Cholesterol synthesis: Determination of synthesis rate utilizing D2O and isotope ratio mass spectrcmetry (original) (raw)
Related papers
Cholesterol metabolism: use of D2O for determination of synthesis rate in cell culture
Journal of Lipid Research, 1985
Cholesterol synthesis in cell culture in the presence of D20 yields a spectrum of enriched molecules having a relative abundance that indicates random substitution of deuterium for hydrogen. Quantitation of the absolute rate of cholesterol synthesis is obtained by isotope ratio mass spectrometry. Mevinolin and 26-hydroxycholestero1 both decrease cholesterol synthesis rate but have a discordant effect on HMG-CoA reductase activity.-Esterman, A. L., B. I. Cohen, and N. B. Javitt. Cholesterol metabolism: use of D20 for determination of synthesis rate in cell culture.
Transfer of cholesterol from Ob1771 cells or LDL to reconstituted, defined high density lipoproteins
Journal of Lipid Research, 1994
We used defined, reconstituted high density lipoproteins (rHDL) to study the effects of structure and composition of these particles on their role as cholesterol acceptors from cell membranes or from low density lipoproteins (LDL). Three discoidal rHDL and one spherical rHDL with distinct apolipoprotein A-I conformations, diameters, and compositions were used in conjunction with Ob1771 cells to measure the rate of [3HH]cholesterol efflux from the cells, direct binding to the cells, and competition with native HDL3 for binding. In addition, the same rHDL particles were used to study the kinetics of cholesterol mass transfer from LDL. e The results show that the rates of cholesterol transfer depend on the nature of the donor (tH 11-19 min from LDL, and t % 5 h from the cells), on the phosphatidylcholine/cholesterol ratio in the acceptors (the closer this ratio is to the equilibrium value, the slower is the rate), and on the diameter of the acceptors (the smallest particles have the lowest t K for cholesterol uptake from LDL, and are the most effective acceptors of [3H]cholesterol from cells after their phospholipid content is taken into account). The cholesterol uptake by the rHDL, both from the cells and from LDL, is determined mostly by the phospholipid pool available in the acceptors. Binding to the cells was equivalent for all the rHDL (& = 38-67 fig/ml) and comparable to HDL3, suggesting that the differences in apoA-I conformation have no effect on the binding to cells. Finally we observed that exposure of rHDL to cells may lead to remodeling of some of the lipoprotein particles.
Journal of lipid …, 1991
Using immunoaffinity chromatography, we sepa-cells, there is little or no oxidative degradation of rated human high density lipoprotein (HDL) into two subfrac-cholesterol, and sterol homeostasis requires that excess tions: LP-AI, in which all particles contain apolipoprotein A-I cholestero~ be removed and transported to the liver. These (apoA-I) but no apoA-11, and LP-AI/AII, in which all particles removal and transport processes collectively are termed contain both apoA-I and apoA-11. To compare LP-AI and LP-AI/AII as acceptors of cell cholesterol, the isolated subfractions reverse cholesterol transport, and are mediated plasma were diluted to 50 pg phospholipid/ml, and then incubated with lipoproteins (reviewed in ref. 2). The initial removal of monolayer cultures of cells in which whole-cell and lysosomal sterol from cells involves the transfer of cholesterol from ~.~~ cholesterol has been labeled with '*c and 3H, respectively. w e used three cell types (Fu5AH rat hepatoma cells, normal human skin fibroblasts, and rabbit aortic smooth muscle cells). When these cells were DreDared to contain normal Dhvsiolocical auan-Abbreviations: HDL, high density lipoprotein; LDL, low density Supplementary key words hepatoma cells fibroblasts smooth lipoprotein; r[SH-COILDL, low density lipoprotein reconstituted with muscle cells apolipoprotein A-I apolipoprotein A-I1 plasma [3H]cholesteryl oleate; apoA-I, apolipoprotein A-I; LP-AI, immembrane lysosomes low density lipoprotein munopurified HDL fraction containing apoA-I, but no apoA-11; LP-AI/AII, immunopurified HDL fraction containing both apoA-I and apoA-11; HEPES, N-Z-hydroxyethylpiperazine-N'-Z-ethanesulfonic acid; BSA, bovine serum albumin; FC, free (unesterified) cholesterol; The major portion of cholesterol in nucleated cells of MEM, Eagle's minimum essential medium; ACAT, acyl-coenzyme mammals is found in the plasma membrane, although A:cholesterol acyltransferase; LCAT, 1ecithin:cholesterol acyltransferase.
Journal of Biological Chemistry
The bidirectional surface transfer of free cholesterol (FC) between Fu5AH rat hepatoma cells and human high density lipoprotein (HDL) was studied. Cells and HDL were prelabeled with [4-14C]FC and [7-3H]FC, respectively. Influx and efflux of FC were measured simultaneously from the appearance of 3H counts in cells and 14C counts in medium. Results were analyzed by a computerized procedure which fitted sets of kinetic data to a model assuming that cell and HDL FC populations each formed a single homogeneous pool and that together the pools formed a closed system. This analysis yielded values for the first-order rate constants of FC influx and efflux (ki and k.), from which influx and efflux of FC mass (Fi and F,) could be calculated. With normal HDL, the uptake and release of FC tracers conformed well to the above-described model; Fi and F, were approximately equal, suggesting an exchange of FC between cells and HDL. HDL was depleted of phospholipid (PL) by treatment with either phospholipase A2 or heparin-releasable rat hepatic lipase, followed by incubation with bovine serum albumin. PL depletion of HDL had little or no effect on ki, but reduced k,, indicating that PL-deficient HDL is a relatively poor acceptor of cell cholesterol. The reduction in k, resulted in initial Fi > F. and, thus, in net uptake of FC by the cells. This result explained previous results demonstrating net uptake of FC from PL-depleted HDL. In the presence of an inhibitor of acyl coenzyme Axholesterol acyltransferase, the steady state distribution of FC mass between cells and HDL was accurately predicted by the ratio of rate constants for FC flux. This result provided additional validation for describing FC flux in terms of first-order rate constants and homogeneous cell and HDL FC pools.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1988
The high plasma cholesterol concentration of the genetically hypercholesterolemic RICO rats fed a low cholesterol base diet (1.28 mg/ml) compared to that of SW rats (0.73 mg/ml) results from an increase in the cholesterol content of the d 2 1.006 lipoproteins. Since the composition of each type of lipoprotein is similar in the two groups of rats, the RICO rat, therefore, is hyperlipoproteinemic with an increase in the number of lipoprotein particles, except VLDL and chylomicrons. Furthermore, the apolipoprotein E (apoE) content in the d < 1.063 lipoproteins is higher in RICO than in SW rats, while that of apoA-I in HDL is lower. In rats fed 0.5% cholesterol base diet, cbolesterolemia doubles in the two groups (SWcH, 1.32 j, 0.10 mg/ml; MC%,, 2.10 f 0.09 mg/ml). This hyperchoiesterolemia is due to an increased cholesterol content in VLDL and chylomicrons. These lipoproteins carry 60% (in SW,,) and 45% (in RICO,,) of the plasma cholesterol and are cholesterol-enact compared with the li~proteins observed in rats fed the base diet. In RICO,,, 24% of the plasma cholesterol is found in apoE-rich LDL, (1.040 < d Q l&63), whereas in SVV,,, this fraction contains only 11% of the plasma cholesterol. Finally, as before with the base diet, RICOcH shows an apoE enrichment of the d< 1.063 lipoproteins and an apoA-1 depletion of HDL compared to SW,". These data suggest that hypercholesterolemia of the RICO rats results from a modification in the turnover of apoE-containing lipoproteins.
The biochemistry of lipoproteins
Journal of Inherited Metabolic Disease, 1988
The triacylglycerol-rich lipoproteins are chylomicrons and verylow-density lipoproteins (VLDL) which are produced by the small intestine and liver, respectively. These lipoproteins mainly carry fatty acids to adipose tissue and muscle where the triacylglycerol is hydrolysed by lipoprotein lipase. The resulting particles that remain in the blood are chylomicron remnants and low-density lipoprotein (LDL), respectively. The remnant is taken up by the liver via endocytosis which is mediated by a specific receptor for apolipoprotein E (apoE). LDL, which are rich in cholesterol, can also be taken up by the liver or extrahepatic tissues by a receptor-mediated endocytosis that specifically recognises apoB or apoE. 'Nascent' high-density lipoprotein (HDL) particles are secreted by the liver and intestine and then undergo modification to become HDL3 and then HDL 2 as they acquire cholesterol ester. They facilitate the reverse transport of cholesterol back to the liver. Little is known of the hormonal regulation of lipoprotein uptake by the liver. Recently, we have shown that insulin and tri-iodothyronine (T3) increase the specific binding of LDL to cultured hepatocytes whereas dexamethasone (a synthetic glucocorticoid) has the opposite effect. The changes in binding produced by insulin and dexamethasone are paralleled by alterations in the rate of degradation of apoB. These findings may in part explain the hypercholesterolaemia and increased risk of premature atherosclerosis that can be associated with poorly controlled diabetes or hypothyroidism.
Characterization of high density lipoprotein particles in familial apolipoprotein A-I deficiency
The Journal of Lipid Research, 2007
Rat adrenocortical cells take up high density lipoprotein cholesterol for use as steroidogenic substrate. To better understand this unique uptake process, we have first characterized HDL binding. Infusion of human '251-labeled HDL into rats pretreated with 4-APP demonstrated that the adrenal and ovary accumulate HDL in a saturable fashion in vivo. Subsequent studies using isolated rat adrenocortical cells demonstrated that cellular uptake of HDL is comprised of two events. One event is characterized by reversible membrane binding and is complete by 60 min = 20 min). The second event is marked by irreversible apoprotein accumulation which continues for at least 3 hr. Reversibly bound material exhibits the same apoprotein distribution as unincubated HDL. Irreversible accumulation could not be attributed to intemalization or lysosomal accumulation inasmuch as it also occurred with partially purified plasma membranes and was not enhanced by addition of chloroquine. Reversible binding of human HDLS exhibited a saturable dependence on concentration (rC, = 27 pg protein/ml; N = 3.0 X lo6 sites/cell) similar to that previously reported for rat liver, ovary, and testis. Cell accumulation of HDL decreased by over 80% at 4°C compared to 37"C, did not require calcium, and was not diminished by prior cell treatment with trypsin or pr0nase.M These results indicate that rat adrenocortical cells possess plasma membrane recognition sites for HDL with different properties than those of the LDL receptor. Moreover, adrenal accumulation of HDL apoproteins does not lead to secondary lysosome formation.-Gwynne, J. T., T. Hughes, and B. H e s r . Characterization of high density lipoprotein binding activity in rat adrenocortical cells.]. Lipid Res. 1984. 2 5 1059-1071. Abbreviations: HDL, high density lipoprotein; LDL, low density lipoprotein; 4-APP, 4-aminopyrazolopyrimidine; MEM, minimum Eagle's medium; TCA. trichloroacetic acid; EDTA, ethylenediiminetetraacetic acid.
The effects of cholesteryl ester transfer protein (CETP) inhibition on the serum lipoprotein profile in both normocholesterolemic and hypercholesterolemic hamsters has been determined following subcutaneous injection of 12.5 mg/kg of the CETP neutralizing monoclonal antibody, TP2. Inhibition of CETP activity was greater than 60% and resulted in a 30-40% increase in high density lipoprotein (HDL) in both normal and hypercholesterolemic animals. These HDL effects were observed 1 day post-injection, were maximal by 4 days, and returned to control values by 14 days. Inhibition of CETP activity resulted in a decrease in both low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol concomitant with HDL increase, and in hypercholesterolemic animals resulted in increased total serum cholesterol. In addition to the quantitative differences in LDL and HDL, there were significant increases in the size of the HDL, a shift to smaller LDL particles, and changes in apolipoprotein (apo) composition as evaluated by FPLC and Western blot analysis. Large apoA-1poor and apoE-containing HDL became prevalent in hypercholesterolemic hamsters after CETP inhibition. In addition, the size of the CETP-containing HDL particles increased with inhibition of transfer activity. While these effects were apparent in normocholesterolemic animals, the changes in apolipoprotein distribution and HDL subspecies as detected on native gels were more significant in the hypercholesterolemic animals. B The changes in the HDL profile and apolipoprotein distribution after CETP inhibition in hamsters were similar to those reported in CETP-deficient Japanese subjects, suggesting the utility of the hypercholesterolemic hamster as an in vivo model for the understanding of the lipoprotein changes associated with CETP inhi-Inhibition of cholesteryl ester transfer protein in normocholesterolemic and hypercholesterolemic hamsters: effects on HDL subspecies, quantity, and apolipoprotein distribution. J . Lipid Res. 1994. 35: 1634-1645. Supplementary key words reverse cholesterol transport apolipoprotein E apolipoprotein A-I lipoproteins CETP monoclonal antibody atherosclerosis Cholesteryl ester transfer protein (CETP) has been implicated in the process of reverse cholesterol transport through studies of CETP-deficient families (1, 2 ) , in rab-Abbreviations: CETP, cholesteryl ester transfer protein; HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; FPLC, fast protein liquid chromatography.