Androgens markedly stimulate the accumulation of neutral lipids in the human prostatic adenocarcinoma cell line LNCaP

Microscopic evaluation of LNCaP cells stained with the lipophilic dye Oil red O revealed that androgens induce a marked stimulation of lipid droplet accumulation. As determined by quantitative analysis of the Oil red O extracted from the stained cells, stimulatory effects of the synthetic androgen R1881 became apparent at concentrations as low as 10(-11) M. Maximal induction (15-fold) was reached at 10(-8) M. Increases were observed 2 days after hormone addition and were maximal 1 day later. Accumulation of lipid droplets was also induced by mibolerone (another synthetic androgen) and by the natural androgens testosterone and dihydrotestosterone. In agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells, stimulation of lipid accumulation was also apparent after treatment with progesterone and estradiol. Cortisol and the synthetic glucocorticoid dexamethasone were ineffective. The androgen antagonist Casodex (bicalutamide) abolished the stimulatory effect of R1881, further supporting the involvement of the androgen receptor. In agreement with this conclusion, no changes in lipid accumulation were observed after androgen treatment of the androgen receptor-negative prostate tumor lines PC-3 and DU-145. To investigate the nature of the lipids affected by androgens, lipid extracts were analyzed by TLC, complemented with enzymatic lipid analyses. Androgens were shown to have major effects on the content of triglycerides and cholesterol esters (33- and 7-fold stimulation, respectively), the two main classes of lipids stained by Oil red O. Phospholipid and cholesterol contents were increased by a factor of 2. Incorporation studies with [2-14C]acetate revealed that androgens caused a major stimulation of 2-14C incorporation into triglycerides and cholesterol esters (11- and 13-fold, respectively), suggesting that androgens act at least in part at the level of lipid synthesis. Taken together, these findings indicate that androgens, besides affecting proliferation and protein secretion, also markedly stimulate the production and accumulation of neutral lipids, revealing a novel interesting aspect of androgen regulation of LNCaP cells.     

Total fatty acid composition of duck fatty tissues

Total lipids extracted from duck fatty tissues were fractionated on thin layer plates into polar lipids and neutral lipids. Neutral lipids were similarly fractionated into their components. Fatty acid methyl esters from total lipids were fractionated by gas-liquid-chromatography. Results indicated that duck fatty tissues are mostly formed by neutral lipids and that triglycerides comprise the vast majority of neutral lipids. Results also indicated that the major fatty acids in duck lipids are: oleic greater than linoleic greater than stearic greater than palmitoleic. About 73% of all fatty acids present belong to the C-18 series. The unsaturation level for duck lipids is about 73%.     

Ultrastructural localization of thyrocyte acid phosphatase in the presence of thyrocyte hyperfunction in rats

Acid phosphatase was found by electron microscopy in the lysosomes which appeared in great numbers in the follicular cells of rat hyperplastic thyroid gland. The other types of granules (mature secretory granules and lipids) whose amounts were also greatly increased in cases of functional thyrocyte strain were also nonreactive. The lysosomes were subdivided into three main groups according to distribution of the reaction product: the lysosomes with dense homogeneous deposit and with deposit in the form of densely or loosely packed dark round granules. The lysosome heterogeneity was apparently connected with their different functions also found within the colloid droplets in the form of inclusions of rarely located dark granules. The authors believe such granules to be the result of the merging of the colloid droplets and lysosomes. The acid phosphatase of the latter participated in the hydrolysis of the product of cell secretion with the formation of active substances.     

Synthesis of a class of DNA-binding proteins in synchronized untransformed and virus-transformed cells

1. Homogenates of Chinese hamster fibroblasts have been fractionated by analytical centrifugation techniques. Lysosomes have been characterised and identified by the distinctive behaviour of specific marker constituents. 2. The acid hydrolases of hamster fibroblasts were located in two distinct organelles, characterised by differences in their equilibrium densities and rates of sedimentation. The lighter population of lysosomes with a modal density of 1.10 g cm-3, contained glycosidases, nucleases and acid phosphatase. The denser population, with a modal density of 1.15 g cm-3, contained proteases and arylsulphatase B. 3. Acid hydrolases in both populations showed similar degrees of latency and structure-linked sedimentability. 4. The heterophagic activity of hamster fibroblasts was investigated by growing the cells in the presence of [14C]sucrose, [14C]inulin and Triton WR 1339. These compounds are taken up by the cells and appear to be located in the denser population of lysosomes. 5. The low density of the lighter population of lysosomes appears to result from a high content of cholesterol esters and triacylglycerols. It is suggested that the lysosomes may be the site of storage of a considerable proportion of these lipids in cultured hamster cells.     

13-hydroxy octadecadienoic acid (13-HODE) inhibits triacylglycerol-rich lipoprotein secretion by CaCo-2 cells

Oxidized lipids present in atherogenic lipoproteins are derived, in part, from the diet. To address the effects of an oxidized lipid on intestinal lipoprotein assembly and secretion, CaCo-2 cells were incubated with 13-HODE or its native fatty acid, linoleic acid, and triacylglycerol-rich lipoprotein synthesis and secretion were investigated. 13-HODE was readily taken up by cells and esterified to lipids. Although both fatty acids were largely esterified to neutral lipids, in comparison to neutral lipids containing linoleic acid, a greater proportion of cellular neutral lipids containing 13-HODE and/or its metabolites was secreted. Compared to linoleic acid, however, 13-HODE caused less triacylglycerol, derived from de novo synthesis, and less triacylglycerol mass to be secreted. Cells incubated with both linoleic acid and 13-HODE together secreted less triacylglycerol mass than did cells incubated with linoleic acid alone. Less newly synthesized apoB and apoB mass were secreted by cells incubated with 13-HODE without altering the abundance of apoB mRNA. The fraction of newly synthesized apoB translocated into the secretory pathway of cells exposed to 13-HODE was significantly less than that observed in cells incubated with linolenic acid, suggesting that 13-HODE interfered with the assembly and secretion of triacylglycerol-rich lipoprotein particles.     

Incorporation in vivo of 1-14C-palmitic acid into placental and fetal liver lipids of the rabbit

The incorporation of free fatty acid into the placental and fetal liver lipids of rabbits was studied after fetal injections of albumin-bound 1-14C-palmitic acid. The fetuses were killed either 5--10 or 10--20 min after the injection. The placentas and livers were extracted for lipids and the specific activities of triglycerides (TG), phospholipids (PL), free fatty acids (FFA), monoglycerides (MG) and diglycerides (DG) measured. The lipids of the liver and placenta took up 17.0 and 3.6% of the dose, respectively, and of that liver TG accounted for 74% and the placental TG 34% of the label in each tissue. Most of the remaining counts were in the PL fraction with the rest more or less evely distributed between the FFA, DG and MG fractions. No activity was recorded in the cholesterol esters. The placental TG, PL, DG and MG specific activities reached the same level as that of the placental FFA, while in the liver these esters had higher specific activities (than the liver FFA). The liver TG, DG and PL had higher specific activities when compared with those of the placenta. The specific activity of the placental FFA was lower at 10--20 min than at 5--10 min; the opposite was seen for the placental TG. No time-related changes were seen in the liver lipids. It is concluded that (i) both placenta and fetal liver incorporate FFA into glycerides and PL; (ii) the liver incorporates FFA more rapidly and to a greater extent than the placenta; (iii) most of the FFA is incorporated into TG and to a lesser extent (PL; (iv) in both organs hydrolysis of PL or TG occurs. These results are discussed with reference to placental transport of FFA and fetal fat metabolism.     

Plasma transport forms of ingested fatty alcohols in the rat

Previous studies have shown that ingested fatty alcohols are absorbed as fatty acids and fatty acid esters, particularly triglycerides. The present study was carried out to determine whether fatty alcohols are also transported as 0-alkyl glyceryl ethers, alk-1-enyl glyceryl ethers, and as wax esters. Oxidation of fatty alcohols to other lipids was assessed by using a mixture of [1-3H] hexadecanol and [1-14C] hexadecanol of predetermined ratio. The results indicate that the absorption of fatty alcohol, and of its transport forms, parallels the absorption of labeled fatty acids. Six to 25% of plasma radioactivity was present as 1-0-alkyl diacylglyceryl ethers with a smaller proportion of ether lipids in the phospholipid fraction. In addition, 4-13% of the ingested hexadecanol appeared in the plasma as a material having the chromatographic properties of wax ester. Fatty alcohols were not detected in the plasma as alk-1-enyl lipids.     

Effect of papain-induced emphysema on the distrubtion of pleural surface pressure

In vivo experiments using 203Pb and radioactively labelled precursors such as [14C] arginine and [3H] tryptophan were performed to identify lead binding components in rat liver. The distribution of lead in 9 tissues and the intracellular distribution in liver and kidney was also investigated. Male rats were injected intravenously with 18 mug of 203Pb/rat and the 203Pb radioactivity was measured in whole tissues as well as in nuclei, mitochondria, lysosomes, microsomes and soluble fractions obtained by centrifugation of liver and kidney homogenates. The subcellular fractions from liver were purified and fractionated into macromolecular components by ultracentrifugation, gel filtration, ion exchange chromatography and solvent extraction. Nuclei were fractionated into membranes, chromatin proteins (histone and residual non-histone proteins) and DNA. Most of the lead was detected in the nuclear membrane fraction bound exclusively to membrane proteins and absent in phospholipids. The intranuclear lead was associated with histone fractions and other basic or very weakly acid proteins as indicated by the incorporation of [14C] arginine and [3H] tryptophan. Lead was present in the chromatographically purified DNA fraction but whether lead was really bound to the nucleic acid was not determined. Mitochondria were fractionated into heavy, soluble and light subfractions representing the inner membranes, the intramitochondrial matrix and the outer membranes respectively. These subfractions contained appreciable quantities of lead. No appreciable lead was present in lipids of the mitochondrial membranes. Significant quantities of lead were associated with the endoplasmic reticulum. Fractionation of microsomes into rough and smooth membranes showed that lead was almost exclusively bound to membranes of rough-surfaced microsomes associated with the heavy rough membrane subfraction. No significant lead was present in the free polysome subfraction or in lipids from the endoplasmic reticulum. More than one lead binding site was identified in the soluble fraction, the high molecular weight components representing the most important lead binding site.     

Fatty acid composition of plasma lipids in hyperlipidemia associated with ischemic heart disease

The authors compared plasma lipid and lipoprotein values and the fatty acid composition in plasma lipids of a group of 38 men with primary hyperlipoproteinaemia (HLP) type II B and IV with a history of myocardial infarction (IM) and in a control group of 63 men with the same type of HLP without a history of ischaemic heart disease (IHD). Hyperlipidaemic subjects after IM differed from controls by the apolipoprotein (apo) B concentration in LDL lipoproteins and by the composition of fatty acids in plasma phosphatidylcholine (PC) and triglycerides (TG). In the discriminating function which makes it possible in the given group of patients to classify correctly hyperlipidaemic subjects after IM and without detectable IHD the independent variables are apo-B concentration in LDL, apo-A-I in plasma, eicosapentaenoic acid in TG, gamma-linolenic acid in cholesterol esters and stearic and oleic acid in PC. These findings confirm the practical value of assessment of apolipoproteins for detection of hyperlipidaemic subjects with a specially high risk of IHD and indicate also the role of essential FA in the pathogenesis of IM.     

Purification and properties of penicillin acylase of Bovista plumbea

Lipids from the insoluble material obtained by pulmonary lavage of 6 patients with alveolar proteinosis and from lamellar organelles of normal rabbit lungs were isolated and characterized. In both types of samples, dipalmitoylphosphatidylcholine was the predominant lipid. Phosphatidylethanolamine, phosphatidylglycerol, lysophosphatidylglycerol, and 2 glycolipids, GM3 and GL1 were also present in both types of preparations. Sphingomyelin, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidyl-N, N-dimethylethanolamine, phosphatidylserine, and lyso(bis)phosphatidic acid were found in the sedimented lavage material from humans but were not detected in lamellar organelles from rabbits. Significant quantities of neutral lipids were present in the lavage material, but only trace amounts, mainly as cholesterol and triglycerides, were detected in lamellar organelles. Phosphatidylcholine and the 2 glycolipids contained mostly saturated fatty acids and essentially no polyunsaturated fatty acids. Sphingomyelin, lysophosphatidycholine, and phosphatidyl-N, N-dimethylethanolamine, found only in the lavage, were also highly saturated. In addition to the fact that several phospholipids found in the lavage were not present in lamellar organelles, another striking difference between the lipids from these 2 sources was that phosphatidylglycerol of lamellar organelles contained predominantly palmitic acid, whereas the phosphatidylglycerol obtained by lavage of humans contained large amounts of stearic and oleic acids.     

Increased saturated triacylglycerol levels in plasma membranes of human neutrophils stimulated by lipopolysaccharide

Neutrophils isolated from patients with bacterial infections or stimulated in vitro with lipopolysaccharide (LPS) produce a high resolution, lipid-dominated spectrum on 1H-NMR spectroscopy (May et al, 1993. J. Infect. Dis. 168: 386-392). We have investigated the origin of this lipid signal using NMR and chemical analyses of both whole neutrophils and purified plasma membranes. Plasma membranes from neutrophils that had been stimulated with 50 microg/ml LPS exhibited the high resolution 1H-NMR signal, and contained double the triacylglycerol (TAG) content of plasma membranes isolated from resting cells. Chemical analysis of the whole cells indicated that the TAG also increased at the cellular level (1.7-fold) after stimulation with LPS. Diradylglycerol increased 2- to 3-fold in both whole cells and plasma membranes after stimulation, but was only a minor component compared with TAG. The plasma membrane protein/phospholipid ratio increased 2.6-fold, whereas cholesterol (free and esterified) was unchanged. The membranes from LPS-stimulated neutrophils exhibited increased fluidity, as judged by increased merocyanine 540 binding, consistent with a 2-fold reduction in cholesterol/phospholipid ratio. LPS induced a shift in fatty acid content of whole cell polar lipids towards more oleic acid and less palmitic acid, whereas the neutral lipid fraction contained increased amounts of palmitic and stearic acids. The TAG fraction of plasma membrane lipids contained increased amounts of palmitic acid when prepared from cells stimulated with LPS. We conclude that the 1H-NMR signal in LPS-stimulated neutrophils arises from increased amounts of plasma membrane TAG with an elevated content of palmitic acid.     

Change in the concentration of total lipids, phospholipids and neutral lipids in rat liver mitochondria and microsomes during chemical carcinogenesis

Sharp increase in the content of total lipids and, especially, neutral lipids, and considerable decrease in the content of phospholipids were observed in liver microsomes and mitochondrias from liver of tumour-bearing rats in the process of the growth of sarcoma, induced by a single injection of 3,4-benzpyrene. The injection of anthracene, a non-cancerogenous hydrocarbon, practically did not affect the lipid composition of rat liver subcellular particles. Thus, the disturbances in normal functioning of subcellular particles under cancerogenesis are due to considerable change in the chemical composition of biomembranes. The data obtained confirm our hypothesis on lipid mobilization under tumour growth.     

Myelin lipids and proteins in experimental global ischemia

The experiments were performed on white rats, in which clinical death was induced according to the method described by Korpatchev et al. (1982). After cardiac arrest and cessation of respiratory function lasting 5 minutes resuscitatory action was performed. The lipids and proteins of the cerebral myelin fraction were studied in animals sacrificed 1, 9 and 14 days after global ischemia. The obtained results lead to the following conclusions: 1. Myelin lipids in postreanimation syndrome are characterized by a marked increase in cholesteryl esters and a mild one of lysophosphatidylcholine content. 2. Myelin lipids in the predemyelinating period demonstrate a general pattern of reaction, notwithstanding to the character of the primary noxious agent. 3. After global ischemia a great fall of small basic protein in the myelin fraction and subsequently of SBP to LBP ratio occurred. 4. Various noxious agents affect different proteins of the myelin membranes and the resulting changes seem to be characteristic for various pathological processes.     

Inclusion of coconut oil in diets for turkey breeders and its effects on embryonic yolk and liver fatty acids

Turkey hens were fed either a standard breeder diet (CON, myristic acid, C14.0, 1.1%; palmitic acid, C16:0, 16.8%; oleic acid, C18:1, 23%; linoleic acid, C18:2, 48.7%) or a diet containing 5% coconut oil (COCO) enriched with medium chain fatty acids (MCFA; lauric acid, C12:0, 22.6%; C14:0, 10.8%; C16:0, 12.5%; C18:1, 14.8%; C18:2, 24.6%). After 10 d on the diets, fresh eggs were collected for yolk lipid and fatty acid (FA) determination. An additional 60 to 95 eggs were incubated and the FA profiles of the neutral lipid (NL) and phospholipid (PL) fractions of yolk sac and liver lipids were determined. The NL fraction of the yolk sac from CON eggs contained less C12:0 (0 vs 0.49%) and C14:0 (0.7 vs 4.6%) and more C18:1 (41.3 vs 37.5%). The PL fraction of the yolk sac from both treatments contained < 1% C14:0, and there was less than a 2% difference between treatments in other FA concentrations. The hepatic NL fraction from both treatments contained < 1% C14:0 and only C18:1 showed > 1% differences between treatments (Control = 59.9%; COCO = 56.62%). There were no dietary effects on the FA profile of hepatic PL. The presence of only minimal quantities of MCFA in hepatic NL and PL suggests that absorbed yolk sac MCFA are extensively metabolized during embryonic development.     

Diester waxes from skin lipids of the feet of biotin depleted and biotin supplemented turkey poults

The neutral lipids of the skin from the feet of turkey poults fed a biotin supplemented or a biotin deficient diet consist mainly of triacyglycerols, and of mono- and diester waxes. Diester waxes from both groups were characterized as fatty acid esters of erythro-2,3-alkanediols. A comparison between fatty acid composition of the two groups, however, revealed the following significant differences. Biotin deficient birds showed a fairly high concentration of very long chain fatty acids (C36-C40) which were completely absent in biotin supplemented birds. Further, almost one-third of the fatty acids of diester waxes in biotin deficient birds were unsaturated while those from biotin supplemented birds were predominantly (96%) saturated.     

Microphase separation in low density lipoproteins. Evidence for a fluid triglyceride core below the lipid melting transition

The structural organization of the neutral lipid core in human low density lipoproteins (LDL) was investigated in physicochemically defined, distinct human LDL subspecies in the density range of 1. 0244-1.0435 g/ml by evaluation of the core lipid transition temperature, chemical composition, and the behavior of spin-labeled core lipids. Calorimetric studies were performed on more than 60 LDL preparations, and the transition temperature, which varied between 19 and 32 degreesC, was correlated to the chemical composition and revealed a discontinuity at a critical cholesteryl ester to triglyceride ratio of approximately 7:1. For electron spin resonance studies, several LDL preparations were probed with spin-labeled cholesteryl esters and triglycerides, respectively. In LDL with a high triglyceride content, both labels exhibited similar mobility behavior. In contrast, in LDL with only small concentrations of triglycerides, the behavior of labeled cholesteryl esters and labeled triglycerides differed distinctly. The cholesteryl esters were strongly immobilized below the transition temperature, whereas the triglycerides remained fluid throughout the measured temperatures. These results suggest that the critical cholesteryl ester to triglyceride mass ratio of 7:1 corresponds to two concentric compartments with a radial ratio of 2:1, where the liquid triglycerides occupy the core, and the cholesteryl esters form the frozen shell. At higher triglyceride contents, the triglyceride molecules insert into the cholesteryl ester shell and depress the peak transition temperature of the LDL core, whereas at lower triglyceride contents, excess cholesteryl esters are dissolved in the core.     

The effect of silymarin-N-methylglucamine salt and silybin-dihemisuccinate on (1-14C)-acetate incorporation in rat liver lipids (author''s transl)

60 min after i.v. application of 140.5 mg silymarin-N-methylglucamine salt/kg body weight dissolved in 4% polyvinylpyrrolidone solution, and 30 min after i.p. administration [1-14C]-acetate, compared to rats treated with solvent only, a statistically significant increase of specific radioactivities in total lipids, triglycerides, total phospholipids as well as in the phosphatidylcholine fraction and a decrease of specific activities in the free cholesterol fraction of the liver can be determined. 70 min after i.p. application of 140.5 mg silybin-dihemisuccinate/kg body weight dissolved in phosphate buffer, and 10 min after i.v. administration of [1-14C]-acetate in comparison with rats treated with solvent only, a statistically significant enhancement of specific activities of total liver lipids, free hepatic cholesterol, liver triglycerides, total liver phospholipids, and the hepatic fraction of phosphatidyl ethanolamine can be measured. Silybin also produces an increased specific radioactivity of the serum triglyceride fraction.     

Host plasma low density lipoprotein particles as an essential source of lipids for the bloodstream forms of Trypanosoma brucei

In contrast to mammalian cells, bloodstream forms of Trypanosoma brucei show no activity for fatty acid and sterol synthesis and critically depend on plasma low density lipoprotein (LDL) particles for their rapid growth. We report here that these parasites acquire such lipids by receptor-mediated endocytosis of LDL, subsequent lysosomal degradation of apoprotein B-LDL, and utilization of these lipids. Uptake of LDL-associated [3H]sphingomyelin and of LDL-associated [3H]cholesteryl oleate paralleled each other, and that of 125I-apoprotein B-LDL showed saturation and could be inhibited by unlabeled LDL or by anti-LDL receptor antibodies. Metabolism of lipids carried by LDL was abolished by chloroquine and by the thiol protease inhibitor, leupeptin. Sphingomyelin was cleaved by an acid sphingomyelinase to yield ceramide, which was itself split up into sphingosine and fatty acids. The latter were further incorporated into phosphatidylcholine, triacylglycerols, or cholesteryl esters. Similarly, cholesteryl oleate was hydrolyzed by an acid lipase to yield free cholesterol, which was reesterified with fatty acids, presumably in the cytosol. Like free cholesterol, LDL provided substrate for cholesterol esterification. In the culture-adapted procyclic form of T. brucei, which is capable of sterol synthesis, exogenous LDL-cholesterol rather than endogenously synthesized sterol was utilized for sterol esterification. Interference with exogenous supply of lipids via receptor-mediated endocytosis of LDL should be explored to fight against trypanosomiasis.     

Plasma lipids, ketone bodies, and glucose concentrations in calves fed high- and low-fat milk replacers

Twenty-four 5-day-old male calves were fed twice daily milk replacers containing either 5% (low-fat) or 25% (high-fat) lard. Plasma lipids, blood glucose, and ketone bodies were determined in jugular blood before feeding and every hour during 8 h after feeding. The high-fat diet caused in the 1st h after feeding a sharp increase of triglycerides and phospholipids followed by a sharp decrease; these two increased slowly during the following 5 h. Within the first 2 h after feeding, there was an increase of cholesterol esters, free cholesterol, and nonesterified fatty acids. With the low-fat diet, triglycerides and cholesterol esters showed a small increase during the 4 h following meal whereas phospholipids, free cholesterol, and nonesterified fatty acids were not affected significantly. With both diets, blood glucose reached a maximum of 110 mg/100ml 1 h after feeding; ketone bodies were not altered significantly. With the high-fat diet, lipid digestion would occur in two phases; firstly, part of the fat would be lipolyzed quickly by pregastric esterase before clot formation in the abomasum; secondly, the rest of the lipids, slowly released by progressive lysis of the coagulum would be digested under the action of gastric and pancreatic lipases. The first phase did not occur with the low-fat diet.     

Polyunsaturated fatty acids in serum lipids of reindeer during the close postnatal period

We examined serum fatty acid composition in reindeer during the close postnatal period (from < 8 h to 3 weeks) by using maternal serum as a reference point and focusing on the proportions of polyunsaturated fatty acids (PUFAs) in serum lipids. A striking dissimilarity was found in the serum PUFAs between the neonatals and their mothers. In particular, the proportions of linoleic acid (18:2) and alpha-linolenic acid in serum cholesteryl esters and phospholipids of the newborn reindeer were significantly lower than those of the mothers. Furthermore, serum phospholipids of the newborns had lower arachidonic acid and docosapentaenoic acid but higher docosahexaenoic acid proportions than the maternal phospholipids. Although the proportions of the principal C18 PUFAs were low in reindeer milk, they increased sharply in serum cholesteryl esters and phospholipids of the calves during the first few days after birth. In particular, there were significant positive correlations in the proportions of 18:2 between serum and milk lipids. We conclude that the proportions of the serum C18 PUFAs are low in the newborn reindeer, but they are increased during the close perinatal period by a rate which suggests an efficient mechanism for selective retention of these fatty acids from milk lipids.