In vitro modulation of rat adipocyte ghost membrane fluidity by cholesterol oxysterols

The effects of cholesterol and cholesterol-derived oxysterols (cholestanone, cholestenone, coprostanone and epicoprostanol) on adipocyte ghost membrane fluidity were studied using a fluorescence depolarization method. The fluorescence anisotropy of the treated membranes was determined using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Cholestanone and cholesterol decreased membranes fluidity at both the concentrations tested (10 & 50 M) while the rest of the sterols did not exert any significant effect on membrane fluidity. In the presence of epinephrine, cholestanone partitioned more towards the lipid core but cholesterol partitioning was not affected. The fusion activation energies (E) obtained for membranes preincubated with cholestanone (8.6 kcal/mol) and cholesterol (8.2 kcal/mol) were not significantly different from that of untreated membranes (8.3 kcal/mol). Membranes preincubated with cholestanone and cholesterol did not exhibit any change in lipid phase throughout the temperature range (10–45°C) tested. The sterols were found to inhibit fisetin-induced phospholipid methylation in isolated rat adipocytes in the rank order of cholesterol > epicoprostanol > cholestanone=cholestenone=coprostanone, while basal methylations was unaffected. When adipocytes were preincubated with the sterols before the addition of fisetin, cholestanone and cholestenone showed 74% and 66% inhibition of maximal methylation respectively. These results indicated that cholesterol oxysterols interact differently with rat adipocyte membranes, with cholestanone interacting more with phospholipids located at the inner lipid bilayer (e.g. phosphatidylethanolamine) while cholesterol interacts more with phosphatidylcholine located at the outer lipid bilayer. This differential interaction may cause selective changes in membrane fluidity at different depths of the bilayer and thus may modulate the activities of membrane-bound proteins such as enzymes and receptors.     

Net transport of cholesterol from cells of the human EA.hy 926 endothelial cell line to high density lipoproteins

EA.hy 926 cells, a human endothelial cell line, show characteristics of differentiated endothelial cells. The cells express saturable binding of apo E-free¹²⁵I-high density lipoprotein₃ (HDL₃). B_max increased from 71 to 226 ng HDL₃ bound/mg cell protein after cholesterol loading of the confluent endothelial cells with cationized low density lipoprotein (LDL). The affinity did not change after cholesterol enrichment (K_d was 37 g HDL₃ protein/ml for control cells and 31 g/ml, for loaded cells). Incubation of cholesterol-loaded EA.hy 926 cells with native HDL and LDL had different effects on cellular cholesterol levels. Incubation with HDL decreased both esterified and unesterified cellular cholesteryl, but LDL did not change total cellular cholesterol However, LDL tended to increase cellular cholesteryl esters, with a concomitant decrease of unesterified, cellular cholesterol. Incubation of endothelial cells with both HDL and LDL also resulted in decreased total cellular cholesterol levels. These data show that cationized LDL-loaded human endothelial EA.hy 926 cells can be used to study the net transport of cellular cholesterol to HDL, the first step in reverse cholesterol transport.     

Amylin given by central or peripheral routes decreases gastric emptying and intestinal transit in the rat

The effect of rat amylin on gastric emptying and intestinal transit in the rat was examined. Amylin administered intracerebroventricularly (1, 2, 2.5 or 4 g/rat) produced the maximal decrease in gastric emptying and intestinal transit at the dose of 2.5 g/rat. Higher doses produced a lower effect. Peripheral administration (25, 50 or 100 g/kg) produced dose-dependent effects. Pre-treatment with neostigmine blocked the effect of amylin when it was centrally injected, while the effect of amylin given peripherally was partially reduced. Pre-treatment with domperidone decreased the inhibitory effect of peripherally injected amylin, but no effect was observed when the peptide was centrally injected.     

Cholesterol feeding to rats does not modulate the expression of binding sites for HDL on liver membranes

Summary The binding of HDL, Apo-E-free, was studied in rats fed a cholesterol rich diet for 2, 4 and 7 days. Plasma cholesterol increased up to 16-fold (from 55 to 900 mg/dl); liver cholesterol was also raised, from 0.5 to 16 mg/g of tissue. The HDL binding to membrane preparations was not affected while the binding of VLDL was reduced to about 50% of the controls. These data show, therefore, that liver binding sites for HDL are refractory to regulation by dietary cholesterol.     

Effects of enterally- and parenterally-administered bombesin on intestinal luminal tryptic activity and protein in the suckling rat

Summary Because of the presence of bombesin-like immunoreactivity in milk we investigated if enteral administration of bombesin affects the intestinal luminal content of trypsin and protein in 12-14-day-old rats. Bombesin (40 g/kg), given either orogastrically or subcutaneously, produced a significant elevation in the intestinal content of trypsin activity. Thus, enterally-administered bombesin can produce acute biologic effects in suckling rats.     

Changes in erythrocyte membrane lipid composition affect the transient decrease in membrane order which accompanies insulin receptor down-regulation

We have recently demonstrated, using electron paramagnetic resonance (EPR) spectroscopy, that insulin receptor internalization in response to insulin incubation (down-regulation) in human erythrocytes is accompanied by a transient decrease in membrane order, as measured by the 2T order parameter. Since membrane lipids play such an important role in receptor internalization, we investigated the possible effects that an alteration of the normally-occurring lipid profile might have on down-regulation and the concomitant transient decrease in membrane order. Consequently, human erythrocytes enriched with cholesterol and erythrocytes from cirrhotic patients were examined, because both of these groups of cells have a higher cholesterol/phospholipid molar ratio (CH/PL) than controls. The 5-nitroxystearate spin label, which inserts into the lipid bilayer of cell membranes, was used to monitor changes in 2T for a 3-h period at 37°C. We report here that both cholesterol-enriched and cirrhotic erythrocytes do not down-regulate, as demonstrated by binding assays, and that they do not show the typical transient decrease in membrane order observed in controls. The results seem to indicate that a more ordered membrane inhibits internalization of the insulin receptor in erythrocytes, and that an increase in membrane disorder is necessary for insulin receptor down-regulation.     

Metabolism in porifera VI. Role of the 5,6 double bond and the fate of the C-4 of cholesterol during the conversion into 3β-hydroxymethyl-A-nor-5α-steranes in the spongeAxinella verrucosa

Summary During the conversion of cholesterol into 3 -hydroxymethyl-A-nor-5-cholestane by the spongeAxinella verrucosa, the carbon-3 of this latter originate from carbon-4 of cholesterol. Cholestanol moreover does not seem an intermediate in this conversion.     

Variability of sterol utilization in stored-products insects

A comparison of sterol utilization by 3 stored-products insects revealed very different capabilities. The fluor beetle,Tribolium castaneum dealkylates and converts dietary sitosterol to about equal amounts of cholesterol (43.7%) and 7-dehydrocholeterol (39.8%), whereas another flour beetle,Tenebrio molitor, produces considerably less 7-dehydrocholesterol (16.8%) and relatively more cholesterol (66.7%) from sitosterol. The lepidopteran,Plodia interpunctella, utilized dietary sterol very similar to plant-feeding lepidoptera, producing primarily cholesterol (86.5%) from sitosterol.     

Die Cholesterin-7α-Hydroxylase-Aktivität der Rattenleber nach Hypophysektomie und nach Substitution mit Hypophysenhormonen

Summary In order to elucidate the role of the hypophysis in the regulation of cholesterol 7-hydroxylase activity, male and female rats hypophysectomized on day 50 of life were treated with different hypophyseal hormones and tested on day 85 of life. Only a crude extract from human hypophysis and ovine prolactin was able to enhance the enzyme activity, thereby restoring the level of intact controls.

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Diese Arbeit wurde im Rahmen des Sonderforschungsbereiches Endokrinologie (SFB 87) von der Deutschen Forschungsgemeinschaft gefördert.     

Effect of administration of estrogen to pregnant rabbits on the lipid content and composition of the fetal brain

Summary Intramuscular injection of 17 -estradiol to pregnant rabbits did not produce any significant change in the phospholipid, neutral lipid and cholesterol content of the fetal brains, nor did it cause any significant difference in their wet and dry weights. It may be inferred that use of estrogen in the gestational period to enhance surfactant production and lung maturation in the fetus would not produce adverse effects on the fetal brain lipids.     

Celiac disease biomarkers identified by transcriptome analysis of small intestinal biopsies

Establishing a celiac disease (CD) diagnosis can be difficult, such as when CD-specific antibody levels are just above cutoff or when small intestinal biopsies show low-grade injuries. To investigate the biological pathways involved in CD and select potential biomarkers to aid in CD diagnosis, RNA sequencing of duodenal biopsies from subjects with either confirmed Active CD (n?=?20) or without any signs of CD (n?=?20) was performed. Gene enrichment and pathway analysis highlighted contexts, such as immune response, microbial infection, phagocytosis, intestinal barrier function, metabolism, and transportation. Twenty-nine potential CD biomarkers were selected based on differential expression and biological context. The biomarkers were validated by real-time polymerase chain reaction of eight RNA sequencing study subjects, and further investigated using an independent study group (n?=?43) consisting of subjects not affected by CD, with a clear diagnosis of CD on either a gluten-containing or a gluten-free diet, or with low-grade intestinal injury. Selected biomarkers were able to classify subjects with clear CD/non-CD status, and a subset of the biomarkers (CXCL10, GBP5, IFI27, IFNG, and UBD) showed differential expression in biopsies from subjects with no or low-grade intestinal injury that received a CD diagnosis based on biopsies taken at a later time point. A large number of pathways are involved in CD pathogenesis, and gene expression is affected in CD mucosa already in low-grade intestinal injuries. RNA sequencing of low-grade intestinal injuries might discover pathways and biomarkers involved in early stages of CD pathogenesis.     

Metabolism in Porifera. IX. Studies on the biological conversion of cholesterol into 19-nor-cholestanol by the spongeAxinella polypoides

Summary The conversion of cholesterol into 19-nor-5-cholestan-3-ol by the spongeAxinella polypoides involves a partial loss (40%) of the 3-hydrogen atom; moreover administration to the sponge of [4-¹⁴C]cholesterol tritiated at C-4 and C-7 showed that the 4- and 7-hydrogen atoms are retained in this conversion. A competitive uptake experiment, [4-¹⁴C]cholesterol vs. [7-³H₂]5-cholestanol, showed that the sponge utilized exclusively cholesterol for the production of 19-nor-5-cholestan-3-ol.Part of this work has been presented at the Nato Conference on Marine Natural Products (Jersey, Great Britain, October, 1976).-This contribution is part of the Programma finalizzato Oceanografia e Fondi marini-sottoprogetto Risorse biologiche C.N.R. Italy.     

Biosynthesis ofα- andβ-ecdysone by the CrayfishOrconectes limosus in vivo and by its Y-Organs in vitro

Summary - and -ecdysone were synthesized from labelled cholesterol by premolt crayfish in vivo and by their Y-organs in vitro.     

Physiological importance of ω-3/ω-6 polyunsaturated fatty acids in man. An overview of still unresolved and controversial questions

Summary The essentiality of (-6) and (-3) fatty acids in mammals is well known. Nevertheless, some important points remain unclear concerning their implication in physiology. After a short discussion about the definition of essential fatty acids deficiency, this brief overview deals with some of these points, pointing out some of the unresolved questions. Different subjects are approached concerning the (-6) and (-3) fatty acids metabolism: desaturases, eicosanoids, production, as well as some of their metabolic effects on cell membranes, intestinal function, glucose and lipid metabolism, haemorheology.     

Cholesterol content and cholesterol esterifying activity of various organs in guinea pigs

Summary In various organs of the guinea pig, the total cholesterol content of an organ was significantly correlated with the percentage of esterified cholesterol present in this organ. Cholesterol esterifying capacity was shown in most organs, with highest activities in the adrenals, the spleen and the liver. The significant correlation found between the cholesteryl ester content of an organ and its acyl cholesterol acyltransferase activity suggests a possible role of this enzyme in determining the level of the total and esterified cholesterol in a tissue.This work was supported by a grant Crédit aux Chercheurs du Fonds National de la Recherche Scientifique of Belgium.Acknowledgments. I am indebted to Prof. C. Harvengt for his stimulating interest and helpful suggestions. I wish to thank Y. van Nieuwenhuyze and J. Costermans for their valuable laboratory assistance and Dr H. Baudon for pathological studies.     

Sur les intermédiaires de la désalkylation en C24 duβ-sitostérol par le criquetLocusta migratoria L.

Summary The locustLocusta migratoria L. transforms-sitosterol into cholesterol. Fucosterol, desmosterol and an unknown sterol (probably in C₂₉) are found to be intermediates, but not 24-methylene cholesterol.     

Peptide-stimulated release of prolactin from the fowl anterior pituitary gland

Summary Anterior pituitary glands from broiler fowl were preincubated for 24 h in either medium 199 only or medium containing estradiol 17, following which they were incubated in medium containing thyrotrophin releasing hormone (TRH), vasoactive intestinal polypeptide (VIP) or substance P (SP), alone or with the dopamine agonist, apomorphine. Estradiol priming stimulated release of prolactin and enhanced apomorphine-inhibition of prolactin release. TRH stimulated prolactin release, an effect reversed by apomorphine, and priming with estradiol potentiated both effects. VIP stimulated prolactin to a lesser degree and again this was inhibited by apomorphine and potentiated by estradiol. SP had little effect on the nonsteroid-primed pituitary, but stimulated release of prolactin after estradiol treatment, though less effectively than TRH or VIP.     

Age-dependent changes of rat liver plasma membrane composition

Summary The chemical composition of liver plasma membrane was studied in Wistar rats aged between 3 and 24 months. Results obtained indicate a significant age-dependent positive correlation of both the protein:phospholipid and cholesterol:phospholipid ratios, whereas the protein:cholesterol ratio seems to remain unaffected. Phospholipid analysis of liver plasma membrane reveals that only the phosphatidylcholine content has a significant negative correlation with age; all other phospholipid species remain basically unchanged.Supported by a grant of the Italian National Research Council, Project Preventive and Rehabilitative Medicine, Subproject Mechanisms of Aging.     

Biosynthèse des hormones stéroïdes

Summary The different pathways of biosynthesis are described, by which steroid hormones are producedin vivo in the gonads, the adrenal cortex and the placenta, orin vitro by corresponding tissue preparations. First of all a short outline of the origin of cholesterol from acetyl coenzyme A through the biological isoprene unit, the pyrophosphate of ³-iso-pentenol, through squalene, lanosterol and zymosterol is given. Partial side-chain degradation of cholesterol, where ACTH plays an important role, leads to pregnenolone and further to progesterone.The latter compounds are the starting material for the other steroid hormones. On the one hand, they are converted in the gonads and adrenals into 17-hydroxy derivatives, in which the side chain is completely removed by enzymes of the said and also of non-endocrine tissues, to form the androgens. Furthermore the adrenals produce their own typical androgens. After hydroxylation of the angular 19-methyl group, androgens are aromatised to estrogens.On the other hand, the adrenal converts progesterone by hydroxylation in 21- and, depending on the case, also in 11- and/or 17-position into the classical adrenocortical hormones. The special feature of an 18-hydroxylation and -dehydrogenation forms part of the biogenesis of aldosterone; a new example is given for the methods used towards its elucidation, consisting in the biosynthetic conversion of progesterone into two virtual intermediates, 18-oxo-progesterone and 21-desoxyaldosterone, which already contain the 18-aldehyde group, characteristic for aldosterone.The regulation of the biogenesis of the different hormones is mentioned and compounds are discussed which block one or other of the biosynthetic steps.

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D'après une conférence faite aux VIes Journées biochimiques latines, Genève (Suisse), 25 mai 1961.

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Publication No. 179Über Steroide. Communication No. 178 voirK. Heusler, P. Wieland etA. Wettstein, Helv. chim. Acta, sous presse.     

Unterschiede in der Carbohydrasenausrüstung der Entwicklungsstadien vonMelolontha melolontha L.

Summary The activities of-xylosidase,-xylanase and inulinase were demonstrated only in the tissues of the larval intestinal canal which also split galactose and arabinose from certain hemicelluloses and-galactan. Several other carbohydrases originating from microorganisms were also detected only in the content of the larval alimentary tract.

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Ein Teil der Arbeit wurd am Institut für Obstbau der Biologischen Bundesanstalt Heidelberg ausgeführt.