Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism最新文献

Carnitine palmitoyltransferase I (CPT-I) catalyzes the rate-determining step in mitochondrial fatty acid β-oxidation. The enzyme has two cognate structural genes (α and β) that are differentially expressed in tissues. We show multiple mature mRNAs in rat heart derived from alternative splicing of CPT-Iβ transcripts. Two novel messages are deleted for regions of the previously described mRNA that encode membrane-spanning and regulatory domains, suggesting that the cognate isozymes will exhibit unique kinetic characteristics.

A cDNA encoding mouse PLC-β3 (mPLC-β3) was identified by screening a mouse kidney cDNA library and using the rapid amplification of cDNA ends (RACE) method. The predicted open reading frame was 3705 bp in length. The deduced 1235 amino acid (aa) sequence shares 95.3% and 92% homology with the sequences of rat and human PLC-β3, respectively. The corresponding mRNA is highly expressed in kidney, skeletal muscle, liver, lung, heart and brain. In spleen, mPLC-β3 mRNA was not detectable, which is in contrast to humans where there is a distinct expression. Using ultrastructural immunocytochemistry, mPLC-β3 expression was detected in the heterochromatin of the nucleus in mouse brain neurons. The observation of PLC-β3 nuclear localization suggests that PLC-β3 may have intranuclear functions.

Ferret lactating mammary gland bile salt-dependent lipase (BSDL, EC 3.1.1.-) has been cloned by RT-PCR. The open reading frame consists of 1869 nucleotides which encode 623 amino acids of the functional enzyme. When compared to other species, the greatest homology is observed between residues 1 and 484, with little or no homology at the C-terminal end where seven repeated segments of similar sequence are located. Ferret mammary gland BSDL retains residues involved in the active site and the tentative heparin binding site at similar positions in comparison to other milk or pancreatic BSDL. Other important items, such as binding peptide to chaperone molecular, phosphorylation site(s) or bile salt binding sites, were also tentatively located in well conserved regions of seven available BSDL sequences.

CTP:phosphocholine cytidylyltransferase (CT) catalyzes a rate-limiting, regulatory step in mammalian biosynthesis of phosphocholine (PC). Anionic phospholipids, fatty acids and diacylglycerol activate CT and promote its intercalation into the lipid bilayer, whereas zwitterionic phospholipids such as phosphatidylcholines do not. We investigated the effectiveness of polyunsaturated phosphatidylcholines as CT activators after hypochlorite oxidation. Detection and quantitation of oxidized PCs were evaluated by thin layer chromatography, high performance liquid chromatography, and conjugated dienes. Purified CT was assayed in the presence of multilamellar vesicles, containing variable concentrations of oxidized and parent PCs. The results demonstrate that particular species of oxidized PCs activate CT as potently as anionic lipids. The greater the number of double bonds available for oxidation in the fatty acid at the sn-2 position of the PC, the more effective was the oxidized PC as an activator of CT. Oxidized phospholipids at 1:1 bleach/lipid activated CT in the following order: PAPC>PL₃PC>PL₂PC compared to unoxidized controls. Since oxidized phospholipids decrease bilayer order (M.L. Wratten et al., Biochemistry 31 (1992) 10901–10907) these results are consistent with the activation of CT by perturbations of lipid bilayer packing.

Probucol, a widely used lipid-lowering agent, is associated with a significant reduction of plasma high density lipoprotein (HDL)-cholesterol levels. To examine the mechanism of probucol HDL-lowering and probucol’s effects on cholesteryl ester transfer protein (CETP) and cholesterol metabolism in cells, we used a Chinese hamster ovary (CHO) cell line that had been stably transfected with a human CETP gene (hCETP-CHO). After this cell line was incubated with various concentrations of probucol (5, 10 and 50 μM) for 24 h, mean intracellular probucol concentrations reached 0.47, 0.67, and 1.52 μg/mg cell protein, respectively. Northern blot analysis showed that cellular CETP mRNA was increased by probucol in a dose-dependent manner (137%, 162%, and 221% of the control, respectively). The specific CET activity in the culture medium, measured as the percentage of [³H]cholesterol oleate transferred from discoidal bilayer particles (which mimic HDL) to LDL, also increased in a dose-dependent manner. Intracellular total cholesterol levels were decreased to 87.5%, 74.9%, and 52.5% of the control, respectively. Probucol had no effects on HMG-CoA reductase activity or cholesterol synthesis from [¹⁴C]acetate in hCETP-CHO. However, ¹⁴C-incorporated cholesterol secretion into the culture medium from hCETP-CHO was increased to 181%, 256% and 354% of the control by 5, 10 and 50 μM probucol, respectively. We concluded that (1) treatment with probucol increased the CETP mRNA level and specific CET activity in the hCETP-CHO cell line, and (2) probucol promoted cholesterol efflux from hCETP-CHO, which resulted in a decrease in intracellular cholesterol levels.

Dietary triglycerides, the major precursors of long chain fatty acids (FA), require hydrolysis by pancreatic enzymes prior to their absorption by the small intestine. Although Caco-2 cells are frequently employed for the study of enterocyte lipid metabolism, the presence of an endogenous lipase activity has never been previously reported. The major goal of this investigation was to determine the presence of endogenous Caco-2 cell lipase activity, to examine its capacity to hydrolyze triglycerides, and to define its intracellular location. Caco-2 cells were found to have an endogenous lipase activity, capable of hydrolyzing [1-¹⁴C]triolein from the apical cell compartment. A time and concentration dependence of lipase activity was observed, with hydrolysis of triolein into free fatty acids and monoglyceride. The majority of the lipase activity was found in the cytosolic cell fraction and, to a lesser extent, in the apical brush border membrane and other organelles. Protamine sulfate markedly reduced the Caco-2 cell lipase activity, yet it remained relatively insensitive to high concentrations of NaCl, taurocholate, calcium, heparin and chloroquine. The addition of exogenous human gastric lipase to the medium of the apical compartment resulted in a significant increased rate of hydrolysis of triolein, followed by enhanced Caco-2 cell fatty acid uptake and basolateral lipid secretion. The major esterified intracellular lipids were triglycerides and phospholipids. We conclude that Caco-2 cells possess an endogenous lipase capable of hydrolyzing cytosolic triglycerides. Furthermore, activity present on the apical membrane and secreted into the apical medium, though quantitatively less important than the cytosolic lipase, may permit an additional route for energy uptake. The addition of gastric lipase to the Caco-2 cell cultures greatly enhanced FA uptake above that seen with the endogenous lipase alone.

2-Acylamino-alkyl phospholipids are potent competitive inhibitors of 14-kDa phospholipases A₂ (e.g., human nonpancreatic secretory PLA₂). As concluded from X-ray studies the amide hydrogen of these inhibitors forms a hydrogen bond to His-48 in the active site of the enzyme. We investigated the quantitative contribution of this hydrogen bond to inhibition using especially designed inhibitors that bear different acyl chains with and without electron withdrawing or donating substituents, thus differing in amide acidity. Relative free enthalpies ΔΔG of enzyme–inhibitor complex formations were calculated from X_i(50) values determined by pH-stat titration using a mixed micelles assay and PLA₂ from Naja mocambique mocambique. A quantitative relationship between amide acidity and ΔΔG values is presented. Comparison of isoacidic and isosteric inhibitors reveals that (i) the hydrogen bond of the amide proton to His-48 is crucial for strong PLA₂ inhibition, (ii) regardless of the headgroup unsubstituted N-acyl groups result in optimal amide acidity for PLA₂ inhibition and (iii) the exceptionally strong inhibition by acetamides and the isosteric fluoroacetamides is due to an additional steric effect.

Transgenic mice were created overproducing a range of human HL (hHL) activities (4–23-fold increase) to further examine the role of hepatic lipase (HL) in lipoprotein metabolism. A 5-fold increase in heparin releasable HL activity was accompanied by moderate (approx. 20%) decreases in plasma total and high density lipoprotein (HDL) cholesterol and phospholipid (PL) but no significant change in triglyceride (TG). A 23-fold increase in HL activity caused a more significant decrease in plasma total and HDL cholesterol, PL and TG (77%, 64%, 60%, and 24% respectively), and a substantial decrease in lipoprotein lipids amongst IDL, LDL and HDL fractions. High levels of HL activity diminished the plasma concentration of apoA-I, A-II and apoE (76%, 48% and 75%, respectively). In contrast, the levels of apoA-IV-containing lipoproteins appear relatively resistant to increased titers of hHL activity. Increased hHL activity was associated with a progressive decrease in the levels and an increase in the density of LpAI and LpB48 particles. The increased rate of disappearance of ¹²⁵I-labeled human HDL from the plasma of hHL transgenic mice suggests increased clearance of HDL apoproteins in the transgenic mice. The effect of increased HL activity on apoB100-containing lipoproteins was more complex. HL-deficient mice have substantially decreased apoB100-containing low density lipoproteins (LDL) compared to controls. Increased HL activity is associated with a transformation of the lipoprotein density profile from predominantly buoyant (VLDL/IDL) lipoproteins to more dense (LDL) fractions. Increased HL activity from moderate (4-fold) to higher (5-fold) levels decreased the levels of apoB100-containing particles. Thus, at normal to moderately high levels in the mouse, HL promotes the metabolism of both HDL and apoB-containing lipoproteins and thereby acts as a key determinant of plasma levels of both HDL and LDL.

Lamin B receptor (LBR), a nuclear protein of avian and mammalian cells, contains an hydrophobic domain that shares extensive structural similarities with the members of the sterol reductase family. To test if the sterol-reductase-like domain of LBR could be enzymatically competent, several sterol reductase-defective strains of Saccharomyces cerevisiae were transformed with a human-LBR expressing vector. LBR production did not change the ergosterol biosynthesis defect in an erg4 mutant impaired in sterol C24(28) reductase. In contrast, the sterol C14 reduction step and ergosterol prototrophy were restored in LBR-producing erg24 transformants which lack endogenous sterol C14 reductase. To test the effects of C14 reductase inhibitors on LBR activity, we constructed EMY54, an ergosterol-requiring strain that is devoid of both sterol C8–C7 isomerase and sterol C14 reductase activities. EMY54 cells recovered the capability of synthesizing ergost-8-en-3β-ol upon transformation with a vector that expressed either yeast sterol C14 reductase or hLBR. In addition, growth in sterol-free medium was restored in these transformants. Sterol biosynthesis and proliferation of LBR-producing cells were found to be highly susceptible to fenpropimorph and tridemorph, but only moderately susceptible to SR 31747. Our results strongly suggest that hLBR is a sterol C14 reductase.

The only endogenous substances isolated and characterised so far that are capable of mimicking the pharmacological actions of the active principle of marijuana, (−)-Δ⁹-tetrahydrocannabinol, are amides and esters of fatty acids. Some of these compounds, like anandamide (N-arachidonoylethanolamine) and 2-arachidonoylglycerol, act as true ‘endogenous cannabinoids’ by binding and functionally activating one or both cannabinoid receptor subtypes present on nervous and peripheral cell membranes. The metabolic pathways and molecular mode of actions of these metabolites, as well as their possible implication in physiopathological responses, are reviewed here.