Pregnenolone and dehydroepiandrosterone accumulate in brain as sulfate and fatty acid esters and unconjugated steroids. The steroid fatty acid ester-synthesizing activity was investigated in rat brain microsomes. Endogenous fatty acids in the microsomal fraction were used for the esterification of steroids. The enzyme system had a pH optimum of 4.5 in acetate buffer with [3H]dehydroepiandrosterone as substrate. The apparent Km was 9.2 +/- 3.1 x 10(-5) M and Vmax was 18.6 +/- 3.4 nmol/h/mg protein (mean +/- SEM). The inhibition constants of pregnenolone and testosterone were 123 and 64 microM, respectively. Results were compatible with a competitive type of inhibition. A high level of synthetic activity was found in the brain of 1- to 3-week-old male rats, which rapidly decreased with aging. Saponification of purified [3H]pregnenolone esters yielded pregnenolone and a mixture of palmitate, oleate, linoleate, stearate, and myristate as the predominant fatty acids. Contrasting with the high rates of esterification of several radioactive delta 5-3 beta-hydroxysteroids or 17 beta-hydroxysteroids, no fatty acid esters of either cholesterol, epitestosterone (with a hydroxyl group at position C-17 alpha), or corticosterone (with hydroxyl groups at C-21 and C-11 beta) were formed in the same incubation conditions.

Children with Alagille syndrome show high serum cholesterol (15-20 mmol/L). To establish correlation of this unusual level of cholesterol with the regulation of cholesterol metabolism, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity and synthesis of cholesterol, fatty acids and acidic steroids from [14C]acetate were determined in cultured skin fibroblasts from 2-3 year old children. Prostaglandin E2 (PGE2) synthesis and nucleic acid synthesis were determined in cells when they were growing in medium containing normal, Alagille or fetal bovine serum. These values were similar to values of controls. HMGR activity was found to be similar in cells of control and children with the syndrome, whether the cells were incubated in lipoprotein-deficient or normal medium. Incorporation of acetate into cholesterol was inhibited to a greater extent by lipoprotein-containing medium in control than in children with the syndrome. Fatty acid synthesis was similar in all conditions. 1-7% of the recovered lipid radioactivity in cells and medium separated as acidic steroids. Serum from a donor patient, when included in the medium, did not affect PGE2 or nucleic acid synthesis compared with normal human or fetal bovine serum. The data suggest that cells of children with Alagille syndrome may have a membrane defect of transfer of cholesterol (LDL receptor defect) leading to excessive cholesterol synthesis. Also, synthesis of acidic steroids (bile acid-like material) and their secretion into the medium occurs in normal fibroblasts and those from children with the syndrome.

UNLABELLED Many publications indicate the beneficial effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) in the control of coronary heart disease and diabetes, although the mechanism is not clear. Some of our previous results suggest that, in contrast to other lipids, n-6 PUFAs could have a permissive effect on carbohydrate oxidation. To check this hypothesis, we determined pyruvate dehydrogenase (PDH, decarboxylase: EC 1.2.4.1) activity in infant skin fibroblasts (ISF) incubated 6 hours in the presence of 0.25 mM linoleic (LI) or arachidonic (AR) acid, compared to oleic acid (OL) and control ISF incubated without addition of fatty acids. The four groups of cells were preincubated 36 hours either in the presence of fetal bovine serum (FBS), or in the presence of lipoprotein-deprived serum (LPDS). RESULTS (1) When the ISF were maintained in the medium containing FBS, the two PUFAs had little inhibitory effect on PDH activity, in contrast with the effect of OL. (2) When the ISF were kept in the lipoprotein-deficient medium, PDH activity was low in controls and in the OL cells, but the addition of LI or AR increased the activity. This suggests the role of n-6 PUFAs in enhancing carbohydrate oxidation, under certain conditions.

In order to determine the incorporation of C1-14C derived from mono- and poly-unsaturated fatty acids into cholesterol of human cells cultured in exponential phase, infant skin fibroblasts (SF) were used at the 5th passage. On Day 6, the SF were preincubated 36 h in a medium containing 5 per cent lipoprotein-deficient serum, and thereafter [1-14C] oleic, -linoleic or -arachidonic acid-without (OL1, LI1 and AR1 group SF), or with the addition of 0.25 mM cold fatty acids (OL2), LI2 and AR2 group SF). Cholesterol specific radioactivity (SRA) peaked 1 h after, and leveled off afterwards in the OL1, LI1 and AR1 groups. Cholesterol-SRA was relatively low in the other groups, but increased progressively, giving a biphasic response: C1-14C derived from from linoleic and arachidonic acids was actively incorporated into cholesterol during the first hours, as compared to C1-14C derived from oleic acid, but stabilized between 6 and 12 h for the LI2 and AR2 group SF incubation. This result appears to be due to the stimulation of pyruvate decarboxylation, observed elsewhere, and consequently to the dilution of the radioactive units in a large pool of non-labeled acetyl-CoA units derived from glucose, when these SF were incubated with 0.25 mM polyunsaturated fatty acids.

ABSTRACT: The present experiment was carried out using the following diets: FF, fat-free, and LP the same diet with 0.7% sunflower oil - given to the progeny of females kept on the FF diet since the mating. After 10 mM Mg2+ activation of the PDH phosphatase, the rate of [1-14C] pyruvate decarboxylation into acetyl-CoA ester units was determined in the liver, brain and adipose-tissue of the pair-fed developing rats. Results: In the male progeny, pyruvate dehydrogenase (PDH) activity was higher (61%) in the LP group livers than in the FF group livers, at the end of the 13 week experiment. Such a difference was not observed in the two group brains up to the 91 days postweaning, but was even larger (94%) between adipose-tissues of the LP and FF groups. In the female progeny kept 12 weeks on the diets, PDH activity in the LP group tissues was also higher than in the FF group tissues: 63% in the liver, 43% in adipose-tissues, and less than 10% in the brain. Therefore, a minute amount of lipids high in linoleic acid appeared to increase PDH activity, and especially in the liver and adipose-tissues of animals kept on a strictly fat-free diet. This stimulation of the PDH activity seems closely related to the phospholipid rehabilitation in the tissues (decrease in the trienoic: tetraenoic acid ratio values).

6-day-old suckling rats, born to females kept on a fat-free diet, were used to determine cholesterol and fatty acid specific radioactivity (SRA) in the liver, kidneys and brain, after injection of 3 microCi uniformly 14C-labeled linoleic acid (ULI) or oleic acid (UOL). 1 h after injection, cholesterol SRA was highest in the liver and kidneys, and then decreased when UOL was injected. Cholesterol SRA peaked 3 h after injection of ULI in liver and kidneys. The delay in appearance of the ULI (over UOL) peak of cholesterol SRA may be due to differences in the rates of oxidation of these two labeled fatty acids into acetyl-CoA ester, according to the structural role of ULI. In the period between 3 and 56 h, cholesterol was more radioactive in the three tissues after injection of ULI than after injection of UOL. The radioactivity of saturated fatty acids was low in the ULI and UOL groups of these very young animals. Therefore, cholesterol synthesis seemed to happen at a faster rate than the other lipid syntheses in liver and kidneys, where the rate of linoleic acid elongation into arachidonic acid was also slower than cholesterol synthesis. Different results were obtained in the brain, where arachidonic acid SRA increased rapidly after ULI injection.

Conference Abstract| April 01 1981 RADIOACTIVITY OF CHOLESTEROL AND OF NON LIPID FRACTIONS AFTER INCORPORATION OF LABELED UNSATURATED FATTY ACIDS IN RAT LIVER AND HUMAN SKIN FIBROBLASTS J. Raulin; J. Raulin 1Université Paris 7, Nutrition Cellulaire, 2 place Jussieu, Paris 05 Search for other works by this author on: This Site PubMed Google Scholar M. Bouchène; M. Bouchène 1Université Paris 7, Nutrition Cellulaire, 2 place Jussieu, Paris 05 Search for other works by this author on: This Site PubMed Google Scholar D. Lapous; D. Lapous 1Université Paris 7, Nutrition Cellulaire, 2 place Jussieu, Paris 05 Search for other works by this author on: This Site PubMed Google Scholar C. Wolfrom; C. Wolfrom +INSERM, Hôpital de Bicêtre, (France) Search for other works by this author on: This Site PubMed Google Scholar M. Gautier M. Gautier +INSERM, Hôpital de Bicêtre, (France) Search for other works by this author on: This Site PubMed Google Scholar Author and article information Publisher: Portland Press Ltd Online ISSN: 1470-8752 Print ISSN: 0300-5127 © 1981 Biochemical Society1981 Biochem Soc Trans (1981) 9 (2): 184P. https://doi.org/10.1042/bst009184pc Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation J. Raulin, M. Bouchène, D. Lapous, C. Wolfrom, M. Gautier; RADIOACTIVITY OF CHOLESTEROL AND OF NON LIPID FRACTIONS AFTER INCORPORATION OF LABELED UNSATURATED FATTY ACIDS IN RAT LIVER AND HUMAN SKIN FIBROBLASTS. Biochem Soc Trans 1 April 1981; 9 (2): 184P. doi: https://doi.org/10.1042/bst009184pc Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1981 Biochemical Society1981 Article PDF first page preview Close Modal You do not currently have access to this content.

Pulse labeling of adipose tissue and liver by injection of [Me3H]thymidine in developing rats receiving 20% dietary lipids suggests a dual function of this molecule in nuclei: tracer incorporation into newly formed DNA, and tracer incorporation into RNA linked to nascent DNA. This conclusion is based on (1) determination of the specific radioactivity (SRA) of long alkali-stable nucleotide sequences which increased dramatically during 1 hr after radioactive injection when rats were given 20% lard (L) containing little linoleic acid, and decreased sharply thereafter; (2) isopycnic centrifugation of the native DNA extracted from nuclei pulse labeled in vivo with [Me3H]thymidine and [6-14C]orotic acid, which indicated the presence of doubly-labeled constituents with the buoyant density of DNA; (3) isopycnic centrifugation after freeze-thawing of the same samples, which indicated the disappearence of 14C-labeled long alkali-stable nucleotide sequences in the tritium labeled material with the buoyant density of DNA.