BBA - Molecular and Cell Biology of Lipids (v.1791, #3)

The sense of taste informs the organism about the quality of ingested food. Five basic taste modalities, e.g., sweet, sour, bitter, salty and umami have so far been identified. Recent compelling evidence from rodent and human studies raise the possibility for an additional sixth taste modality devoted to the perception of lipids. Recent studies strongly suggest that lingual CD36, being implicated in the perception of dietary fat, may act as a gustatory lipid sensor. Knocking down of CD36 gene decreases the spontaneous preference for long chain fatty acids (LCFA) in mice subjected to a free choice situation. Lingual CD36, after activation by LCFA, is able to trigger specific signalling mechanisms, e.g., increase in free intracellular calcium concentrations, ([Ca2+]i), phosphorylation of protein-tyrosine kinase (PTK) and release of the neurotransmitters like serotonin and nor-adrenaline into synaptic clefts. This signalling cascade is likely responsible for physiologic responses, induced by the detection of lipids in the oral cavity (i.e., lingual fat preference and cephalic phase of digestion). This review provides recent insights into the molecular mechanisms involved in the oro-sensory perception of lipids.
Keywords: CD36; Lipid; Gustatory perception; Taste;

Cytosolic phospholipase A2-driven PGE2 synthesis within unsaturated fatty acids-induced lipid bodies of epithelial cells by Luciana S. Moreira; Bruno Piva; Luciana B. Gentile; Fabio P. Mesquita-Santos; Heloisa D'Avila; Clarissa M. Maya-Monteiro; Patrícia T. Bozza; Christianne Bandeira-Melo; Bruno L. Diaz (156-165).
Cytoplasmic lipid bodies (also known as lipid droplets) are intracellular deposits of arachidonic acid (AA), which can be metabolized for eicosanoid generation. PGE2 is a major AA metabolite produced by epithelial cells and can modulate restoration of epithelium homeostasis after injury. We studied lipid body biogenesis and their role in AA metabolic pathway in an epithelial cell line derived from normal rat intestinal epithelium, IEC-6 cells. Lipid bodies were virtually absent in confluent IEC-6 cells. Stimulation of confluent IEC-6 cells with unsaturated fatty acids, including AA or oleic acid (OA), induced rapid lipid body assembly that was independent on its metabolism to PGE2, but dependent on G-coupled receptor-driven signaling through p38, PKC, and PI3K. Newly formed lipid bodies compartmentalized cytosolic phospholipase (cPL)A2-α, while facilitated AA mobilization and synthesis of PGE2 within epithelial cells. Thus, both lipid body-related events, including highly regulated biogenesis and functional assembly of cPLA2-α-driven enhanced AA mobilization and PGE2 production, may have key roles in epithelial cell-driven inflammatory functions, and may represent relevant therapeutic targets of epithelial pathologies.
Keywords: Lipid droplet; Epithelial cell; cPLA2-α; PGE2; Arachidonic acid; Oleic acid; p38 MAP kinase;

Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris by T. Wriessnegger; E. Leitner; M.R. Belegratis; E. Ingolic; G. Daum (166-172).
Here we describe for the first time isolation and biochemical characterization of highly purified mitochondrial inner and outer membranes from Pichia pastoris and systematic lipid analysis of submitochondrial fractions. Mitochondria of this yeast are best developed during growth on glycerol or sorbitol, but also on methanol or fatty acids. To obtain organelle membranes at high quality, methods of isolation and subfractionation of mitochondria originally developed for Saccharomyces cerevisiae were adapted and employed. A characteristic feature of the outer mitochondrial membrane of P. pastoris is the higher phospholipid to protein ratio and the lower ergosterol to phospholipid ratio compared to the inner membrane. Another marked difference between the two mitochondrial membranes is the phospholipid composition. Phosphatidylcholine and phosphatidylethanolamine are major phospholipids of both membranes, but the inner membrane is enriched in cardiolipin, whereas the outer membrane contains a high amount of phosphatidylinositol. The fatty acid composition of both mitochondrial membranes is similar. Variation of the carbon source, however, leads to marked changes of the fatty acid pattern both in total and mitochondrial membranes. In summary, our data are the first step to understand the P. pastoris lipidome which will be prerequisite to manipulate membrane components of this yeast for biotechnological purposes.
Keywords: Pichia pastoris; Yeast; Mitochondria; Phospholipid; Sterol; Fatty acid;

PC is made via the CDP-choline pathway, in which CTP:phosphocholine cytidylyltransferase α (CTα), encoded by Pcyt1a, is the rate-limiting enzyme whose mRNA expression is strictly regulated. Previously, we reported that Ets1 enhanced and Net repressed CTα transcription by binding at the Ets binding site (− 49/− 47) in the Pcyt1a promoter. In this study, we asked if an Ets1 analogue, Ets2, also regulates CTα transcription and investigated the importance of its nuclear localization signal (NLS) and nuclear export signal (NES). Ets2 is primarily detected in the nucleus. Various mutated Ets2 proteins fused with enhanced green fluorescent protein were constructed to identify the NLS and NES in Ets2. Mutation of Ets2 at amino acids 404–410 results in a protein that is evenly distributed in the cell. Interestingly, an Ets2 protein deleted at the C-terminus (amino acids 1–392 present) was localized to the cytoplasm and site-specific mutation in the region 364–372 of this construct resulted in cytoplasmic and nuclear distribution. These results suggest that the NLS in Ets2 is between amino acids 404 and 410, and that the NES is between amino acids 364 and 372. Ets2 enhanced, but the mutant forms of Ets2 had little effects on the transcription of a CTα-reporter construct. When RAW264 cells, murine macrophage cell-line, were stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) or macrophage-colony stimulating factor, the transcription of CTα was enhanced accompanied by increased mRNA of Ets2. These results suggest that the induction of Ets2 is important for CTα transcription by TPA and macrophage-colony stimulating factor.
Keywords: CTP:phosphocholine cytidylyltransferase α; Ets1; Ets2; Macrophage-colony stimulating factor; Nuclear export signal; Nuclear localization signal; 12-O-tetradecanoylphorbol-13-acetate;

Functional expression of a humanized gene for an ω-3 fatty acid desaturase from scarlet flax in transfected bovine adipocytes and bovine embryos cloned from the cells by Yoriko Indo; Atsuhiro Tatemizo; Yuki Abe; Iwane Suzuki; Kazuya Matsumoto; Yoshihiko Hosoi; Mikio Kinoshita; Koji Mikami; Norio Murata; Akira Iritani; Kazuhiro Saeki (183-190).
Long-chain n-3 fatty acids can lower the risk of lifestyle-related diseases, therefore, we introduced a plant fatty acid desaturation3 (FAD3) gene into mammalian cells. The FAD3 cDNA was isolated from the immature seeds of scarlet flax and optimized to human high-frequency codon usage for enhancement of its expression levels in mammalian cells (hFAD3). We introduced the gene into bovine muscle satellite cells, which can be differentiated into multilocular adipocytes in vitro. After hFAD3 transfection, the cells were differentiated into adipocytes and their fatty acid composition was analyzed by gas chromatography. The level of α-linolenic acid (18:3n-3) in transfected adipocytes increased about ten-fold compared with non-transfected adipocytes. In addition, the levels of docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in transfected adipocytes were significantly higher than those in non-transfected adipocytes. Moreover, we produced bovine cloned embryos from the hFAD3 cells by somatic cell nuclear transfer. Blastocyst rates of hFAD3 clones were the same as the control clones using the non-transfected cells (21% vs 27%, P  > 0.05). hFAD3 transcripts were detected in all of the blastocysts. These results demonstrate the functional expression of a plant hFAD3 in mammalian adipocytes, and normal development of cloned embryos carrying the hFAD3 gene.
Keywords: Fatty acid desaturation3; Transfection; Bovine muscle satellite cell; n-3 polyunsaturated fatty acid; Nuclear transplantation; Humanization;

PXR agonism decreases plasma HDL levels in ApoE⁎3-Leiden.CETP mice by Willeke de Haan; Jitske de Vries-van der Weij; Isabel M. Mol; Menno Hoekstra; Johannes A. Romijn; J. Wouter Jukema; Louis M. Havekes; Hans M.G. Princen; Patrick C.N. Rensen (191-197).
Pregnane X receptor (PXR) agonism has been shown to affect multiple steps in both the synthesis and catabolism of HDL, but its integrated effect on HDL metabolism in vivo remains unclear. The aim of this study was to evaluate the net effect of PXR agonism on HDL metabolism in ApoE⁎3-Leiden (E3L) and E3L.CETP mice, well-established models for human-like lipoprotein metabolism. Female mice were fed a diet with increasing amounts of the potent PXR agonist 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN). In E3L and E3L.CETP mice, PCN increased liver lipids as well as plasma cholesterol and triglycerides. However, whereas PCN increased cholesterol contained in large HDL-1 particles in E3L mice, it dose-dependently decreased HDL-cholesterol in E3L.CETP mice, indicating that CETP expression dominates the effect of PCN on HDL metabolism. Analysis of the hepatic expression of genes involved in HDL metabolism showed that PCN decreased expression of genes involved in HDL synthesis (Abca1, Apoa1), maturation (Lcat, Pltp) and clearance (Sr-b1). The HDL-increasing effect of PCN, observed in E3L mice, is likely caused by a marked decrease in hepatic SR-BI protein expression, and completely reversed by CETP expression. We conclude that chronic PXR agonism dose-dependently reduces plasma HDL-cholesterol in the presence of CETP.
Keywords: Cholesteryl ester transfer protein; Gene expression; High density lipoprotein; Pregnane X Receptor; Transgenic mice; Triglycerides;

ABHD5/CGI-58 facilitates the assembly and secretion of apolipoprotein B lipoproteins by McA RH7777 rat hepatoma cells by Jorge M. Caviglia; Janet D. Sparks; Nikhil Toraskar; Anita M. Brinker; Terry C. Yin; Joseph L. Dixon; Dawn L. Brasaemle (198-205).
Lipolysis of stored triacylglycerols provides lipid precursors for the assembly of apolipoprotein B (apoB) lipoproteins in hepatocytes. Abhydrolase domain containing 5 (ABHD5) is expressed in liver and facilitates the lipolysis of triacylglycerols. To study the function of ABHD5 in lipoprotein secretion, we silenced the expression of ABHD5 in McA RH7777 cells using RNA interference and studied the metabolism of lipids and secretion of apoB lipoproteins. McA RH7777 cells deficient in ABHD5 secreted reduced amounts of apoB, triacylglycerols, and cholesterol esters. Detailed analysis of liquid chromatography-mass spectrometry data for the molecular species of secreted triacylglycerols revealed that deficiency of ABHD5 significantly reduced secretion of triacylglycerols containing oleate, even when oleate was supplied in the culture medium; the ABHD5-deficient cells partially compensated by secreting higher levels of triacylglycerols containing saturated fatty acids. In experiments tracking the metabolism of [14C]oleate, silencing of ABHD5 reduced lipolysis of cellular triacylglycerols and incorporation of intermediates derived from stored lipids into secreted triacylglycerols and cholesterol esters. In contrast, the incorporation of exogenous oleate into secreted triacylglycerols and cholesterol esters was unaffected by deficiency of ABHD5. These findings suggest that ABHD5 facilitates the use of lipid intermediates derived from lipolysis of stored triacylglycerols for the assembly of lipoproteins.
Keywords: Lipolysis; Triacylglycerol; Cholesterol ester; RNA interference; McArdle hepatoma; Liquid chromatography-mass spectrometry;

Human plasma phospholipid transfer protein specific activity is correlated with HDL size: Implications for lipoprotein physiology by Marian C. Cheung; Gertrud Wolfbauer; Hiroshi Deguchi; José A. Fernández; John H. Griffin; John J. Albers (206-211).
To gain further insights into the relationship between plasma phospholipid transfer protein (PLTP) and lipoprotein particles, PLTP mass and phospholipid transfer activity were measured, and their associations with the level and size of lipoprotein particles examined in 39 healthy adult subjects. No bivariate correlation was observed between PLTP activity and mass. PLTP activity was positively associated with cholesterol, triglyceride, apo B and VLDL particle level (r s  = 0.40–0.56, p  ≤ 0.01) while PLTP mass was positively associated with HDL-C, large HDL particles, and mean LDL and HDL particle sizes (r s  = 0.44–0.52, p  < 0.01). Importantly, plasma PLTP specific activity (SA) was significantly associated with specific lipoprotein classes, positively with VLDL, IDL, and small LDL particles (r s  = 0.42–0.62, p  ≤ 0.01) and inversely with large LDL, large HDL, and mean LDL and HDL particle size (r s  = − 0.42 to − 0.70, p  ≤ 0.01). After controlling for triglyceride levels, the correlation between PLTP mass or SA and HDL size remained significant. In linear models, HDL size explained 45% of the variability of plasma PLTP SA while triglyceride explained 34% of the PLTP activity. Thus, in healthy adults a significant relationship exists between HDL size and plasma PLTP SA (r s  = − 0.70), implying that HDL particle size may modulate PLTP SA in the vascular compartment.
Keywords: Phospholipid transfer protein specific activity; PLTP mass; PLTP activity; LDL; HDL; Lipoprotein particle size;

Crucial role for LKB1 to AMPKα2 axis in the regulation of CD36-mediated long-chain fatty acid uptake into cardiomyocytes by Daphna D.J. Habets; Will A. Coumans; Mohammed El Hasnaoui; Elham Zarrinpashneh; Luc Bertrand; Benoit Viollet; Bente Kiens; Thomas E. Jensen; Erik A. Richter; Arend Bonen; Jan F.C. Glatz; Joost J.F.P. Luiken (212-219).
Enhanced contractile activity increases cardiac long-chain fatty acid (LCFA) uptake via translocation of CD36 to the sarcolemma, similarly to increase in glucose uptake via GLUT4 translocation. AMP-activated protein kinase (AMPK) is assumed to mediate contraction-induced LCFA utilization. However, which catalytic isoform (AMPKα1 versus AMPKα2) is involved, is unknown. Furthermore, no studies have been performed on the role of LKB1, a kinase with AMPKK activity, on the regulation of cardiac LCFA utilization. Using different mouse models (AMPKα2-kinase-dead, AMPKα2-knockout and LKB1-knockout mice), we tested whether LKB1 and/or AMPK are required for stimulation of LCFA and glucose utilization upon treatment of cardiomyocytes with compounds (oligomycin/AICAR/dipyridamole) which induce CD36 translocation similar to that seen upon contraction. In AMPKα2- kinase-dead cardiomyocytes, the stimulating effects of oligomycin and AICAR on palmitate and deoxyglucose uptake and palmitate oxidation were almost completely lost. Moreover, in AMPKα2- and LKB1-knockout cardiomyocytes, oligomycin-induced LCFA and deoxyglucose uptake were completely abolished. However, the stimulatory effect of dipyridamole on palmitate uptake and oxidation was preserved in AMPKα2-kinase-dead cardiomyocytes. In conclusion, in the heart there is a signaling axis consisting of LKB1 and AMPKα2 which activation results in enhanced LCFA utilization, similarly to enhanced glucose uptake. In addition, an unknown dipyridamole-activated pathway can stimulate cardiac LCFA utilization by activating signaling components downstream of AMPK.
Keywords: AMPK; Cardiac substrate utilization; Cardiomyocyte; Contraction signalling; LKB1;

The involvement of cytochrome p450 (CYP) 26 in the retinoic acid metabolism of human epidermal keratinocytes by Elizabeth Pavez Loriè; Hao Li; Anders Vahlquist; Hans Törmä (220-228).
All-trans retinoic acid (RA) levels are controlled by enzymes of the vitamin A metabolism (RDH16, RalDH2, and LRAT) and RA catabolism (CYP26 and CYP2S1). Here, the mRNA expression of these enzymes was investigated in human keratinocytes at different Ca2+concentrations and after exposure to RA and CYP26 inhibitors. Cellular differentiation (high Ca2+) increased the expression of LRAT, RDH16 and RalDH2, and decreased CYP26B1. RA (1 μM) induced CYP26A1, CYP26B1, CYP2S1, CRABPII and LRAT mRNA. The CYP26 inhibitor talarozole altered CYP26A1 and LRAT mRNA expression in a similar way as RA, increased the cellular accumulation of [3H]RA, and induced a punctate CRABPII staining, also observed after siRNA knock-down of CYP26B1 (but not after RA exposure). Furthermore, CYP26B1 siRNA increased the accumulation of [3H]RA and the CRABPII mRNA, suggesting an augmented retinoid signalling. Thus CYP26B1 appears essential for RA catabolism under physiological conditions, whereas CYP26A1 might play a greater role during RA excess.
Keywords: All-trans retinoic acid; Keratinocytes; Epidermis; Metabolism; Retinol; Cytochrome P450;