BBA - Molecular and Cell Biology of Lipids (v.1831, #2)

Role of palmitate-induced sphingoid base-1-phosphate biosynthesis in INS-1 β-cell survival by Julien Véret; Nicolas Coant; Irina A. Gorshkova; Paola Giussani; Magali Fradet; Elena Riccitelli; Anastasia Skobeleva; Jonathan Goya; Nadim Kassis; Viswanathan Natarajan; Bernard Portha; Evgeny V. Berdyshev; Hervé Le Stunff (251-262).
Sphingoid base-1-phosphates represent a very low portion of the sphingolipid pool but are potent bioactive lipids in mammals. This study was undertaken to determine whether these lipids are produced in palmitate-treated pancreatic β cells and what role they play in palmitate-induced β cell apoptosis. Our lipidomic analysis revealed that palmitate at low and high glucose supplementation increased (dihydro)sphingosine-1-phosphate levels in INS-1 β cells. This increase was associated with an increase in sphingosine kinase 1 (SphK1) mRNA and protein levels. Over-expression of SphK1 in INS-1 cells potentiated palmitate-induced accumulation of dihydrosphingosine-1-phosphate. N,N-dimethyl-sphingosine, a potent inhibitor of SphK, potentiated β-cell apoptosis induced by palmitate whereas over-expression of SphK1 significantly reduced apoptosis induced by palmitate with high glucose. Endoplasmic reticulum (ER)-targeted SphK1 also partially inhibited apoptosis induced by palmitate. Inhibition of INS-1 apoptosis by over-expressed SphK1 was independent of sphingosine-1-phosphate receptors but was associated with a decreased formation of pro-apoptotic ceramides induced by gluco-lipotoxicity. Moreover, over-expression of SphK1 counteracted the defect in the ER-to-Golgi transport of proteins that contribute to the ceramide-dependent ER stress observed during gluco-lipotoxicity. In conclusion, our results suggest that activation of palmitate-induced SphK1-mediated sphingoid base-1-phosphate formation in the ER of β cells plays a protective role against palmitate-induced ceramide-dependent apoptotic β cell death.► Palmitate increases sphingoid base phosphate levels in β cells. ► SphK1 contributes to the accumulation of DHS1P induced by palmitate. ► Sphingoid base phosphates partially inhibit palmitate-induced β cell apoptosis. ► Potective role of SphK1 is independent of S1P receptors. ► Protective role of SphK1 is mediated by down-regulating ceramide synthesis.
Keywords: (dihydro)Sphingosine-1-phosphate; Ceramides; Type 2 diabetes; Gluco-lipotoxicity; Cell death; Pancreatic β cells;

Cholesterol efflux is differentially regulated in neurons and astrocytes: Implications for brain cholesterol homeostasis by Jing Chen; Xiaolu Zhang; Handojo Kusumo; Lucio G. Costa; Marina Guizzetti (263-275).
Disruption of cholesterol homeostasis in the central nervous system (CNS) has been associated with neurological, neurodegenerative, and neurodevelopmental disorders. The CNS is a closed system with regard to cholesterol homeostasis, as cholesterol-delivering lipoproteins from the periphery cannot pass the blood–brain-barrier and enter the brain. Different cell types in the brain have different functions in the regulation of cholesterol homeostasis, with astrocytes producing and releasing apolipoprotein E and lipoproteins, and neurons metabolizing cholesterol to 24(S)-hydroxycholesterol. We present evidence that astrocytes and neurons adopt different mechanisms also in regulating cholesterol efflux. We found that in astrocytes cholesterol efflux is induced by both lipid-free apolipoproteins and lipoproteins, while cholesterol removal from neurons is triggered only by lipoproteins. The main pathway by which apolipoproteins induce cholesterol efflux is through ABCA1. By upregulating ABCA1 levels and by inhibiting its activity and silencing its expression, we show that ABCA1 is involved in cholesterol efflux from astrocytes but not from neurons. Furthermore, our results suggest that ABCG1 is involved in cholesterol efflux to apolipoproteins and lipoproteins from astrocytes but not from neurons, while ABCG4, whose expression is much higher in neurons than astrocytes, is involved in cholesterol efflux from neurons but not astrocytes. These results indicate that different mechanisms regulate cholesterol efflux from neurons and astrocytes, reflecting the different roles that these cell types play in brain cholesterol homeostasis. These results are important in understanding cellular targets of therapeutic drugs under development for the treatments of conditions associated with altered cholesterol homeostasis in the CNS.► ABC cholesterol transporters are differentially expressed in neurons and astroglia. ► ABCA1/ABCG1 induction increases cholesterol efflux from glia but not from neurons. ► ABCA1 silencing and inhibition reduce cholesterol efflux from glia but not neurons. ► ABCG1 silencing inhibits cholesterol efflux from astrocytes but not from neurons. ► ABCG4 silencing reduces cholesterol efflux from neurons but not from astrocytes.
Keywords: Neurons; Astrocytes; ABCA1; ABCG1; ABCG4; Cholesterol efflux;

The G0/G1 switch gene 2 (G0S2): Regulating metabolism and beyond by Bradlee L. Heckmann; Xiaodong Zhang; Xitao Xie; Jun Liu (276-281).
The G0/G1 switch gene 2 (G0S2) was originally identified in blood mononuclear cells following induced cell cycle progression. Translation of G0S2 results in a small basic protein of 103 amino acids in size. It was initially believed that G0S2 mediates re-entry of cells from the G0 to G1 phase of the cell cycle. Recent studies have begun to reveal the functional aspects of G0S2 and its protein product in various cellular settings. To date the best-known function of G0S2 is its direct inhibitory capacity on the rate-limiting lipolytic enzyme adipose triglyceride lipase (ATGL). Other studies have illustrated key features of G0S2 including sub-cellular localization, expression profiles and regulation, and possible functions in cellular proliferation and differentiation. In this review we present the current knowledge base regarding all facets of G0S2, and pose a variety of questions and hypotheses pertaining to future research directions.► In this review we describe the G0/G1 switch gene 2 (G0S2) and its functions. ► We illustrate the key features of G0S2's localization, expression, and regulation. ► We present the currently known functions of G0S2 in lipid metabolism and beyond. ► We pose further questions and hypotheses for future research directions of G0S2.
Keywords: G0/G1 switch gene 2; Lipolysis; Cell proliferation; Differentiation; Cell cycle; Apoptosis;

Lipidome and proteome of lipid droplets from the methylotrophic yeast Pichia pastoris by Vasyl A. Ivashov; Karlheinz Grillitsch; Harald Koefeler; Erich Leitner; Dominic Baeumlisberger; Michael Karas; Günther Daum (282-290).
Lipid droplets (LD) are the main depot of non-polar lipids in all eukaryotic cells. In the present study we describe isolation and characterization of LD from the industrial yeast Pichia pastoris. We designed and adapted an isolation procedure which allowed us to obtain this subcellular fraction at high purity as judged by quality control using appropriate marker proteins. Components of P. pastoris LD were characterized by conventional biochemical methods of lipid and protein analysis, but also by a lipidome and proteome approach. Our results show several distinct features of LD from P. pastoris especially in comparison to Saccharomyces cerevisiae. P. pastoris LD are characterized by their high preponderance of triacylglycerols over steryl esters in the core of the organelle, the high degree of fatty acid (poly)unsaturation and the high amount of ergosterol precursors. The high phosphatidylinositol to phosphatidylserine of ~ 7.5 ratio on the surface membrane of LD is noteworthy. Proteome analysis revealed equipment of the organelle with a small but typical set of proteins which includes enzymes of sterol biosynthesis, fatty acid activation, phosphatidic acid synthesis and non-polar lipid hydrolysis. These results are the basis for a better understanding of P. pastoris lipid metabolism and lipid storage and may be helpful for manipulating cell biological and/or biotechnological processes in this yeast.► We isolated and characterized lipid droplets from P. pastoris for the first time. ► Lipidome and proteome analysis of P. pastoris lipid droplets were performed. ► Lipid droplets from P. pastoris are different from S. cerevisiae lipid droplets. ► P. pastoris lipid droplets contain much triacylglycerols but little steryl esters. ► A large number of P. pastoris lipid droplet proteins are involved in lipid metabolism.
Keywords: Lipid droplet; Triacylglycerol; Steryl ester; Lipidome; Proteome; Pichia pastoris;

Dietary eicosapentaenoic acid supplementation accentuates hepatic triglyceride accumulation in mice with impaired fatty acid oxidation capacity by Zhen-Yu Du; Tao Ma; Bjørn Liaset; Alison H. Keenan; Pedro Araujo; Erik-Jan Lock; Laurent Demizieux; Pascal Degrace; Livar Frøyland; Karsten Kristiansen; Lise Madsen (291-299).
Reduced mitochondrial fatty acid (FA) β-oxidation can cause accumulation of triglyceride in liver, while intake of eicosapentaenoic acid (EPA) has been recommended as a promising novel therapy to decrease hepatic triglyceride content. However, reduced mitochondrial FA β-oxidation also facilitates accumulation of EPA. To investigate the interplay between EPA administration, mitochondrial activity and hepatic triglyceride accumulation, we investigated the effects of EPA administration to carnitine-deficient mice with impaired mitochondrial FA β-oxidation. C57BL/6J mice received a high-fat diet supplemented or not with 3% EPA in the presence or absence of 500 mg mildronate/kg/day for 10 days. Liver mitochondrial and peroxisomal oxidation, lipid classes and FA composition were determined. Histological staining was performed and mRNA level of genes related to lipid metabolism and inflammation in liver and adipose tissue was determined. Levels of pro-inflammatory eicosanoids and cytokines were measured in plasma. The results showed that mildronate treatment decreased hepatic carnitine concentration and mitochondrial FA β-oxidation and induced severe triglyceride accumulation accompanied by elevated systemic inflammation. Surprisingly, inclusion of EPA in the diet exacerbated the mildronate-induced triglyceride accumulation. This was accompanied by a considerable increase of EPA accumulation while decreased total n-3/n-6 ratio in liver. However, inclusion of EPA in the diet attenuated the mildronate-induced mRNA expression of inflammatory genes in adipose tissue. Taken together, dietary supplementation with EPA exacerbated the triglyceride accumulation induced by impaired mitochondrial FA β-oxidation. Thus, further thorough evaluation of the potential risk of EPA supplementation as a therapy for NAFLD associated with impaired mitochondrial FA oxidation is warranted.► Dietary EPA supplementation exacerbates mildronate-induced NAFLD in mice. ► Dietary EPA supplementation alleviates NAFLD-related inflammation in mice. ► EPA supplementation as a therapy for NAFLD with impaired FA β-oxidation is warranted.
Keywords: Eicosapentaenoic acid; Hepatic triglyceride accumulation; Mildronate; Mitochondrial fatty acid β-oxidation; Carnitine; Inflammation;

The transport of DDT from chylomicrons to adipocytes does not mimic triacylglycerol transport by Alison B. Kohan; Abbey E. Vandersall; Qing Yang; Min Xu; Ronald J. Jandacek; Patrick Tso (300-305).
Despite being banned in the U.S., organochlorine toxins such as DDT are frequently detected in human adipose tissue. The main route of exposure is through the consumption of contaminated foods and subsequent intestinal packaging of DDT into chylomicrons. These chylomicrons, which also contain dietary triacylglycerol (TG), are delivered directly to peripheral tissues without first being metabolized by the liver. The physiological process by which these compounds are delivered from chylomicrons to adipose is not well understood, but is clinically relevant since it bypasses first-pass metabolism. Based on its highly lipophilic nature, it has been assumed that DDT is transferred to peripheral tissues similar to TG; however, this has not been measured. Here, we use the lymph fistula rat to isolate chylomicrons containing both DDT and TG. These chylomicrons are the in vivo DDT delivery vehicle. Using 3T3-L1 adipocytes, we investigated the rate at which DDT transfers from chylomicrons to adipocytes, and mediators of this process. This novel approach closely approximates the in vivo DDT exposure route. We show that: 1) DDT repartitions from chylomicrons to adipocytes, 2) this transport does not require hydrolysis of TG within the chylomicron, and is stimulated by the inhibition of LPL, 3) albumin does not inhibit DDT uptake, 4) DDT dissolved in DMSO does not appropriately mimic in vivo DDT transport; and most importantly, 5) DDT uptake from chylomicrons does not mimic the uptake of TG from the same particles. Understanding these factors is important for designing interventions for human populations exposed to DDT.► DDT is transported from the intestine to adipose, but the mechanism is unclear. ► DDT transport to adipocytes does not require hydrolysis of chylomicron TG. ► Albumin does not sequester or inhibit DDT transport. ► In vitro delivery of DDT dissolved in DMSO does not mimic in vivo transport. ► We conclude: DDT transport from chylomicrons to adipocytes is independent from TG.
Keywords: Chylomicrons; Organochlorines; DDT; Xenobiotics; Triacylglycerol; 3T3-L1 adipocytes;

Inhibition of constitutive Akt (PKB) phosphorylation by docosahexaenoic acid in the human breast cancer cell line MDA-MB-453 by Satoshi B. Sato; Jungha Park; Jun Kawamoto; Sho Sato; Tatsuo Kurihara (306-313).
Many breast cancer cells express aberrantly activated receptor tyrosine kinases and are associated with deregulated phosphorylation of Akt (PKB). They are also often associated with a high level of free monounsaturated (MUFA) and saturated (SFA) fatty acids. We studied the effect of DHA and other polyunsaturated fatty acids (PUFAs) on these anomalies in a human breast cancer cell line, MDA-MB-453. Inhibitors of the Akt T308 kinase (PDK1) or S473 kinase (mTORC2, DNA-dependent protein kinase and integrin-linked kinase) and combinations of two of them incompletely inhibited, or even enhanced, the phosphorylation in this cell line. In contrast, it was found that DHA as well as other PUFAs inhibited Akt phosphorylation on T308 after 24 h. These PUFAs also blocked phosphorylation of S473, although certain omega-6 PUFAs were ineffective. After 48 h, only DHA inhibited Akt phosphorylation on the both residues. DHA, and other PUFAs though less efficiently, also elevated the expression of a mitochondrial enzyme, 2,4-dienoyl-CoA reductase, which catalyzes process necessary for β-oxidation of PUFAs. These PUFAs were present in the cells at high concentrations and reduced the amount of free and phospholipid-bound MUFAs. DHA most efficiently blocked deregulated cell proliferation while the effects of other PUFAs were moderate. These results suggest that DHA suppressed the growth of the cancer cell through its specifically persistent block of Akt phosphorylation in conjunction with modulation of fatty acid metabolism.► Common effect of polyunsaturated fatty acids (PUFAs) on breast cancer properties. ► Transient suppression of Akt phosphorylation by many PUFAs. ► Uniquely sustained suppression of Akt phosphorylation by docosahexaenoic acid (DHA). ► Unique suppression of deregulated growth of cancer cells by DHA.
Keywords: Polyunsaturated fatty acids; Docosahexaenoic acids; Fatty acids; Breast cancer; Akt;

Yeast and cancer cells – common principles in lipid metabolism by Klaus Natter; Sepp D. Kohlwein (314-326).
One of the paradigms in cancer pathogenesis is the requirement of a cell to undergo transformation from respiration to aerobic glycolysis – the Warburg effect – to become malignant. The demands of a rapidly proliferating cell for carbon metabolites for the synthesis of biomass, energy and redox equivalents, are fundamentally different from the requirements of a differentiated, quiescent cell, but it remains open whether this metabolic switch is a cause or a consequence of malignant transformation. One of the major requirements is the synthesis of lipids for membrane formation to allow for cell proliferation, cell cycle progression and cytokinesis. Enzymes involved in lipid metabolism were indeed found to play a major role in cancer cell proliferation, and most of these enzymes are conserved in the yeast, Saccharomyces cerevisiae. Most notably, cancer cell physiology and metabolic fluxes are very similar to those in the fermenting and rapidly proliferating yeast. Both types of cells display highly active pathways for the synthesis of fatty acids and their incorporation into complex lipids, and imbalances in synthesis or turnover of lipids affect growth and viability of both yeast and cancer cells. Thus, understanding lipid metabolism in S. cerevisiae during cell cycle progression and cell proliferation may complement recent efforts to understand the importance and fundamental regulatory mechanisms of these pathways in cancer.► Warburg effect of cancer and Crabtree effect of yeast result in similar physiology. ► Proliferating cells have a high demand for membrane biogenesis. ► Pathways for fatty acid synthesis are highly active in cancer. ► Phospholipid remodeling is important for membrane homeostasis in growing cells.
Keywords: Malignant transformation; Warburg effect; Fatty acids; Membrane lipids; Storage lipids; Saccharomyces cerevisiae;

Nuclear lipid droplets: A novel nuclear domain by J.P. Layerenza; P. González; M.M. García de Bravo; M.P. Polo; M.S. Sisti; A. Ves-Losada (327-340).
We investigated nuclear neutral-lipid (NL) composition and organization, as NL may represent an alternative source for providing fatty acids and cholesterol (C) to membranes, signaling paths, and transcription factors in the nucleus. We show here that nuclear NL were organized into nonpolar domains in the form of nuclear-lipid droplets (nLD). By fluorescent confocal microscopy, representative nLD were observed in situ within the nuclei of rat hepatocytes in vivo and HepG2 cells, maintained under standard conditions in culture, and within nuclei isolated from rat liver. nLD were resistant to Triton X-100 and became stained with Sudan Red, OsO4, and BODIPY493/503. nLD and control cytosolic-lipid droplets (cLD) were isolated from rat-liver nuclei and from homogenates, respectively, by sucrose-gradient sedimentation. Lipids were extracted, separated by thin-layer chromatography, and quantified. nLD were composed of 37% lipids and 63% proteins. The nLD lipid composition was as follows: 19% triacylglycerols (TAG), 39% cholesteryl esters, 27% C, and 15% polar lipids; whereas the cLD composition contained different proportions of these same lipid classes, in particular 91% TAG. The TAG fatty acids from both lipid droplets were enriched in oleic, linoleic, and palmitic acids. The TAG from the nLD corresponded to a small pool, whereas the TAG from the cLD constituted the main cellular pool (at about 100% yield from the total homogenate). In conclusion, nLD are a domain within the nucleus where NL are stored and organized and may be involved in nuclear lipid homeostasis.► Nuclear neutral lipids are organized in nLD within the nucleus in the nuclear matix. ► nLD is a nuclear domain present under normal growth conditions. ► nLD are randomly distributed within the nucleus. ► nLD have a lipid composition different than that of cLD. ► nLD TAG and CE are a small pool whereas the main cellular pool of these lipids is in cLD.
Keywords: Nuclear neutral lipid; Nuclear domain; Lipid droplet; Endonuclear lipid; Triacylglycerol; Oleic acid;

Spatially resolved investigation of the oil composition in single intact hyphae of Mortierella spp. with micro-Raman spectroscopy by Ute Münchberg; Lysett Wagner; Eike T. Spielberg; Kerstin Voigt; Petra Rösch; Jürgen Popp (341-349).
Zygomycetes are well known for their ability to produce various secondary metabolites. Fungi of the genus Mortierella can accumulate highly unsaturated lipids in large amounts as lipid droplets. However, no information about the spatial distribution or homogeneity of the oil inside the fungi is obtainable to date due to the invasive and destructive analytical techniques applied so far. Raman spectroscopy has been demonstrated to be well suited to investigate biological samples on a micrometre scale. It also has been shown that the degree of unsaturation of lipids can be determined from Raman spectra. We applied micro-Raman spectroscopy to investigate the spatial distribution and composition of lipid vesicles inside intact hyphae. For Mortierella alpina and Mortierella elongata distinct differences in the degree of unsaturation and even the impact of growth conditions are determined from the Raman spectra. In both species we found that the fatty acid saturation in the vesicles is highly variable in the first 600 μm of the growing hyphal tip and fluctuates towards a constant composition and saturation ratio in all of the remaining mycelium. Our approach facilitates in vivo monitoring of the lipid production and allows us to investigate the impact of cultivation parameters on the oil composition directly in the growing hyphae without the need for extensive extraction procedures.► Lipid vesicles inside intact hyphae of Mortierella are investigated. ► The degree of unsaturation of the oil is determined with Raman spectroscopy. ► The vesicle composition is highly variable in the first 600 μm of the hyphal tips. ► Lipid composition is homogeneous in all older hyphal parts throughout the mycelium.
Keywords: Oleaginous fungi; Mortierellales; Mortierellomycotina; Arachidonic acid production; Polyunsaturated fatty acids (PUFAs); Lipid bodies;

C16-Ceramide-induced F-actin regulation stimulates mouse embryonic stem cell migration: Involvement of N-WASP/Cdc42/Arp2/3 complex and cofilin-1/α-actinin by Su Shin Park; Mi Ok Kim; Seung Pil Yun; Jung Min Ryu; Jae Hong Park; Bit Na Seo; Ji Hoon Jeon; Ho Jae Han (350-360).
Ceramide, a major structural element in the cellular membrane, is a key regulatory factor in various cellular behaviors that are dependent on ceramide-induced association of specific proteins. However, molecular mechanisms that regulate ceramide-induced embryonic stem cell (ESC) migration are still not well understood. Thus, we investigated the effect of ceramide on migration and its related signal pathways in mouse ESCs. Among ceramide species with different fatty acid chain lengths, C16-Cer increased migration of mouse ESCs in a dose- (≥ 1 μM) and time-dependent (≥ 8 h) manners, as determined by the cell migration assay. C16-Cer (10 μM) increased protein-kinase C (PKC) phosphorylation. Subsequently, C16-Cer increased focal adhesion kinase (FAK) and Paxillin phosphorylation, which were inhibited by PKC inhibitor Bisindolylmaleimide I (1 μM). When we examined for the downstream signaling molecules, C16-Cer activated small G protein (Cdc42) and increased the formation of complex with Neural Wiskott-Aldrich Syndrome Protein (N-WASP)/Cdc42/Actin-Related Protein 2/3 (Arp2/3). This complex formation was disrupted by FAK- and Paxillin-specific siRNAs. Furthermore, C16-Cer-induced increase of filamentous actin (F-actin) expression was inhibited by Cdc42-, N-WASP-, and Arp2/3-specific siRNAs, respectively. Indeed, C16-Cer increased cofilin-1/F-actin interaction or F-actin/α-actinin-1 and α-actinin-4 interactions in the cytoskeleton compartment, which was reversed by Cdc42-specific siRNA. Finally, C16-Cer-induced increase of cell migration was inhibited by knocking down each signal pathway-related molecules with siRNA or inhibitors. In conclusion, C16-Cer enhances mouse ESC migration through the regulation of PKC and FAK/Paxillin-dependent N-WASP/Cdc42/Arp2/3 complex formation as well as through promoting the interaction between cofilin-1 or α-actinin-1/-4 and F-actin.► C16-Cer increases mouse ESC migration. ► C16-Cer regulates PKC and FAK/Paxillin-dependent Cdc42 activation. ► C16-Cer treatment leads to formation of N-WASP/Cdc42/Arp2/3 complex. ► F-actin expression is increased following C16-Cer treatment. ► C16-Cer promotes the interaction between cofilin-1 or α-actinin-1/-4 and F-actin.
Keywords: Mouse embryonic stem cell; Ceramide; Filamentous actin (F-actin); Cofilin-1; α-Actinin; Migration;

Characterization of a mutation that results in independence of oxidosqualene cyclase (Erg7) activity from the downstream 3-ketoreductase (Erg27) in the yeast ergosterol biosynthetic pathway by Jacob V. Layer; Brett M. Barnes; Yuji Yamasaki; Robert Barbuch; Liangtao Li; Silvia Taramino; Gianni Balliano; Martin Bard (361-369).
In yeast, deletion of ERG27, which encodes the sterol biosynthetic enzyme, 3-keto-reductase, results in a concomitant loss of the upstream enzyme, Erg7p, an oxidosqualene cyclase (OSC). However, this phenomenon occurs only in fungi, as mammalian Erg27p orthologues are unable to rescue yeast Erg7p activity. In this study, an erg27 mutant containing the mouse ERG27 orthologue was isolated that was capable of growing without sterol supplementation (FGerg27). GC/MS analysis of this strain showed an accumulation of squalene epoxides, 3-ketosterones, and ergosterol. This strain which was crossed to a wildtype and daughter segregants showed an accumulation of squalene epoxides as well as ergosterol indicating that the mutation entailed a leaky block at ERG7. Upon sequencing the yeast ERG7 gene an A598S alteration was found in a conserved alpha helical region. We theorize that this mutation stabilizes Erg7p in a conformation that mimics Erg27p binding. This mutation, while decreasing OSC activity still retains sufficient residual OSC activity such that the strain in the presence of the mammalian 3-keto reductase enzyme functions and no longer requires the yeast Erg27p. Because sterol biosynthesis occurs in the ER, a fusion protein was synthesized combining Erg7p and Erg28p, a resident ER protein and scaffold of the C-4 demethyation complex. Both FGerg27 and erg27 strains containing this fusion plasmid and the mouse ERG27 orthologue showed restoration of ergosterol biosynthesis with minimal accumulation of squalene epoxides. These results indicate retention of Erg7p in the ER increases its activity and suggest a novel method of regulation of ergosterol biosynthesis.► We identify a novel mutation allowing independence of Erg7 from Erg27. ► We sequenced the ERG27 mutation and found a non synonymous alteration at position 598. ► We constructed a fusion plasmid that allowed retention of Erg7 in the ER.
Keywords: Saccharomyces cerevisiae; Ergosterol; Erg7; Erg27; 3-keto reductase; Oxidosqualene cyclase;

A high-fat diet increases l-carnitine synthesis through a differential maturation of the Bbox1 mRNAs by Caroline Rigault; Françoise Le Borgne; Bassim Tazir; Alexandre Benani; Jean Demarquoy (370-377).
l-carnitine is a key molecule in both mitochondrial and peroxisomal lipid metabolisms. l-carnitine is biosynthesized from gamma-butyrobetaine by a reaction catalyzed by the gamma-butyrobetaine hydroxylase (Bbox1). The aim of this work was to identify molecular mechanisms involved in the regulation of l-carnitine biosynthesis and availability. Using 3′ RACE, we identified four alternatively polyadenylated Bbox1 mRNAs in rat liver. We utilized a combination of in vitro experiments using hybrid constructs containing the Bbox1 3′ UTR and in vivo experiments on rat liver mRNAs to reveal specificities in the different Bbox1 mRNA isoforms, especially in terms of polyadenylation efficiency, mRNA stability and translation efficiency. This complex maturation process of the Bbox1 mRNAs in the liver was studied on rats fed a high-fat diet. High-fat diet selectively increased the level of three Bbox1 mRNA isoforms in rat liver and the alternative use of polyadenylation sites contributed to the global increase in Bbox1 enzymatic activity and l-carnitine levels. Our results show that the maturation of Bbox1 mRNAs is nutritionally regulated in the liver through a selective polyadenylation process to adjust l-carnitine biosynthesis to the energy supply.► Bbox1 mRNA maturation involves an alternative polyadenylation process. ► A high fat diet alters the polyadenylation of Bbox1 mRNAs. ► Alternative polyadenylation of Bbox1 affects l-carnitine bioavailability. ► Polyadenylation process can be controlled by the nature of the diet.
Keywords: l-carnitine; Fatty acid metabolism; Polyadenylation; mRNA stability;

Macrophages are centrally involved during atherosclerosis development and are the predominant cell type that accumulates cholesterol in the plaque. Macrophages however, are heterogeneous in nature reflecting a variety of microenvironments and different phenotypes may be more prone to contribute towards atherosclerosis progression. Using primary human monocyte-derived macrophages, we sought to evaluate one aspect of atherogenic potential of different macrophage phenotypes by determining their propensity to associate with and accumulate oxidized low density lipoprotein (oxLDL). Classically-activated macrophages treated simultaneously with interferon γ (IFNγ) and tumor necrosis factor α (TNFα) associated with less oxLDL and accumulated less cholesterol compared to untreated controls. The combined treatment of IFNγ and TNFα reduced the mRNA expression of CD36 and the expression of both cell surface CD36 and macrophage scavenger receptor 1 (MSR1) protein. Under oxLDL loaded conditions, IFNγ and TNFα did not reduce macrophage protein expression of the transcription factor peroxisome proliferator-actived receptor γ (PPARγ) which is known to positively regulate CD36 expression. However, macrophages treated with IFNγ attenuated the ability of the PPARγ-specific agonist rosiglitazone from upregulating cell surface CD36 protein expression. Our results demonstrate that the observed reduction of cholesterol accumulation in macrophages treated with IFNγ and TNFα following oxLDL treatment was due at least in part to reduced cell surface CD36 and MSR1 protein expression.► We polarize human monocyte-derived macrophages (MDMs) to various sub-phenotypes. ► We test the propensity of different MDM sub-phenotypes to accumulate cholesterol. ► IFNγ or TNFα treatment reduces total cholesterol accumulation. ► Treatment with either IFNγ or TNFα reduces the expression of both CD36 and SR-AI. ► IFNγ attenuates the PPARγ-mediated upregulation of CD36.
Keywords: Macrophage; Cholesterol; Interferon γ; CD36; Macrophage scavenger receptor I; Peroxisome proliferator-activated receptor γ;

Characterization of lipid and lipoprotein metabolism in primary human hepatocytes by Ji Ling; Jamie Lewis; Donna Douglas; Norman M. Kneteman; Dennis E. Vance (387-397).
Primary rodent hepatocytes and hepatoma cell lines are commonly used as model systems to elucidate and study potential drug targets for metabolic diseases such as obesity and atherosclerosis. However, if therapies are to be developed, it is essential that our knowledge gained from these systems is translatable to that of human. Here, we have characterized lipid and lipoprotein metabolism in primary human hepatocytes for comparison to rodent primary hepatocytes and human hepatoma cell lines. Primary human hepatocytes were maintained in collagen coated dishes in confluent monolayers for up to 3 days. We found primary human hepatocytes were viable, underwent lipid synthesis, and were able to secret lipoproteins up to 3 days in culture. Furthermore, the lipoprotein profile secreted by primary human hepatocytes was comparable to that found in human plasma; this contrasts with primary rodent hepatocytes and human hepatoma cells. We also investigated the pharmacological effects of nicotinic acid (niacin, NA), a potent dyslipidemic drug, on hepatic lipid synthesis and lipoprotein secretion. We found NA increased the expression of ATP-binding cassette transporter A1 in primary human hepatocytes, which may potentially explain how NA increases plasma high-density lipoproteins in humans. In conclusion, primary human hepatocytes are a more relevant model to study lipid synthesis and lipoprotein secretion than hepatoma cells or rodent primary hepatocyte models. Further research needs to be done to maintain liver specific functions of primary human hepatocytes in prolonged cultures for these cells to be a viable model.► Primary human hepatocytes were maintained in confluent monolayers for 3 days. ► Lipoproteins secreted by primary human hepatocytes are similar to human plasma. ► Nicotinic acid did not change the labeling of triacylglycerol or phosphatidylcholine. ► Apo B100, Apo A1 and major lipids in hepatocyte medium were similar ± nicotinic acid.
Keywords: High density lipoprotein; Very low density lipoprotein; Nicotinic acid; Cholesteryl ester; Triacylglcyerol; Phospholipid;

Cyclosporine A and PSC833 inhibit ABCA1 function via direct binding by Kohjiro Nagao; Minami Maeda; Noralyn B. Mañucat; Kazumitsu Ueda (398-406).
ATP-binding cassette protein A1 (ABCA1) plays a key role in generating high-density lipoprotein (HDL). However, the detailed mechanism of HDL formation remains unclear; in order to reveal it, chemicals that specifically block each step of HDL formation would be useful. Cyclosporine A inhibits ABCA1-mediated cholesterol efflux, but it is not clear whether this is mediated via inhibition of calcineurin. We analyzed the effects of cyclosporine A and related compounds on ABCA1 function in BHK/ABCA1 cells. Cyclosporine A, FK506, and pimecrolimus inhibited ABCA1-mediated cholesterol efflux in a concentration-dependent manner, with IC50 of 7.6, 13.6, and 7.0 μM, respectively. An mTOR inhibitor, rapamycin also inhibited ABCA1, with IC50 of 18.8 μM. The primary targets for these drugs were inhibited at much lower concentrations in BHK/ABCA1 cells, suggesting that they were not involved. Binding of [3H] cyclosporine A to purified ABCA1 could be clearly detected. Furthermore, a non-immunosuppressive cyclosporine, PSC833, inhibited ABCA1-mediated cholesterol efflux with IC50 of 1.9 μM, and efficiently competed with [3H] cyclosporine A binding to ABCA1. These results indicate that cyclosporine A and PSC833 inhibit ABCA1 via direct binding, and that the ABCA1 inhibitor PSC833 is an excellent candidate for further investigations of the detailed mechanisms underlying formation of HDL.► No inhibitors for HDL formation are clear in their mechanisms. ► Cyclosporine A and PSC833 directly bind to ABCA1 and inhibit HDL formation. ► Non-toxic PSC833 is an excellent inhibitor of ABCA1 to analyze HDL formation.
Keywords: ABCA1; HDL; Inhibitor; Cyclosporine A; PSC833; Cholesterol;

Adenosine A2A receptor activation supports an atheroprotective cholesterol balance in human macrophages and endothelial cells by Iryna Voloshyna; Steven Carsons; Michael J. Littlefield; Jayson M. Rieger; Robert Figler; Allison B. Reiss (407-416).
The adenosine A2A receptor (A2AR) plays an important role in the regulation of inflammatory and immune responses. Our previous work has demonstrated that A2AR agonists exhibit atheroprotective effects by increasing expression of reverse cholesterol transport proteins in cultured human macrophages. This study explores the impact of pharmacologic activation/inhibition and gene silencing of A2AR on cholesterol homeostasis in both THP-1 human monocytes/macrophages and primary human aortic endothelial cells (HAEC).THP-1 human monocytes/macrophages and HAEC exposed to the A2AR-specific agonist ATL313 exhibited upregulation of proteins responsible for cholesterol efflux: the ABCA1 and G1 transporters. Further, activation of A2AR led to upregulation of the cholesterol metabolizing enzyme P450 27-hydroxylase, accompanied by intracellular changes in level of oxysterols. We demonstrate that anti-atherogenic properties of A2AR activation are not limited to the regulation of lipid efflux in vasculature, but include protection from lipid overload in macrophages, particularly via suppression of the CD36 scavenger receptor. The reduced lipid accumulation manifests directly as a diminution in foam cell transformation. In THP-1 macrophages, either A2AR pharmacological blockade or gene silencing promote lipid accumulation and enhance foam cell transformation.Our pre-clinical data provides evidence suggesting that A2AR stimulation by ATL313 has the potential to be a viable therapeutic strategy for cardiovascular disease prevention, particularly in patients with elevated risk due to immune/inflammatory disorders.► ATL313 stimulates cholesterol efflux through increased expression of ABCA1 and G1 transporters. ► ATL313 upregulates 27-hydroxylase expression and activity, with concomitant changes in the level of oxysterols. ► ATL313 inhibits cholesterol influx and scavenger receptors expression ► ATL313 protects from lipid accumulation and foam cell formation. ► A2AR blockade promotes lipid accumulation and augments foam cell transformation
Keywords: Atherosclerosis; Adenosine 2A receptor; Foam Cell Formation; Reverse Cholesterol Transport; Scavenger Receptors;

The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): Structure drives preference for simple neutral glycosphingolipids by Ravi Kanth Kamlekar; Dhirendra K. Simanshu; Yong-guang Gao; Roopa Kenoth; Helen M. Pike; Franklyn G. Prendergast; Lucy Malinina; Julian G. Molotkovsky; Sergei Yu Venyaminov; Dinshaw J. Patel; Rhoderick E. Brown (417-427).
Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a key role in glycosphingolipid (GSL) production using its C-terminal domain to transport newly synthesized glucosylceramide away from the cytosol-facing glucosylceramide synthase in the cis-Golgi for further anabolic processing. Structural homology modeling against human glycolipid transfer protein (GLTP) predicts a GLTP-fold for FAPP2 C-terminal domain, but no experimental support exists to warrant inclusion in the GLTP superfamily. Here, the biophysical properties and glycolipid transfer specificity of FAPP2-C-terminal domain have been characterized and compared with other established GLTP-folds. Experimental evidence for a GLTP-fold includes: i) far-UV circular dichroism (CD) showing secondary structure with high alpha-helix content and a low thermally-induced unfolding transition (~ 41 °C); ii) near-UV-CD indicating only subtle tertiary conformational change before/after interaction with membranes containing/lacking glycolipid; iii) Red-shifted tryptophan (Trp) emission wavelength maximum (λmax  ~ 352 nm) for apo-FAPP2-C-terminal domain consistent with surface exposed intrinsic Trp residues; iv) ‘signature’ GLTP-fold Trp fluorescence response, i.e., intensity decrease (~ 30%) accompanied by strongly blue-shifted λmax (~ 14 nm) upon interaction with membranes containing glycolipid, supporting direct involvement of Trp in glycolipid binding and enabling estimation of partitioning affinities. A structurally-based preference for other simple uncharged GSLs, in addition to glucosylceramide, makes human FAPP2-GLTP more similar to fungal HET-C2 than to plant AtGLTP1 (glucosylceramide-specific) or to broadly GSL-selective human GLTP. These findings along with the distinct mRNA exon/intron organizations originating from single-copy genes on separate human chromosomes suggest adaptive evolutionary divergence by these two GLTP-folds.► FAPP2 enables glycosphingolipid synthesis in the Golgi by transfer of glucosylceramide. ► FAPP2 contains a modified GLTP-fold that can also transfer other neutral glycosphingolipids. ► Glycolipid selectivity of FAPP2-GLTP is more focused than human GLTP. ► Glycolipid compartmentation appears to mute evolutionary selection pressure and divergence.
Keywords: Glycosphingolipid binding and transfer; GLTP superfamily; Membrane interaction; Tryptophan fluorescence; Near-UV and far-UV circular dichroism; Divergent evolution;

Import of phosphatidylserine to and export of phosphatidylethanolamine molecular species from mitochondria by Ville Kainu; Martin Hermansson; Satu Hänninen; Kati Hokynar; Pentti Somerharju (429-437).
Heavy isotope-labeled ethanolamine and serine as well as exogenous PE and PS species were used to study trafficking of phosphatidylethanolamine (PE) and -serine (PS) molecular species between the endoplasmic reticulum (ER) and mitochondria in HeLa cells. Import of both endogenous and exogenous PS to IMM was a relatively slow process (T1/2 = several hours), but depended on the acyl chains. In particular, the 38:4 and 38:5 species were imported more efficiently compared to the other PS species. Knock-down of Mitofusin 2 or Mitostatin had no detectable effect on PS import to mitochondria, suggesting that the ER–mitochondria contacts regulated by these proteins are not essential. Knock-down of PS synthase 1 inhibited PS decarboxylation, suggesting that import of PS to mitochondria is coupled to its synthesis. Also the export of PE from IMM to microsomes is a relatively slow process, but again depends markedly on the acyl chain structure. Most notably, the polyunsaturated 38:4 and 38:5 PE species were less efficiently exported, which together with rapid import of the PS precursors most probably explains their enrichment in IMM. PE synthesized via the CDP-ethanolamine was also imported to IMM, but most of the PE in this membrane derives from imported PS. In contrast to PS, all PC species made in Golgi/ER translocated similarly and rapidly to IMM. In conclusion, selective translocation of PS species and PS-derived PE species between ER and mitochondria plays a major role in phospholipid homeostasis of these organelles.► Mass spectrometry was used to study PE and PS trafficking to and from mitochondria. ► The acyl chains have a major effect on translocation of both PS and PE. ► KD of Mitofusin 2 and Mitostatin had no effect on PS translocation to mitochondria. ► Inhibition of PS synthesis by KD of PS synthase 1 markedly inhibited PS translocation. ► The CDP-EA and PSD pathways both contribute significantly to the PE pools of IMM and ER.
Keywords: Phospholipid; Contact site; Spontaneous diffusion; Mass-spectrometry; Heavy isotope; MAM;

Sugar availability modulates polyisoprenoid and phytosterol profiles in Arabidopsis thaliana hairy root culture by Adam Jozwiak; Magdalena Ples; Karolina Skorupinska-Tudek; Magdalena Kania; Marta Dydak; Witold Danikiewicz; Ewa Swiezewska (438-447).
Sugars are recognized as signaling molecules regulating the biosynthesis of secondary metabolites in plants. Here, a modulatory effect of sugars on dolichol and phytosterol profiles was noted in the hairy roots of Arabidopsis thaliana. Arabidopsis roots contain a complex dolichol mixture comprising three groups (‘families’) of dolichols differing in the chain-length. These dolichols, especially the longest ones are accompanied by considerable amounts of polyprenols of the same length. The spectrum of polyisoprenoid alcohols, i.e. dolichols and polyprenols, was dependent on sugar type (glucose or sucrose) and its concentration in the medium. Among the long-chain dolichols Dol/Pren-20 (dolichol or prenol molecule composed of 20 isoprene residues) and Dol/Pren-23 were the main components at 0.5% and 2% glucose, respectively. Moreover, the ratio of polyprenols versus respective dolichols was also modulated by sugar in this group of polyisoprenoids, with polyprenols dominating at 3% sucrose and dolichols at 2% glucose. Glucose concentration affected the expression level of genes encoding cis-prenyltransferases, enzymes responsible for elongation of the polyisoprenoid chain. The most abundant phytosterols of the A. thaliana roots, β-sitosterol, stigmasterol and campesterol, were accompanied by corresponding stanols and traces of brassicasterol, stigmast-4,22-dien-3-one and stigmast-4-en-3-one. Similar to the polyisoprenoids, sterol profile responded to the sugar present in the medium, β-sitosterol dominating in roots grown on 3% or lower glucose concentrations and stigmasterol in 3% sucrose. These results indicate an involvement of sugar signaling in the regulation of cis-prenyltransferases and phytosterol pathway enzymes.Display Omitted► Lipid profile of A. thaliana is modulated by sugar composition and concentration. ► Expression level of cis-prenyltransferases is affected by glucose concentration. ► Complex polyisoprenoid and sterol composition of A. thaliana roots is described.
Keywords: Dolichol; Phytosterol; Dolichyl ester; Steryl ester; cis-prenyltransferase; Arabidopsis thaliana;

Regulation of lung surfactant phospholipid synthesis and metabolism by Victoria Goss; Alan N. Hunt; Anthony D. Postle (448-458).
The alveolar type II epithelial (ATII) cell is highly specialised for the synthesis and storage, in intracellular lamellar bodies, of phospholipid destined for secretion as pulmonary surfactant into the alveolus. Regulation of the enzymology of surfactant phospholipid synthesis and metabolism has been extensively characterised at both molecular and functional levels, but understanding of surfactant phospholipid metabolism in vivo in either healthy or, especially, diseased lungs is still relatively poorly understood. This review will integrate recent advances in the enzymology of surfactant phospholipid metabolism with metabolic studies in vivo in both experimental animals and human subjects. It will highlight developments in the application of stable isotope-labelled precursor substrates and mass spectrometry to probe lung phospholipid metabolism in terms of individual molecular lipid species and identify areas where a more comprehensive metabolic model would have considerable potential for direct application to disease states.Display Omitted► Normal lung surfactant phosphatidylcholine homeostasis is tightly regulated. ► Regulated steps include synthesis, molecular selection, intracellular transport, secretion, reuptake and recycling. ► Aberrant regulation is involved in many surfactant pathologies. ► Dynamic lipidomic analyses using stable isotopes provide novel insights into the underlying mechanisms. ► Lung phospholipid synthesis and turnover are integrated with extra-pulmonary whole body phospholipid metabolism.
Keywords: Pulmonary surfactant; Phospholipid synthesis; Stable isotopes; Mass spectrometry;

Disruption of Stard10 gene alters the PPARα-mediated bile acid homeostasis by Masanori Ito; Yoshihide Yamanashi; Yu Toyoda; Hiroko Izumi-Nakaseko; Satoko Oda; Atsushi Sugiyama; Masaru Kuroda; Hiroshi Suzuki; Tappei Takada; Satomi Adachi-Akahane (459-468).
STARD10, a member of the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) protein family, is highly expressed in the liver and has been shown to transfer phosphatidylcholine. Therefore it has been assumed that STARD10 may function in the secretion of phospholipids into the bile. To help elucidate the physiological role of STARD10, we produced Stard10 knockout mice (Stard10 −/−) and studied their phenotype. Neither liver content nor biliary secretion of phosphatidylcholine was altered in Stard10 −/− mice. Unexpectedly, the biliary secretion of bile acids from the liver and the level of taurine-conjugated bile acids in the bile were significantly higher in Stard10 −/− mice than wild type (WT) mice. In contrast, the levels of the secondary bile acids were lower in the liver of Stard10 −/− mice, suggesting that the enterohepatic cycling is impaired. STARD10 was also expressed in the gallbladder and small intestine where the expression level of apical sodium dependent bile acid transporter (ASBT) turned out to be markedly lower in Stard10 −/− mice than in WT mice when measured under fed condition. Consistent with the above results, the fecal excretion of bile acids was significantly increased in Stard10 −/− mice. Interestingly, PPARα-dependent genes responsible for the regulation of bile acid metabolism were down-regulated in the liver of Stard10 / mice. The loss of STARD10 impaired the PPARα activity and the expression of a PPARα-target gene such as Cyp8b1 in mouse hepatoma cells. These results indicate that STARD10 is involved in regulating bile acid metabolism through the modulation of PPARα-mediated mechanism.► Knockout of Stard10 enhanced biliary secretion and fecal excretion of bile acids. ► ASBT expression was impaired in the gallbladder and small intestine of Stard10 /. ► PPARα-target genes such as Cyp8b1 were down-regulated in the liver of Stard10 /. ► The PPARα activity was affected by the expression of STARD10 in mouse hepatoma cells. ► STARD10 is involved in the PPARα-mediated regulation of bile acid homeostasis.
Keywords: Bile acid; Lipid transfer protein; Stard10; CYP8B1; ASBT; PPARα;