Vitamin E reduces cholesterol esterification and uptake of acetylated low density lipoprotein in macrophages

The effects of vitamin E on cholesteryl ester (CE) metabolism in 1774 cells were examined. Pretreatment of 1774 cells with vitamin E at concentrations above 50 μM significantly decreased acetylated low density lipoprotein (LDL)-induced incorporation of [¹⁴C]oleate into CF in cells in a dose-dependent manner. This was partly due to vitamin E Also significantly inhibiting the uptake of [³H]CE-labeled acetylated LDL by 1774 cells. A trend existed toward suppression of acyl-CoA:cholesterol acyltransferase (ACAT) activity in the cell lysate at high vitamin E concentration, but there was no effect on hydrolysis of CE. These data indicate that vitamin E reduces the uptake of modified LDL and suppresses ACAT activity, resulting in less cholesterol esterification in macrophages; a novel mechanism underlying the antiatherogenic properties of vitamin E.     

Upregulation of low density lipoprotein receptor activity by tumor necrosis factor,a process independent of tumor necrosis factor-induced lipid synthesis and secretion

It has been shown that tumor necrosis factor (TNF) rapidly upregulates expression of the low density lipoprotein (LDL) receptors on Hep G2 cells and acutely stimulates hepatic lipid synthesis and secretionin vivo. It may thus be possible that TNF-induced expression of LDL receptors is secondary to a decrease in cellular cholesterol content caused by TNF-stimulated lipid secretion. In order to know whether TNF upregulates LDL receptors by depletion of the cellular cholesterol content, the present experiments were designed to study the temporal relationship between TNF-stimulated expression of LDL receptor activity and TNF-induced changes in lipid synthesis and secretion in anin vitro setting by using Hep G2 cells (a highly differentiated human hepatoma cell line) as a hepatocyte model. Hep G2 cells were incubated with TNF (usually 2.5 nmol/L) for certain periods, and LDL receptor activity was evaluated by measuring [¹²⁵I]LDL binding at 4°C; lipid synthesis and secretion were assayed by measuring [³H]glycerol incorporation into triglycerides and phospholipids as well as [¹⁴C]acetate incorporation into cholesterol. We found that a 30-h exposure of the cells to TNF was needed for the effect of TNF to be seen on lipid synthesis and secretion as measured by incorporation of [³H]glycerol into triglycerides and phospholipids, whereas TNF rapidly (in several hours) upregulated LDL receptor activity. TNF stimulated triglyceride synthesis, but did not stimulate phospholipid synthesis. On the other hand, TNF stimulated phospholipid secretion, but did not stimulate triglyceride secretion. Exposure of the cells to TNF for 16 or 24 h neither decreased cholesterol synthesis nor stimulated cholesterol secretion as measured by [¹⁴C]acetate incorporation into cholesterol. Upregulation of LDL receptor activity through inhibition of cellular cholesterol synthesis with compactin (a competitive inhibitor of the 3-hydroxyl-3-methylglutaryl-CoA reductase) was augmented by TNF, whereas downregulation of LDL receptor activity through stimulation of cellular cholesterol synthesis with mevalonolactone almost completely blocked the upregulatory effect of TNF. In conclusion, TNF-stimulated expression of LDL receptor activity is not secondary to a depletion of cellular cholesterol content through TNF-stimulated lipid secretion or inhibition of cholesterol synthesis.     

The processing of exogenous cholesterol in the alveolar compartment of the rat lung

We have examined the esterification of [³H]cholesterol following the intratracheal instillation of a tracer amount into the isolated rat lung perfused with Krebs bicarbonate containing 4.5% albumin. At 5, 30 and 60 min after instillation, lungs were lavaged at 2°C with 3×10 ml of 0.15 M NaCl, each volume instilled and withdrawn three times. Each lung was lavaged at only one time point. The saline recovered was centrifuged at 150 g (5 min) to sediment the macrophage-rich fraction, leaving the surfactant in the supernatant. The amounts and specific activity of cholesterol and cholesteryl ester were measured following isolation by high performance liquid chromatography of the free cholesterol and the hydrolyzed ester-derived cholesterol. There was a rapid fall in [³H]cholesterol in the surfactant fraction, accompanied by a reciprocal increase in [³H]cholesteryl ester. Likewise, there was a rapid increase in [³H]cholesteryl ester in the macrophage-rich fraction, while the level of free [³H]cholesterol in that fraction remained very low. These data are consistent with exogenous cholesterol being rapidly esterified in the alveolus, and the ester then being cleared by the macrophages. We were unable to locate the actual site of esterification. Lipids     

Effect of 25-hydroxycholesterol on cholesteryl ester formation in Caco-2 cells

Incubation of Caco-2 cells, a human intestinal cell line, with 25-hydroxycholesterol (25-HOC) markedly enhanced cellular cholesteryl ester formation determined by incorporation of [¹⁴C]oleic acid into intracellular cholesteryl [¹⁴C]oleate. The stimulation by 25-HOC of cholesteryl ester formation was suppressed by staurosporine, a kinase inhibitor, but not by cycloheximide or actinomycin D. The specific activity of microsomal acyl-coenzyme A:cholesterol acyltransferase (ACAT) increased two-fold in cells treated with 10 μM 25-HOC for 5 h. ACAT activity decreased when microsomes were incubated without sodium fluoride, a phosphatase inhibitor, but the decrease in ACAT activity in cells stimulated with 25-HOC was more pronounced. The results suggest that protein phosphorylation may be involved in the stimulation of cholesteryl ester formation by 25-HOC in Caco-2 cells.     

Mifepristone Treatment Results in Differential Regulation of Glycerolipid Biosynthesis in Baby Hamster Kidney Cells Expressing a Mifepristone-Inducible ABCA1

ATP binding cassette A1 (ABCA1) transports cholesterol, phospholipids and lipophilic molecules to and across cellular membranes. We examined if ABCA1 expression altered cellular de novo glycerolipid biosynthesis in growing Baby hamster kidney (BHK) cells. Mock BHK cells or cells expressing a mifepristone-inducible ABCA1 (ABCA1) were incubated plus or minus mifepristone and then with [³H]serine or [³H]inositol or [³H]ethanolamine or [methyl-³H]choline or [³H]glycerol or [¹⁴C]oleate and radioactivity incorporated into glycerolipids determined. Mifepristone did not affect [1,3-³H]glycerol or [¹⁴C]oleate or [³H]ethanolamine or [methyl-³H]choline uptake in BHK cells. In contrast, [³H]glycerol and [¹⁴C]oleate incorporated into phosphatidylserine (PtdSer) were elevated 2.4-fold (p < 0.05) and 54% (p < 0.05), respectively, upon ABCA1 induction confirming increased PtdSer biosynthesis from these precursors. However, mifepristone inhibited [³H]serine uptake and incorporation into PtdSer indicating that PtdSer synthesis from serine in BHK cells is dependent on serine uptake. Mifepristone stimulated [³H]inositol uptake in mock and ABCA1 cells but not its incorporation into phosphatidylinositol indicating that its synthesis from inositol is independent of inositol uptake in BHK cells. [³H]glycerol and [¹⁴C]oleate incorporated into triacylglycerol were reduced and into diacylglycerol elevated only in mifepristone-induced ABCA1 expressing cells due to a decrease in diacylglycerol acyltransferase-1 (DGAT-1) activity. The presence of trichostatin A, a class I and II histone deacetylase inhibitor, reversed the ABCA1-mediated reduction in DGAT-1 activity but did not affect DGAT-1 mRNA expression. Thus, mifepristone has diverse effects on de novo glycerolipid synthesis. We suggest that caution should be exercised when using mifepristone-inducible systems for studies of glycerolipid metabolism in cells expressing glucocorticoid responsive receptors.     

Hydrocarbon transport in chylomicrons and high-density lipoproteins in rat

A lipoprotein system is described that transports gut hydrocarbons of low polarity in chylomicrons of intestinal lymph and plasma to plasma high density lipoproteins (HDL) in rat. Four highly lipophilic aryl and alkyl hydrocarbons [benzo(α)pyrene; 1,1,1-trichloro-2,2-bis(p-chlorophenol)ethane (DDT), hexadecane and octadecane] were selected to give a graded range of polarity. Chylomicrons were labeled doubly with radioisotopes in triacylglycerol and a single hydrocarbon by feeding [³H]-glycerol and [¹⁴C]hydrocarbon. All hydrocarbons were transported in the triacylglycerol oil phase of chylomicrons. Injected chylomicron triacylglycerol and 3 of 4 hydrocarbons were cleared simultaneously from plasma consistent with lipoprotein-lipase dependent hydrocarbon clearance but DDT was cleared more rapidly. HDL was the major plasma acceptor of all labeled hydrocarbons. Plasma chemical fluxes were measured for octadecane and DDT and both showed net fluxes from chylomicrons to HDL. HDL selectively concentrated chylomicron hydrocarbons from chylomicron triacylglycerol. Lipoprotein lipase stimulation by intravenous heparin significantly increased transfer of alkanes from chylomicrons to HDL. These results indicate that (a) chylomicrons transport gut-derived hydrocarbons with a wide range of structure and polarity as triacylglycerol solutes; (b) HDL are a major plasma acceptor of all these hydrocarbons, demonstrating both selective solute uptake from triacylglycerol and net chemical uptake for the 2 hydrocarbons studied and (c) efflux of these chylomicron hydrocarbons from plasma and into HDL is regulated partly by hydrolysis of chylomicron triacylglycerol.     

Metabolic behavior in rats of a nonprotein microemulsion resembling low-density lipoprotein

A protein-free microemulsion (LDE) with a lipid composition resembling that of low-density lipoprotein (LDL) was used in metabolic studies in rats to compare LDE with the native lipoprotein. LDE labeled with radioactive lipids was injected into the bloodstream of male Wistar rats, and plasma kinetics of the labeled lipids were followed on plasma samples collected at regular intervals for 12 h after injection. The 24-h LDE uptake by different tissues was also measured in tissue samples excised after the animals had been sacrificed. We found that LDE plasma kinetics were similar to those described for native LDL [fractional clearance rate (FCR) of cholesteryl ester, 0.42±0.11 h⁻¹]. The major site for LDE uptake was the liver, and the tissue distribution of the LDE injected radioactivity was as one would expect for LDL. To test whether LDE was taken up by the specific LDL receptors, the LDE emulsion was injected into rats treated with 17α-ethinylestradiol, which is known to increase the activity of these receptors; as expected, removal of LDE from the bloodstream increased (FCR=0.90±0.35 h⁻¹). On the other hand, saturation of the receptors that remove remnants by prior infusion of massive amounts of lymph chylomicrons did not change LDE plasma kinetics. These results indicate that LDE is cleared from plasma by B,E receptors and not by the E receptors that remove remnants. Incorporation of free cholesterol into LDE increased LDE plasma clearance. Incubation studies also showed that LDE incorporates a variety of apolipoproteins, including apo E, a ligand for recognition of lipoproteins by specific receptors. Our data suggest that LDE can be a useful tool to test LDL metabolism and B,E receptor function.     

Effect of chylomicron remnants on cholesterol metabolism in cultured rabbit hepatocytes: Very low density lipoprotein and bile acid production

The interrelationship between very low density lipoprotein (VLDL) secretion and bile acid production was studied in primary culture of rabbit hepatocytes. Chylomicron remnants (CR) were added to the cultures to study their effect on VLDL secretion and bile acid production. After 24 hr preincubation of cells with CR (10–50 μg protein/mL), intercellular neutral lipid content was increased 1.5–4-fold in a dose-dependent manner. Neutral lipid accumulation was accompanied by a 70–90% reduction of [¹⁴C]acetate incorporation into cholesterol, while no stimulation of [¹⁴C]oleate incorporation into cholesteryl esters was observed. Incubation of cells with CR increased secretion of free cholesterol, triacylglycerol and apoproteins B and E in VLDL. Stimulation of VLDL cholesterol secretion was accompanied by a reduction of taurocholic acid synthesis. These data demonstrate the existence of an inverse relationship between secretion of VLDL cholesterol and bile acid production under conditions of effective uptake of triacylglycerol-rich CR by hepatocytes.     

Extraction of Alkali Metal Cations by Lipophilic Dibenzo-14-crown-4-carboxylic Acids

Abstract

The extraction of alkali metal cations by the lipophilic crown ether, bis-t-octylbenzo-14-crown-4 (BOB14C4), three derivatives of BOB14C4 having pendant carboxylic acid sidearms, and a lipophilic carboxylic acid, 2-methyl-2-heptylnonanoic acid (HMHN) was studied by two-phase potentiometric titration and ion-chromatography. The lipophilic, ionizable crown ethers, BOB14C4-acetic acid (BOB14C4AA), BOB14C4-propanoic acid (BOB14C4PA), and BOB14C4-oxyacetic acid (BOB14C4OAA) extract cations efficiently from aqueous mixed alkali metal chloride solutions into 1-octanol by an ion-exchange mechanism in the range p[H] > 7, as does HMHN. The mode of attachment of the ionizable sidearm, via an ether linkage (BOB14C4OAA) versus a carbon linkage (BOB14C4AA and BOB14C4PA), has a significant effect on the cation selectivity and extraction efficiency of these extractants. BOB14C4 exhibits no p[H] dependent extraction behavior and has no significant effect on the extraction of alkali metal cations by HMHN in a mixture of these two compounds. Although BOB14C4AA and BOB14C4PA extract cations at lower p[H] than HMHN, all three compounds exhibit similar selectivity for Li⁺ over Na⁺, K⁺, Rb⁺ and Cs⁺. A significant reversal in selectivity is observed with BOB14C4OAA, which extracts Na⁺ and K⁺ selectively over Li⁺, Rb⁺, and Cs⁺ and at significanty lower p[H] than BOB14C4AA, BOB14C4PA, or HMHN. The unique behavior of BOB14C4OAA may be attributed to the presence of the ether linkage between the crown ether and the pendant carboxylic acid.     

The relative incorporation of arachidonic and eicosapentaenoic acids into human platelet phospholipids

The incorporation of arachidonic acid (AA) as compared to eicosapentaenoic acid (EPA) into human platelet phospholipids was tested by incubating washed platelets with a known mixture of [³H]AA and [¹⁴C]EPA. Following incubation, the platelet lipids were extracted, the individual phospholipids—phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE)—were separated by thin layer chromatography, and their corresponding [³H]/[¹⁴C] ratios were determined. Based on a [³H]/[¹⁴C] ratio of unity for the substrate mixture, the PC, PS, PI and PE exhibited ratios of 0.55, 0.93, 1.12 and 0.74, respectively, which were significantly different from 1.00 in all instances except in the case of PS. These results indicate that PC and PE selectively incorporated EPA, while PI showed preference toward AA. These selectivities may account partly for the differing AA/EPA mass ratios that have been observed among the individual phospholipids of human subjects consuming fish oils.     

[3H]cholesterol transfer from microemulsion particles of different sizes to human fibroblasts

A new technique for preparing microemulsion particles of well-defined sizes and compositions is presented. Utilization of these microemulsions is advocated as lipoprotein models in studies of lipid transport and metabolism, rather than the currently used phospholipid-cholesterol vesicles. The emulsion particles consisted of egg phosphatidylcholine and cholesterol as surface lipids and cholesteryl oleate as core lipid. They were prepared by a combined injection and sonication technique and size-separated by a two-step procedure of gel filtration chromatography and density gradient centrifugation. By varying the ratios of core and surface material, particles covering a size range of 20–200 nm in diameter could be produced. The adequacy of these microemulsions as lipoprotein models was tested by studying the transfer of [³H]cholesterol and [¹⁴C]cholesterol oleate from the particles to cultured human fibroblasts. Up to a particle size of 100 nm, there was a slight increase of [³H]cholesterol transfer. The transfer of [¹⁴C]cholesteryl oleate was very slow, yet measurable. Studies of the exchangeability of cholesterol between the microemulsion core and surface phases indicated that all cholesterol can be transferred from microemulsions to cultured cells as a single pool.     

DDT absorption and chylomicron transport in rat

Male rats were fed 100 nM dichlorodiphenyltrichloroethane-¹⁴C in oil by gastric tube. Recovery of dichlorodiphenyltrichloroethane-¹⁴C in thoracic duct lymph was 60% in 12 hr. Lymph dichlorodiphenyltrichloroethane-¹⁴C (97%) occurred in lipoproteins of d<1.006, designated chylomicrons. Mechanical separation of chylomicron triglyceride core (labeled with triglyceride-³H) from chylomicron membrane (labeled with phospholipid-³²P) showed that 97% dichlorodiphenyltrichloroethane-¹⁴C was present in triglyceride core. To investigate possible association of plasma clearance of the two core lipids, rats were pulse injected with chylomicrons, doubly labeled with triglyceride-³H and dichlorodiphenyltrichloroethane-¹⁴C. The decay of dichlorodiphenyltrichloroethane-¹⁴C in sequential serum samples was rapid (T^1/2=2 min) and was independent of triglyceride-³H decay. In tissues removed 14 min after injection of chylomicrons, 30% administered dichlorodiphenyltrichloroethane-¹⁴C was found in liver but only 1% in adipose tissue. In hepatectomized (eviscerated) rats, the decay of serum dichlorodiphenyltrichloroethane-¹⁴C (T^1/2=10 min) was also independent of and more rapid than triglyceride-³H decay. With sucrose density gradients, it was shown that chylomicron dichlorodiphenyltrichloroethane-¹⁴C transferred to higher density serum proteins in vitro and in vivo and to bovine albumin in vitro. Thus, dichlorodiphenyltrichloroethane was transported from intestine largely in the triglyderide phase of chylomicrons; disappearance of chylomicron-dichlorodiphenyltrichloroethane from the systemic circulation was rapid and partly independent of the presence of the liver and of triglyceride hydrolysis; some dichlorodiphenyltrichloroethane was transported from serum chylomicrons to albumin or other plasma proteins before tissue uptake.     

Aerobic Exercise Improves Reverse Cholesterol Transport in Cholesteryl Ester Transfer Protein Transgenic Mice

We analyzed the effect of a 6-week aerobic exercise training program on the in vivo macrophage reverse cholesterol transport (RCT) in human cholesteryl ester transfer protein (CETP) transgenic (CETP-tg) mice. Male CETP-tg mice were randomly assigned to a sedentary group or a carefully supervised exercise training group (treadmill 15 m/min, 30 min sessions, five sessions per week). The levels of plasma lipids were determined by enzymatic methods, and the lipoprotein profile was determined by fast protein liquid chromatography (FPLC). CETP activity was determined by measuring the transfer rate of ¹⁴C-cholesterol from HDL to apo-B containing lipoproteins, using plasma from CETP-tg mice as a source of CETP. The reverse cholesterol transport was determined in vivo by measuring the [³H]-cholesterol recovery in plasma and feces (24 and 48 h) and in the liver (48 h) following a peritoneal injection of [³H]-cholesterol labeled J774-macrophages into both sedentary and exercise trained mice. The protein levels of liver receptors were determined by immunoblot, and the mRNA levels for liver enzymes were measured using RT-PCR. Exercise training did not significantly affect the levels of plasma lipids or CETP activity. The HDL fraction assessed by FPLC was higher in exercise-trained compared to sedentary mice. In comparison to the sedentary group, a greater recovery of [³H]-cholesterol from the injected macrophages was found in the plasma, liver and feces of exercise-trained animals. The latter occurred even with a reduction in the liver CYP7A1 mRNA level in exercised trained animals. Exercise training increased the liver LDL receptor and ABCA-1 protein levels, although the SR-BI protein content was unchanged. The RCT benefit in CETP-tg mice elicited by exercise training helps to elucidate the role of exercise in the prevention of atherosclerosis in humans.     

Effects of non-β-oxidizable sulfur-substituted fatty acid analogues on synthesis and secretion of triacylglycerol and cholesterol in cultured rat hapatocytes

The mechanisms behind the hypolipidemic effect of two sulfur-substituted fatty acid analogues, 3-thiadicarboxylic acid and tetradecylthioacetic acid, have been investigated in cultured hepatocytes. There was a dose-dependent reduction in incorporation of [³H]water into triacylglycerol and diacylglycerol when tetradecylthioacetic acid was added to rat hepatocytes cultured in the presence of 200 μM oleic acid. Tetradecylthioacetic acid also increased the oxidation of [¹⁴C]palmitic acid compared to oleic acid, inhibited the incorporation of radiolabeled precursors into diacylglycerol to a greater extent than into triacylglycerol, and reduced the secretion of triacylglycerol more than its synthesis. A stimulation, rather than a reduction, in glycerolipid synthesis and secretion by tetradecylthioacetic acid was observed when oleic acid was omitted from the culture medium. When 3-thiadicarboxylic acid was added to cultured hepatocytes, the effects on glycerolipid synthesis were generally similar to those observed with tetradecylthioacetic acid, but 3-thiadicarboxylic acid did not increase the oxidation of [¹⁴C]palmitic acid. The two fatty acid analogues also had different effects on the synthesis and secretion of cholesterol and cholesteryl esters—3-thiadicarboxylic acid reduced the incorporation of [³H]water into synthesized and secreted cholesterol and cholesteryl esters, whereas tetradecylthioacetic acid only reduced the secretion of cholesteryl esters without affecting its synthesis. It is concluded that tetradecylthioacetic acid increases the oxidation of fatty acids and reduces the synthesis and secretion of glycerolipids. 3-Thiadicarboxylic acid reduces the synthesis and secretion of both glycerolipids and cholesterol to approximately the same extent without a concomitant increase in the oxidation of fatty acids.     

Chylomicron remnant and asialoglycoprotein metabolism are independent

Because of the considerable similarities between the hepatic metabolism of chylomicron remnants and asialoglycoproteins, the hypothesis that they might share a cell surface receptor or a common step in internalization was tested. Unlabeled chylomicron remnants did not reduce the binding of¹²⁵I-asiaglycoprotein to plasma membranes, but did compete for¹²⁵I-chylomicron remanant binding. The converse also was true. This suggested the receptors were distinct. The two substances did not compete with each other for removal by the isolated perfused rat liver. This suggests that no potentially common post binding events can become rate limiting. In conclusion, despite similarities in their removal and metabolism, chylomicron remnants and asialoglycoproteins are metabolized independently.     

Metabolism of erucic acid in adipocytes isolated from rat epididymal fat

The metabolism of [14-¹⁴C]erucic acid and [U-¹⁴C]palmitic acid has been investigated in adipocytes isolated from rat epididymal fat. The rate of acylation of [¹⁴C]erucic acid in cellular lipids and oxidation to CO₂ and acid-soluble activity was ca. 1/3 of the rate with [¹⁴C]palmitic acid as substrate. A maximal incorporation of fatty acids in triacylglycerol was found at a fatty acid concentration of 0.8 mM in the medium, both with [¹⁴C]erucic acid and [¹⁴C]palmitic acid as substrate. Glucose added to the medium increased the esterification and decreased the oxidation of both fatty acids. No significant chain-shortening of [¹⁴C]erucic acid to shorter monoenes was identified in the fat cells. Increasing concentrations of unlabeled palmitic acid in the incubation medium markedly inhibited the esterification of [¹⁴C]erucic acid, whereas unlabeled erucic acid had little effect on the rate of esterification of [¹⁴C]palmitic acid.     

Modulation of phorbol ester-associated events in epidermal cells by linoleate and arachidonate

To elucidate the events elicited by the skin tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), which are modulated by linoleic acid (LA) and arachidonic acid (AA), the activity of these fatty acids in cultured mouse epidermal cells was compared. Approximately 94% of either exogenous radiolabelled fatty acid was incorporated into the total phospholipid pool over 15 h. The relative distribution among the phospholipid classes differed, however, such that approximately 70% of phospholipid-associated [¹⁴C]-LA was found in phosphatidylcholine, compared to approximately 30% for [¹⁴C]AA. Phosphatidylethanolamine and phosphatidylinositol/phosphatidylserine contained 17 and 13% of the phospholipid [¹⁴C]LA, and 34 and 30% of [¹⁴C]AA, respectively. Prostaglandin (PG) E₂ production was low but similar in unstimulated cultures prelabelled with either [¹⁴C]LA or [¹⁴C]AA. However, in cultures treated with TPA (1.6 μM), [¹⁴C]AA-prelabelling resulted in approximately three times the amount of [¹⁴C]PGE₂ compared with cultures prelabelled with [¹⁴C]LA. Cultured cells were found to contain significant δ6 desaturase activity, which may enable conversion of LA to AA, and thus may account for the observed PGE₂ production from [¹⁴C]LA treated cells. AA-Supplemented (1.6 μM) cultures supported approximately twice the induction of ornithine decarboxylase activity by TPA compared with cultures treated with 1.8 μM LA. Activation of partially purified protein kinase C was similar for either fatty acid tested over a 10–300 μM dose range. Overall, the results suggest that LA does not have the same biological activity as AA with regard to several TPA-associated events known to be important in skin tumor promotion. This reduced biological activity of LA may be partly responsible for the known inhibition of mouse skin tumor promotion by high dietary levels of LA [Leyton, J., Lee, M.L., Locniskar, M.F., Belury, M.A., Slaga, T.J., Bechtel, D., and Fischer, S.M. (1991)Cancer Res. 51, 907–915].     

Biochemical and functional abnormalities in hypercholesterolemic rabbit platelets

This study was designed to elucidate changes in rabbit platelet lipids induced by a cholesterol rich diet and to explore the possible correlation of these lipid changes with platelet abnormalities. Pronounced biochemical alterations were observed when serum cholesterol levels of 700–1000 mg% were reached. Hypercholesterolemic (HC) platelets contained 37% more neutral lipids and 16% less phospholipids than the controls. Lysolecithin, cholesterol esters and phosphatidylinositol (PI) levels were increased in HC platelets, and the levels of phosphatidylcholine (PC) were decreased. The cholesterol/phospholipid molar ratio of lipidemic platelets increased from 0.55±0.011 to 0.89±0.016 (P<0.01) in eight weeks. HC platelets had 90% more arachidonic acid (AA) in the PI than normal platelets. No significant changes in AA of PC were observed. Platelet function was monitored by the uptake and release of [¹⁴C]serotonin in platelet rich plasma (PRP), using varying concentrations of collagen as an aggregating agent. The uptake of [¹⁴C]serotonin in HC and normal platelets ranged from 78–94%. The percent of [¹⁴C]serotonin released from normal and HC platelets was proportional to the concentration of collagen. However, lipidemic platelets were hyperreactive to low concentrations of collagen. Incorporation of 50 μM acetylsalicylic acid into the aggregating medium suppressed the release of [¹⁴C]serotonin in normal PRP by more than 90%, but had only a partial effect on lipidemic PRP.     

Cholesterol transport and uptake in miniature swine fed vegetable and animal fats and proteins. 2. LDL uptake and cholesterol distribution in tissues

In a 2×2 factorial arrangement, miniature pigs were fed four diets containing vegetable protein/fat (soybean) and animal protein (egg white)/fat (beef tallow) to demonstrate the effects of protein and fat source on tissue cholesterol concentrations, uptake of intact low density lipoproteins (LDL) and free cholesterol exchange from LDL to tissues. Soybean oil feeding, compared with beef tallow feeding, resulted in greater concentrations of cholesterol in aorta, heart, and large and small intestines. Similar trends were seen in liver, adipose tissue and skeletal muscle. Dietary protein source had little or no effect on tissue cholesterol concentrations. Uptake of intact LDL, as measured by using [¹⁴C]sucrose-LDL, tended to be greater in pigs fed soybean oil or soy protein. Net exchange of free cholesterol from LDL, as measured with [³H]cholesterol, tended to be greater when vegetable products were fed. Relative contributions of whole tissues to total uptake by either mechanism were not influenced by diet. Mechanisms in addition to uptake of cholesterol from LDL seem to be involved in the greater accumulation of tissue cholesterol resulting from polyunsaturated fat feeding. Data taken from a dissertation submitted to Iowa State University by L. S. Walsh Hentges as partial fulfillment of the requirements for the Ph.D. degree. A preliminary paper was presented at the 68th Annual Meeting of the Federation of American Societies for Experimental Biology in St. Louis, Missouri, April 1984 (Fed. Proc. 43:796).     

A Phytosterolemic Mixture of Sterols Inhibits Cholesterol Synthesis,Esterification, and Low-Density Lipoprotein Receptor mRNA Abundance in HepG2 Cells

HepG2 cells were incubated with a 16.5:1.7:1 ratio of cholesterol:sitosterol:campesterol (CSC), a ratio of the major sterols observed in the plasma of phytosterolemia patients, or with cholesterol alone in combination with [¹⁴C]acetate for 24 h and the radioactivity incorporated into lipids determined. Cells incubated with CSC exhibited a 40% reduction in cholesterol esterification (p < 0.05) compared to cells incubated with cholesterol alone. In addition, a 17.5-fold reduction (p < 0.05) in total cholesterol (cholesterol plus cholesteryl ester) synthesis from [¹⁴C]acetate was observed in cells incubated with CSC compared to cholesterol alone. Low-density lipoprotein receptor (LDLR) mRNA abundance was lower in cells incubated with CSC compared to cells incubated with cholesterol alone. Our results suggest that incubation of HepG2 cells with a ratio of sterols that mimic the plasma concentration seen in phytosterolemia patients reduces cholesterol esterification, total cholesterol synthesis, and inhibits LDLR mRNA abundance. We suggest that future cell and animal-based work on phytostosterolemia might employ this methodology to serve as a novel paradigm of the disease.