localized to the cytoplasm and the endoplasmic reticulum (ER). More specifically, in THP-1 macrophages StarD4 co-localized to areas of the ER enriched in Acyl-CoA:cholesterol acyltransferase-1 (ACAT-1), and was closely associated with budding lipid droplets. The addition of purified StarD4 recombinant protein to an in vitro assay increased ACAT activity 2-fold, indicating that StarD4 serves as a rate-limiting step in cholesteryl ester formation by delivering cholesterol to ACAT-1-enriched ER. In addition, StarD4 protein was found to be highly regulated and to redistribute in response to sterol levels. In summary, these observations, together with our previous findings demonstrating the ability of increased StarD4 expression to increase bile acid synthesis and cholesteryl ester formation, provide strong evidence for StarD4 as a highly regulated, non-vesicular, directional, intracellular transporter of cholesterol which plays a key role in the maintenance of intracellular cholesterol homeostasis.► StarD4 is a START domain protein with a cholesterol-specific lipid binding pocket. ► StarD4 is found in hepatocytes, macrophages and fibroblast in the cytoplasm and ER. ► StarD4 is regulated by sterols and upregulated during macrophage differentiation. ► The protein is able to increase ACAT activity in an in vitro assay. ► StarD4 is a highly regulated, non-vesicular, directional cholesterol transporter.

Keywords: Macrophage; Cholesterol; Metabolism; Steroidogenic acute regulatory protein; Cholesterol transporter; START domain

The thermoacidophilic archaeon Sulfolobus acidocaldarius contains an unsually short, highly reduced dolichyl phosphate by Ziqiang Guan; Benjamin H. Meyer; Sonja-Verena Albers; Jerry Eichler (pp. 607-616).

Polyprenoids, polymers containing varied numbers of isoprene subunits, serve numerous roles in biology. In Eukarya, dolichyl phosphate, a phosphorylated polyprenol bearing a saturated α-end isoprene subunit, serves as the glycan carrier during N-glycosylation, namely that post-translational modification whereby glycans are covalently linked to select asparagine residues of a target protein. As in Eukarya, N-glycosylation in Archaea also relies on phosphorylated dolichol. In this report, LC-ESI/MS/MS was employed to identify a novel dolichyl phosphate (DolP) in the thermoacidophilic archaeon, Sulfolobus acidocaldarius. The unusually short S. acidocaldarius DolP presents a degree of saturation not previously reported. S. acidocaldarius DolP contains not only the saturated α- and ω-end isoprene subunits observed in other archaeal DolPs, but also up to five saturated intra-chain isoprene subunits. The corresponding dolichol and hexose-charged DolP species were also detected. The results of the present study offer valuable information on the biogenesis and potential properties of this unique DolP. Furthermore, elucidation of the mechanism of α-isoprene unit reduction in S. acidocaldarius dolichol may facilitate the identification of the alternative, as yet unknown polyprenol reductase in Eukarya.► A novel dolichol phosphate was identified in the thermoacidophilic archaeon, Sulfolobus acidocaldarius using LC-ESI/MS/MS. ► The unusually short S. acidocaldarius dolichol phosphate presents a degree of saturation not previously reported. ► S. acidocaldarius dolichol phosphate α- and ω-position isoprenes are saturated, as are intra-chain isoprenes. ► Hexose-charged dolichol phosphate species were also detected.

Keywords: Abbreviations; Acetate; Ac; Chloroform; CHCl; ₃; cis; -polyprenyl diphosphate synthase; CPDS; Collision-induced dissociation; CID; Deoxyxylulose phosphate; DOXP; Dimethylallyl diphosphate; DMAPP; Dolichyl phosphate; DolP; Geranylgeranyl diphosphate; GGPP; Geranylgeranyl reductase; GGR; Isopentenyl diphosphate; IPP; Methanol; MeOH; Mevalonate; MVA; Multiple reaction monitoring; MRM; Tandem mass spectrometry; MS/MS; Undecaprenol phosphate; UndPArchaea; Dolichol; Electrospray ionization mass spectrometry; Polyprenol; Polyprenol reductase; Sulfolobus acidocaldarius

Involvement of CTP:phosphocholine cytidylyltransferase-β2 in axonal phosphatidylcholine synthesis and branching of neurons by Jana Strakova; Laurent Demizieux; Robert B. Campenot; Dennis E. Vance; Jean E. Vance (pp. 617-625).

In the brain, phosphatidylcholine (PC) is synthesized by the CDP-choline pathway in which the rate-limiting step is catalyzed by two isoforms of CTP:phosphocholine cytidylyltransferase (CT): CTα and CTβ2. In mice, CTβ2 mRNA is more highly expressed in the brain than in other tissues, and several observations suggest that CTβ2 plays an important role in the nervous system. We, therefore, investigated the importance of CTβ2 for PC synthesis as well as for axon formation, growth and branching of primary sympathetic neurons. We show that in cultured primary neurons nerve growth factor increases the amount of CTβ2, but not CTα, mRNA and protein. The brains of mice lacking CTβ2 had normal PC content despite having 35% lower CT activity than wild-type brains. CTβ2 mRNA and protein are abundant in distal axons of mouse sympathetic neurons whereas CTα mRNA and protein were not detected. Moreover, CTβ2 deficiency in distal axons reduced the incorporation of [³H]choline into PC by 95% whereas PC synthesis in cell bodies/proximal axons was unaltered. These data suggest that CTβ2 is the major CT isoform involved in PC synthesis in axons. Axons of CTβ2-deficient sympathetic neurons contained 32% fewer branch points than did wild-type neurons although the number of axons/neuron and the rate of axon extension were the same as in wild-type neurons. We conclude that in distal axons of primary sympathetic neurons CTβ2 is a major contributor to PC synthesis and promotes axon branching, whereas CTα appears to be the major CT isoform involved in PC synthesis in cell bodies/proximal axons.► Expression of CTß2, but not CTα, in primary neurons is increased by NGF. ► CTß2, but not CTα, mRNA and protein are abundant in distal axons. ► Elimination of CTß2 reduces phosphatidylcholine synthesis in distal axons. ► Elimination of CTß2 impairs axon branching.

Keywords: Abbreviations; CT; CTP:phosphocholine cytidylyltransferase; NGF; nerve growth factor; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PEMT; phosphatidylethanolamine; N; -methyltransferaseNeuron; Astrocyte; Axon branching; Phosphatidylcholine; CTP:phosphocholine cytidylyltransferase; Nerve growth factor

Lipid accumulation impairs adiponectin-mediated induction of activin A by increasing TGFbeta in primary human hepatocytes by Josef Wanninger; Markus Neumeier; Claus Hellerbrand; Doris Schacherer; Sabrina Bauer; Thomas S. Weiss; Hanna Huber; Schaffler Andreas Schäffler; Charalampos Aslanidis; Scholmerich Jürgen Schölmerich; Christa Buechler (pp. 626-633).

Fatty liver is commonly detected in obesity and has been identified as a risk factor for the progression of hepatic fibrosis in a wide range of liver diseases. Transforming growth factor beta (TGFβ) and activin A, both members of the TGFβ superfamiliy, are central regulators in liver fibrosis and regeneration, and the effect of hepatocyte lipid accumulation on the release of these proteins was studied. Primary human hepatocytes (PHH) were incubated with palmitic acid or oleic acid to increase lipid storage. Whereas activin A and its natural inhibitor follistatin were not affected, TGFβ was 2-fold increased. The hepatoprotective adipokine adiponectin dose-dependently induced activin A while lowering follistatin but did not alter TGFβ. Activin A was markedly reduced in hepatocyte cell lines compared to PHH and was not induced upon adiponectin incubation demonstrating significant differences of primary and transformed cells. In free fatty acid (FFA)-incubated PHH adiponectin-mediated induction of activin A was impaired. Inhibition of TGFβ receptors ALK4/5 and blockage of SMAD3 phosphorylation rescued activin A synthesis in FFA and in TGFβ incubated cells suggesting that FFA inhibit adiponectin activity by inducing TGFβ. To evaluate whether serum levels of activin A and its antagonist are altered in patients with hepatic steatosis, both proteins were measured in the serum of patients with sonographically diagnosed fatty liver and age- and BMI-matched controls. Systemic adiponectin was significantly reduced in patients with fatty liver but activin A and follistatin were not altered. In summary the current data demonstrate that lipid accumulation in hepatocytes induces TGFβ which impairs adiponectin bioactivity, and thereby may contribute to liver injury.► Adiponectin induces activin A and lowers its natural inhibitor follistatin in primary human hepatocytes. ► Palmitic and oleic acid increase TGFβ in primary human hepatocytes. ► Impaired adiponectin signaling in fat loaded cells is related to increased TGFβ. ► Systemic adiponectin but not activin A and follistatin are significantly reduced in patients with fatty liver.

Keywords: Lipid accumulation; Hepatocyte; Activin A; Adiponectin

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BBA - Molecular and Cell Biology of Lipids (v.1811, #10)