BBA - Molecular and Cell Biology of Lipids (v.1580, #1)
1-O-Hexadecyl-2-desoxy-2-amino-sn-glycerol, a substrate for human sphingosine kinase by Sofie Gijsbers; Stanny Asselberghs; Piet Herdewijn; Paul P. Van Veldhoven (1-8).
The substrate specificity of human sphingosine kinase was investigated using a bacterially expressed poly(His)-tagged protein. Only the D-erythro isomer of the sphingoid bases, sphinganine and sphingenine, was effectively phosphorylated. Long chain 1-alkanols, alkane-1,2-diols, 2-amino-1-alkanol or 1-amino-2-alkanol and short chain 2-amino-1,3-alkanediols were very poor substrates, indicating that the kinase is recognizing the chain length and the position of the amino and secondary hydroxy group. A free hydroxy group at carbon 3 is not a prerequisite, however, since 1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol was an efficient substrate with an apparent K m value of 3.8 μM (versus 15.7 μM for sphingenine). This finding opens new perspectives to design sphingosine kinase inhibitors. It also calls for some caution since it cannot be excluded that this ether lipid analogue is formed from precursors that are frequently used in research on platelet activating factor or from phospholipid analogues which are less prone to degradation.
Keywords: Sphingenine; Ceramide; Signaling; Platelet activating factor; Ether phospholipid; Sphingolipid;
Functional implications of post-translational modifications of phospholipases D1 and D2 by Zhi Xie; Wan-Ting Ho; John H Exton (9-21).
Our previous studies showed that truncation of the N-terminal 168 amino acids of rat brain phospholipase D1 (rPLD1) abolishes its response to protein kinase C (PKC) and greatly diminishes its palmitoylation and Ser/Thr phosphorylation. In this study, we show that the response to PKC as well as the palmitoylation and Ser/Thr phosphorylation were restored when the truncated rPLD1 mutant (rPLD1(ΔN168)) was coexpressed with a fragment containing the N-terminal 168 amino acids. Immunoprecipitation experiments showed that the N-terminal fragment associated with rPLD1(ΔN168) when coexpressed in COS 7 cells and that palmitoylation of Cys240 and Cys241 was not necessary for the association. In addition, we found that rat PLD2 (rPLD2) was palmitoylated on Cys223 and Cys224 in COS 7 cells. Mutation of both these cysteines reduced the basal activity of rPLD2, however its response to PMA stimulation in vivo was retained. As in the case of rPLD1, loss of palmitoylation weakened membrane association of rPLD2. In summary, the N-terminal 168-amino-acid fragment of rPLD1 can associate with truncated rPLD1(ΔN168) to restore its palmitoylation, Ser/Thr phosphorylation and PKC response. Although rPLD2 differs from rPLD1 in many properties, it is palmitoylated at the corresponding conserved cysteine residues in COS 7 cells.
Keywords: Phospholipase D; Protein kinase C; Palmitoylation; Phosphorylation; Membrane association;
Active taurocholic acid flux through hepatoma cells increases the cellular pool of unesterified cholesterol derived from lipoproteins by Q. Li; S. Yokoyama; L.B. Agellon (22-30).
The effect of bile acid flux on the fate of lipoprotein-derived cholesterol was studied in bile acid-transporting McNtcp.18 hepatoma cells. The intracellular unesterified cholesterol (UC) concentration rose when McNtcp.18 cells grown in the presence of either high density lipoproteins (HDL) or low density lipoproteins (LDL) were incubated with taurocholic acid (TCA). This effect was more pronounced when the exogenous source of cholesterol was HDL. The presence of TCA in the culture medium of McNtcp.18 cells had no discernible effect on the uptake of cholesteryl esters (CE) from either lipoprotein. TCA treatment of cells preincubated with either lipoprotein did not affect cholesterol synthesis but antagonized the stimulation of cholesterol esterification in cells that were incubated with LDL. The CE concentration in cells treated with TCA was decreased, relative to cells not incubated with TCA, suggesting that cellular CE stores were also hydrolyzed. The TCA treatment reduced the amount of total cholesterol released into the medium by the lipoprotein-treated cells, which was coincident with the reduction in the amount of apolipoprotein B in the culture medium. However, the proportion of UC released into the medium by the lipoprotein-treated cells was increased in cells capable of active bile acid transport. The results indicate that active bile acid flux through hepatoma cells increases the cellular pool of UC derived from lipoproteins. The UC released by the cells into the culture medium under this condition may represent cholesterol destined for direct biliary secretion.
Keywords: Bile acid; Cholesterol; Enterohepatic circulation; Hepatoma cell; Lipoprotein;
Comparative cytotoxic and cytoprotective effects of taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA) in HepG2 cell line by Francesca Carubbi; Maria Eugenia Guicciardi; Mauro Concari; Paola Loria; Marco Bertolotti; Nicola Carulli (31-39).
This study was performed to compare the effects of two hydrophilic bile acids, taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA), on HepG2 cells. Cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations (50–800 μmol/l) of either bile acid, while their cytoprotective effect was tested in comparison with deoxycholic acid (DCA) (350 μmol/l and 750 μmol/l)-induced cytotoxicity. Culture media, harvested at the end of each incubation period, were analyzed to evaluate aspartate transaminase (AST), alanine transaminase and γ-glutamyltranspeptidase release. In addition, the hemolytic effect of THDCA and TUDCA on human red blood cells was also determined. At 24 h of incubation neither THDCA nor TUDCA was cytotoxic at concentrations up to 200 and 400 μmol/l. At 800 μmol/l both THDCA and TUDCA induced a slight increase in AST release. At this concentration and with time of exposure prolonged up to 72 h, THDCA and TUDCA induced a progressive increase of AST release significantly (P<0.05) higher than that of controls being AST values for THDCA (2.97±0.88 time control value (tcv) at 48 h and 4.50±1.13 tcv at 72 h) significantly greater than those of TUDCA (1.50±0.20 tcv at 48 h and 1.80±0.43 tcv at 72 h) (P<0.01). In cytoprotection experiments, the addition of 50 μmol/l THDCA decreased only slightly (−5%) AST release induced by 350 μmol/l DCA, while the addition of 50 μmol/l TUDCA was significantly effective (−23%; P<0.05). Higher doses of THDCA or TUDCA did not reduce toxicity induced by 350 μmol/l DCA, but were much less toxic than an equimolar dose of DCA alone. At the concentration used in this experimental model neither THDCA nor TUDCA was hemolytic; however at a very high concentration (6 mmol/l) both bile acids induced 5–8% hemolysis. We conclude that bile acid molecules with a similar degree of hydrophilicity may show different cytotoxic and cytoprotective properties.
Keywords: Bile acid; Hepatocyte; Hepatotoxicity; Hepatoprotection; Hydrophilic–hydrophobic balance;
Δ6-, Stearoyl CoA-, and Δ5-desaturase enzymes are expressed in β-cells and are altered by increases in exogenous PUFA concentrations by Sasanka Ramanadham; Sheng Zhang; Zhongmin Ma; Mary Wohltmann; Alan Bohrer; Fong-Fu Hsu; John Turk (40-56).
In the evolution of Type II diabetes, an initial period of hyper-fatty acidemia leads to an insulin secretory defect which triggers overt hyperglycemia and frank diabetes. The mechanism by which elevated free fatty acids contribute to β-cell dysfunction, however, is not clearly understood. We recently reported that arachidonic acid (20:4) or linoleic acid (18:2) supplementations result in increases in abundances of long chain polyunsaturated fatty acids in INS-1 β-cell membrane lipids, suggesting that β-cells express desaturases that catalyze generation of unsaturated fatty acids. As expression of desaturases by β-cells has not yet been addressed, we initiated studies to examine this issue using INS-1 β-cells and find that they express messages for the Δ6-, stearoyl CoA-, and Δ5-desaturase. Supplementation of the INS-1 β-cells with arachidonic acid leads to decreased expression of all three desaturases, presumably in response to the decreased need for endogenous generation of unsaturated fatty acids. In contrast, linoleic acid supplementation promoted minimal changes in the three desaturases. These findings demonstrate for the first time that β-cells express regulatable desaturases. Additionally, reverse transcriptase–polymerase chain reaction analyses reveal expression of the desaturases in native pancreatic islets. It might be speculated that long-term elevations in fatty acids can also adversely influence desaturase activity in β-cells and affect PUFA composition in β-cell membranes contributing to β-cell membrane structural abnormalities and altered secretory function.
Keywords: β-Cell; Desaturase; Polymerase chain reaction; Mass spectrometry; Arachidonic acid;
Torpor-associated fluctuations in surfactant activity in Gould’s wattled bat by Jonathan R Codd; Samuel Schürch; Christopher B Daniels; Sandra Orgeig (57-66).
The primary function of pulmonary surfactant is to reduce the surface tension (ST) created at the air–liquid interface in the lung. Surfactant is a complex mixture of lipids and proteins and its function is influenced by physiological parameters such as metabolic rate, body temperature and breathing. In the microchiropteran bat Chalinolobus gouldii these parameters fluctuate throughout a 24 h period. Here we examine the surface activity of surfactant from warm–active and torpid bats at both 24°C and 37°C to establish whether alterations in surfactant composition correlate with changes in surface activity. Bats were housed in a specially constructed bat room at Adelaide University, at 24°C and on a 8:16 h light:dark cycle. Surfactant was collected from bats sampled during torpor (25<T b<28°C), and while active (T b>35°C). Alterations in the lipid composition of surfactant occur with changes in the activity cycle. Most notable is an increase in surfactant cholesterol (Chol) with decreases in body temperature [Codd et al., Physiol. Biochem. Zool. 73 (2000) 605–612]. Surfactant from active bats was more surface active at higher temperatures, indicated by lower STmin and less film area compression required to reach STmin at 37°C than at 24°C. Conversely, surfactant from torpid bats was more active at lower temperatures, indicated by lower STmin and less area compression required to reach STmin at 24°C than at 37°C. Alterations in the Chol content of bat surfactant appear to be crucial to allow it to achieve low STs during torpor.
Keywords: Surfactant; Surface activity; Torpor; Bat; Cholesterol;
Both isoforms of mammalian phosphatidylinositol transfer protein are capable of binding and transporting sphingomyelin by Hong Li; Jacqueline M Tremblay; Lynwood R Yarbrough; George M Helmkamp (67-76).
The structurally related mammalian α and β isoforms of phosphatidylinositol (PtdIns) transfer protein (PITP) bind reversibly a single phospholipid molecule, preferably PtdIns or phosphatidylcholine (PtdCho), and transport that lipid between membrane surfaces. PITPβ, but not PITPα, is reported extensively in the scientific literature to exhibit the additional capacity to bind and transport sphingomyelin (CerPCho). We undertook a detailed investigation of the lipid binding and transfer specificity of the soluble mammalian PITP isoforms. We employed a variety of donor and acceptor membrane lipid compositions to determine the sensitivity of recombinant rat PITPα and PITPβ isoforms toward PtdIns, PtdCho, CerPCho, and phosphatidate (PtdOH). Results indicated often striking differences in protein–phospholipid and protein–membrane interactions. We demonstrated unequivocally that both isoforms were capable of binding and transferring CerPCho; we confirmed that the β isoform was the more active. The order of transfer specific activity was similar for both isoforms: PtdIns>PtdCho>CerPCho≫PtdOH. Independently, we verified the binding of CerPCho to both isoforms by showing an increase in holoprotein isoelectric point following the exchange of protein-bound phosphatidylglycerol for membrane-associated CerPCho. We conclude that PITPα and PITPβ are able to bind and transport glycero- and sphingophospholipids.
Keywords: Phosphatidylinositol transfer protein; Phosphatidylinositol; Phosphatidylcholine; Sphingomyelin; Small unilamellar vesicle;
The identification of intestinal scavenger receptor class B, type I (SR-BI) by expression cloning and its role in cholesterol absorption by Scott W Altmann; Harry R Davis; Xiaorui Yao; Maureen Laverty; Douglas S Compton; Li-ji Zhu; James H Crona; Mary Ann Caplen; Lizbeth M Hoos; Glen Tetzloff; Tony Priestley; Duane A Burnett; Catherine D Strader; Michael P Graziano (77-93).
The molecular mechanisms of cholesterol absorption in the intestine are poorly understood. With the goal of defining candidate genes involved in these processes a fluorescence-activated cell sorter-based, retroviral-mediated expression cloning strategy has been devised. SCH354909, a fluorescent derivative of ezetimibe, a compound which blocks intestinal cholesterol absorption but whose mechanism of action is unknown, was synthesized and shown to block intestinal cholesterol absorption in rats. Pools of cDNAs prepared from rat intestinal cells enriched in enterocytes were introduced into BW5147 cells and screened for SCH354909 binding. Several independent clones were isolated and all found to encode the scavenger receptor class B, type I (SR-BI), a protein suggested by others to play a role in cholesterol absorption. SCH354909 bound to Chinese hamster ovary (CHO) cells expressing SR-BI in specific and saturable fashion and with high affinity (K d∼18 nM). Overexpression of SR-BI in CHO cells resulted in increased cholesterol uptake that was blocked by micromolar concentrations of ezetimibe. Analysis of rat intestinal sections by in situ hybridization demonstrated that SR-BI expression was restricted to enterocytes. Cholesterol absorption was determined in SR-B1 knockout mice using both an acute, 2-h, assay and a more chronic fecal dual isotope ratio method. The level of intestinal cholesterol uptake and absorption was similar to that seen in wild-type mice. When assayed in the SR-B1 knockout mice, the dose of ezetimibe required to inhibit hepatic cholesterol accumulation induced by a cholesterol-containing ‘western’ diet was similar to wild-type mice. Thus, the binding of ezetimibe to cells expressing SR-B1 and the functional blockade of SR-B1-mediated cholesterol absorption in vitro suggest that SR-B1 plays a role in intestinal cholesterol metabolism and the inhibitory activity of ezetimibe. In contrast studies with SR-B1 knockout mice suggest that SR-B1 is not essential for intestinal cholesterol absorption or the activity of ezetimibe.
Keywords: Cholesterol; Absorption; Enterocyte; Expression; Cloning; Scavenger receptor class B; Type I;