BBA - Molecular and Cell Biology of Lipids (v.1861, #2)

High density lipoprotein efficiently accepts surface but not internal oxidised lipids from oxidised low density lipoprotein by Aliki A. Rasmiena; Christopher K. Barlow; Theodore W. Ng; Dedreia Tull; Peter J. Meikle (69-77).
Oxidised low density lipoprotein (oxLDL) contributes to atherosclerosis, whereas high density lipoprotein (HDL) is known to be atheroprotective due, at least in part, to its ability to remove oxidised lipids from oxLDL. The molecular details of the lipid transfer process are not fully understood. We aimed to identify major oxidised lipid species of oxLDL and investigate their transfer upon co-incubation with HDL with varying levels of oxidation.A total of 14 major species of oxidised phosphatidylcholine and oxidised cholesteryl ester from oxLDL were identified using an untargeted mass spectrometry approach. HDL obtained from pooled plasma of normolipidemic subjects (N = 5) was oxidised under mild and heavy oxidative conditions. Non-oxidised (native) HDL and oxidised HDL were co-incubated with oxLDL, re-isolated and lipidomic analysis was performed. Lipoprotein surface lipids, oxidised phosphatidylcholines and oxidised cholesterols (7-ketocholesterol and 7β-hydroxycholesterol), but not internal oxidised cholesteryl esters, were effectively transferred to native HDL. Saturated and monounsaturated lyso-phosphatidylcholines were also transferred from the oxLDL to native HDL. These processes were attenuated when HDL was oxidised under mild and heavy oxidative conditions. The impaired capacities were accompanied by an increase in a ratio of sphingomyelin to phosphatidylcholine and a reduction in phosphatidylserine content in oxidised HDL, both of which are potentially important regulators of the oxidised lipid transfer capacity of HDL.Our study has revealed the differential transfer efficiency of surface and internal oxidised lipids from oxLDL and their acceptance onto HDL. These capacities were modulated when HDL was itself oxidised.
Keywords: Lipidomic; Cholesteryl ester; Oxysterols; Phosphatidylcholine; Transfer; Anti-oxidative;

Increased synthesis of hyaluronic acid (HA) is often associated with increased metastatic potential and invasivity of tumor cells. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis, and has been studied as a potential anti-tumor drug to inhibit the growth of primary tumors and distant metastasis of tumor cells. Although several studies reported that the anticancer effects of MU are mediated by inhibition of HA signaling, the mechanism still needs to be clarified. In a previous study we demonstrated the regulation of HA synthesis by ceramide, and now show how MU activated neutral sphingomyelinase2 (NSMase2) generates ceramides and mediates MU induced inhibition of HA synthesis, cell migration and invasion, and apoptosis of tumor cells. Using a HA enriched mouse oligodendroglioma cell line G26-24 we found that MU elevated the activity of NSMase2 and increased ceramide levels, which in turn increased phosphatase PP2A activity. Further, the activated PP2A reduced phosphorylation of Akt, decreased activities of HA synthase2 (HAS2) and calpains, and inhibited both the synthesis of HA, and the migration and invasion of G26-24 tumor cells. In addition, MU mediated ceramide stimulated activation of p53 and caspase-3, reduced SIRT1 expression and decreased G26-24 viability. The mechanism of the MU anticancer therefore initially involves NSMase2/ceramide/PP2A/AKT/HAS2/caspase-3/p53/SIRT1 and the calpain signaling pathway, suggesting that ceramides play a key role in the ability of a tumor to become aggressively metastatic and grow.
Keywords: 4-Methylumbelliforone; NSMase2; Ceramide; Hyaluronic acid; Anticancer;

Palmitic acid (16:0) competes with omega-6 linoleic and omega-3 ɑ-linolenic acids for FADS2 mediated Δ6-desaturation by Hui Gyu Park; Kumar S.D. Kothapalli; Woo Jung Park; Christian DeAllie; Lei Liu; Allison Liang; Peter Lawrence; J. Thomas Brenna (91-97).
Sapienic acid, 16:1n-10 is the most abundant unsaturated fatty acid on human skin where its synthesis is mediated by FADS2 in the sebaceous glands. The FADS2 product introduces a double bond at the Δ6, Δ4 and Δ8 positions by acting on at least ten substrates, including 16:0, 18:2n-6, and 18:3n-3. Our aim was to characterize the competition for accessing FADS2 mediated Δ6 desaturation between 16:0 and the most abundant polyunsaturated fatty acids (PUFA) in the human diet, 18:2n-6 and 18:3n-3, to evaluate whether competition may be relevant in other tissues and thus linked to metabolic abnormalities associated with FADS2 or fatty acid levels. MCF7 cells stably transformed with FADS2 biosynthesize 16:1n-10 from exogenous 16:0 in preference to 16:1n-7, the immediate product of SCD highly expressed in cancer cell lines, and 16:1n-9 via partial β-oxidation of 18:1n-9. Increasing availability of 18:2n-6 or 18:3n-3 resulted in decreased bioconversion of 16:0 to 16:1n-10, simultaneously increasing the levels of highly unsaturated products. FADS2 cells accumulate the desaturation-elongation products 20:3n-6 and 20:4n-3 in preference to the immediate desaturation products 18:3n-6 and 18:4n-3 implying prompt/coupled elongation of the nascent desaturation products. MCF7 cells incorporate newly synthesized 16:1n-10 into phospholipids. These data suggest that excess 16:0 due to, for instance, de novo lipogenesis from high carbohydrate or alcohol consumption, inhibits synthesis of highly unsaturated fatty acids, and may in part explain why supplemental preformed EPA and DHA in some studies improves insulin resistance and other factors related to diabetes and metabolic syndrome aggravated by excess calorie consumption.
Keywords: Palmitic acid; Sapienic acid; Δ6 desaturase; Fatty acid desaturases; Monounsaturated fatty acids; Polyunsaturated fatty acids;

Itinerary of high density lipoproteins in endothelial cells by Damir Perisa; Lucia Rohrer; Andres Kaech; Arnold von Eckardstein (98-107).
High density lipoprotein (HDL) and its main protein component apolipoprotein A-I (ApoA-I) have multiple anti-atherogenic functions. Some of them are exerted within the vessel wall, so that HDL needs to pass the endothelial barrier. To elucidate their itinerary through endothelial cells (ECs), we labelled ApoA-I and HDL either fluorescently or with 1.4 nm nanogold and investigated their cellular localization by using immunofluorescent microscopy (IFM) and electron microscopy (EM). HDL as well as ApoA-I is taken up by ECs into the same route of intracellular trafficking. Time kinetics and pulse chase experiments revealed that HDL is trafficked through different vesicles. HDL partially co-localized with LDL, albumin, and transferrin. HDL did not co-localize with clathrin and caveolin-1. Fluorescent HDL was recovered at small proportions in early endosomes and endosome to trans-golgi network vesicles but not at all in recycling endosomes, in late endosomes or lysosomes. EM identified HDL mainly in large filled vesicles which however upon IFM did not colocalize with markers of multivesicular bodies or autophagosomes. The uptake or cellular distribution of HDL was altered upon pharmacological interference with cytochalasine D, colchicine and dynasore. Blockage of fluid phase uptake with Amiloride or EIPA did not reduce the uptake of HDL. Neither did we observe any co-localization of HDL with dextran as the marker of fluid phase uptake. In conclusion, HDL and ApoA-I are internalized and trafficked by endothelial cells through a non-classical endocytic route.
Keywords: High density lipoproteins; Endothelial cells; Endocytosis; Multivesicular bodies;

The genome of Aspergillus niger codes for a fusion protein (EHA25900), which can be aligned with ~ 50% sequence identity to 9S-dioxygenase (DOX)-allene oxide synthase (AOS) of Fusarium oxysporum, homologues of the Fusarium and Colletotrichum complexes and with over 62% sequence identity to homologues of Aspergilli, including (DOX)-9R-AOS of Aspergillus terreus. The aims were to characterize the enzymatic activities of EHA25900 and to identify crucial amino acids for the stereospecificity. Recombinant EHA25900 oxidized 18:2n-6 sequentially to 9R-hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HPODE) and to a 9R(10)-allene oxide. 9S- and 9R-DOX-AOS catalyze abstraction of the pro-R hydrogen at C-11, but the direction of oxygen insertion differs. A comparison between twelve 9-DOX domains of 9S- and 9R-DOX-AOS revealed conserved amino acid differences, which could contribute to the chirality of products. The Gly616Ile replacement of 9R-DOX-AOS (A. niger) increased the biosynthesis of 9S-HPODE and the 9S(10)-allene oxide, whereas the Phe627Leu replacement led to biosynthesis of 9S-HPODE and the 9S(10)-allene oxide as main products. The double mutant (Gly616Ile, Phe627Leu) formed over 90% of the 9S stereoisomer of HPODE. 9S-HPODE was formed by antarafacial hydrogen abstraction and oxygen insertion, i.e., the original H-abstraction was retained but the product chirality was altered. We conclude that 9R-DOX-AOS can be altered to 9S-DOX-AOS by replacement of two amino acids (Gly616Ile, Phe627Leu) in the DOX domain.
Keywords: Fatty acid oxidation; Mass spectrometry; Class III cytochrome P450; Heme peroxidase; Linoleate diol synthase; Site-specific mutagenesis;

Lack of phosphatidylethanolamine N-methyltransferase in mice does not promote fatty acid oxidation in skeletal muscle by Guergana Tasseva; Jelske N. van der Veen; Susanne Lingrell; René L. Jacobs; Dennis E. Vance; Jean E. Vance (119-129).
Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected from high-fat diet-induced obesity and insulin resistance, and exhibit increased whole-body energy expenditure and oxygen consumption. Since skeletal muscle is a major site of fatty acid oxidation and energy utilization, we determined if rates of fatty acid oxidation/oxygen consumption in muscle are higher in Pemt / mice than in Pemt +/+ mice. Although PEMT is abundant in the liver, PEMT protein and activity were undetectable in four types of skeletal muscle. Moreover, amounts of PC and PE in the skeletal muscle were not altered by PEMT deficiency. Thus, we concluded that any influence of PEMT deficiency on skeletal muscle would be an indirect consequence of lack of PEMT in liver. Neither the in vivo rate of fatty acid uptake by muscle nor the rate of fatty acid oxidation in muscle explants and cultured myocytes depended upon Pemt genotype. Nor did PEMT deficiency increase oxygen consumption or respiratory function in skeletal muscle mitochondria. Thus, the increased whole body oxygen consumption in Pemt / mice, and resistance of these mice to diet-induced weight gain, are not primarily due to increased capacity of skeletal muscle for utilization of fatty acids as an energy source.
Keywords: Phosphatidylcholine; Mitochondria; High-fat diet; Myotubes; Fatty acid oxidation; Malonyl-CoA;

Angiopoietin-like protein 8 (betatrophin) is a stress-response protein that down-regulates expression of adipocyte triglyceride lipase by Yuan Zhang; Shiwu Li; William Donelan; Chao Xie; Hai Wang; Qi Wu; Daniel L. Purich; Westley H. Reeves; Dongqi Tang; Li-Jun Yang (130-137).
Atypical angiopoietin-like 8 (ANGPTL8), also known as betatrophin, is known to regulate lipid metabolism. However, its mechanism of action remains elusive.HepG2, 3T3-L1, and NIT-1 cells were cultured in amino acid-complete MEM or histidine-free MEM to detect ANGPTL8 expression. The three cell types were treated with or without recombinant ANGPTL8 to investigate its role in lipid metabolism. Hydrodynamic tail vein gene delivery was also used to examine the role of ANGPTL8 in mice.ANGPTL8 is significantly up-regulated in amino acid-deprived cultured cells in vitro. The activation of ANGPTL8 gene transcription was mediated through the RAS/c-RAF/MAPK signaling pathway rather than the general GCN2/ATF4 pathways. ANGPTL8 activated the ERK signal transduction pathway in hepatocytes, adipocytes, and pancreatic β-cells, up-regulating early growth response transcription factor (Egr1) and down-regulating adipose triglyceride lipase (ATGL).ANGPTL8 is a stress-response protein that regulates fat metabolism by suppressing ATGL expression, revealing a mechanistic connection between ANGPTL8 and lipid homeostasis in mammalian cells.
Keywords: ANGPTL8; Betatrophin; Stress protein; Histidine deprivation; ATGL;

Deletion of sphingosine kinase 1 ameliorates hepatic steatosis in diet-induced obese mice: Role of PPARγ by Jinbiao Chen; Wei Wang; Yanfei Qi; Dominik Kaczorowski; Geoffrey W. McCaughan; Jennifer R. Gamble; Anthony S. Don; Xin Gao; Mathew A. Vadas; Pu Xia (138-147).
Sphingolipid metabolites have emerged playing important roles in the pathogenesis of nonalcoholic fatty liver disease, whereas the underlying mechanism remains largely unknown. In the present study, we provide both in vitro and in vivo evidence showing a pathogenic role of sphingosine kinase 1 (SphK1) in hepatocellular steatosis. We found that levels of SphK1 expression were significantly increased in steatotic hepatocytes. Enforced overexpression of SphK1 or treatment with sphingosine 1-phosphate (S1P) markedly enhanced hepatic lipid accumulation. In contrast, the siRNA-mediated knockdown of SphK1 or S1P receptors, S1P2 and S1P3, profoundly inhibited lipid accumulation in hepatocytes. Moreover, Sphk1 −/− mice exhibited a significant amelioration of hepatosteatosis under diet-induced obese (DIO) conditions, compared to wild-type littermates. In addition, DIO-induced up-regulation of PPARγ and its target genes were significantly reduced by SphK1 deficiency. Furthermore, treatment of hepatocytes with S1P induces a dose-dependent increase in PPARγ expression at the transcriptional level. Blockage of S1P receptors and the Akt-mTOR signaling profoundly inhibited S1P-induced PPARγ expression. Notably, down-regulation of PPARγ by using its siRNA significantly diminished the pro-steatotic effect of SphK1/S1P. Thus, the study demonstrates a new pathway connecting SphK1 and PPARγ involved in the pathogenesis of hepatocellular steatosis.
Keywords: Sphingosine kinase 1; Sphingolipids; Hepatocytes; Nonalcoholic fatty liver disease; PPARγ;