Paulette Bournot

Hepatic farnesyl diphosphate synthase expression is suppressed by polyunsaturated fatty acids

Dietary vegetable oils and fish oils rich in PUFA (polyunsaturated fatty acids) exert hypocholesterolaemic and hypotriglyceridaemic effects in rodents. The plasma cholesterol-lowering properties of PUFA are due partly to a diminution of cholesterol synthesis and of the activity of the rate-limiting enzyme HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase). To better understand the mechanisms involved, we examined how tuna fish oil and individual n-3 and n-6 PUFA affect the expression of hepatic FPP synthase (farnesyl diphosphate synthase), a SREBP (sterol regulatory element-binding protein) target enzyme that is subject to negative-feedback regulation by sterols, in co-ordination with HMG-CoA reductase. Feeding mice on a tuna fish oil diet for 2 weeks decreased serum cholesterol and triacylglycerol levels, by 50% and 60% respectively. Hepatic levels of FPP synthase and HMG-CoA reductase mRNAs were also decreased, by 70% and 40% respectively. Individual n-3 and n-6 PUFA lowered FPP synthase and HMG-CoA reductase mRNA levels in H4IIEC3 rat hepatoma cells to a greater extent than did stearate and oleate, with the largest inhibitory effects occurring with arachidonate, EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). We observed a similar inhibitory effect on protein levels of FPP synthase. The suppressive effect of PUFA on the FPP synthase mRNA level was not due to a decrease in mRNA stability, but to transcription inhibition. Moreover, a lower nuclear availability of both SREBP-1 and SREBP-2 mature forms was observed in HepG2 human hepatoblastoma cells treated with arachidonate, EPA or DHA. Taken together, these data suggest that PUFA can down-regulate hepatic cholesterol synthesis through inhibition of HMG-CoA reductase and FPP synthase, at least in part through impairment of the SREBP pathway.

Synthesis and gas chromatographic-mass spectrometric analysis of reduced compounds derived from 6α- and 6β-hydroxy-11-deoxycorticosterone

Abstract A series of thirty two 6-hydroxylated steroids were synthesized by selective reduction of the 4–5 double bond, the 3-oxo group, and/or the 20-oxo group of 6α- and 6β-hydroxyDOC. The different reactions leading to the production of specific isomers are discussed. The gas chromatographic and spectrometric characteristics of the methoxime-trimethylsilyl (MO-TMS) or trimethylsilyl (TMS) derivatives of the isomers obtained are given. The gas chromatographic separation of the syn- and anti -isomers of the methoxime in position 3 was found to be characteristic of the configuration of the hydroxyl in position 6. The difference between methylene unit values of syn- and anti- isomers is much larger for the 6α-series than for the 6β-series. The mass spectral analysis showed that many ions are specific of the MO-TMS derivatives of steroids with 3,6-dihydroxy-4-ene or 3-oxo-6-hydroxy-4-ene structure. In the case of steroids with a satureated ring A no significant ions characteristic of the presence of a 6-trimethylsilyloxy substituent were found. This work provides previously unavailable reference data on 6-hydroxylated steroids which should facilitate the study of corticosteroid metabolism.

The role of ACTH in determining the metabolic pathways of deoxycorticosterone by newborn rat adrenal cells in primary culture

The metabolism of deoxycorticosterone (DOC) by newborn rat adrenal cells in primary culture at various times after culture, with and without ACTH, was studied. After 5 days in culture before addition of ACTH, the main products of the metabolism of DOC were corticosterone and 18-hydroxy-11-deoxycorticosterone in a 2:1 ratio. Smaller amounts of 20 alpha-dihydrocorticosterone and 18-hydroxycorticosterone were also found. No reduced metabolites of DOC were detected. Without ACTH the conversion of DOC to corticosterone and 18-hydroxyDOC declined rapidly. After 13 days in culture, this conversion accounted for only half the metabolites. The reductive metabolism of DOC which yields products reduced at 20 alpha and/or 3 alpha/beta and 5 alpha accounted for the other half. When ACTH (22 mU/ml) was added to the culture daily for several weeks, the primary metabolism of DOC remained that of 11 beta- and 18-hydroxylation yielding corticosterone and 18-hydroxyDOC. A minor reductive metabolism was found. Both cultures produced 6 beta-hydroxyDOC. These results demonstrate that ACTH is needed to maintain the efficiency of the 11 beta/18-hydroxylating system. They also show that ACTH controls the type of metabolism predominant in the rat adrenal cell and may be responsible for the balance between the biosynthesis of glucocorticoids and their reductive catabolism in the fasciculata zone of the adrenal gland.

Bioconversion of 16α-hydroxyprogesterone to 16α-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture

Abstract The bioconversion of 2α-hydroxyprogesterone into 2-hydroxylated steroids was accomplished using newborn rat adrenal cells in primary culture. The products were purified using column and thin-layer chromatography, and identified by GC-MS. They resulted principally from the enzymatic reactions of 21-hydroxylation, 11β-hydroxylation, reduction of 20-oxo and 3-oxo groups, and epimerization of the substrate. In addition, minor metabolites resulted from 18-hydroxylation, 6β-hydroxylation and reduction of the 3-oxo-4-ene group. The identification of these compounds allowed us to conclude that the metabolism of 2α-hydroxyprogesterone is similar to that of progesterone in this cellular system. Assuming that the 2β-epimers of the different metabolites arose principally from the transformation of 2β-hydroxyprogesterone, the specificity of the various enzyme systems seems to be similar for both epimers except in the case of the 11β-hydroxylation where the reaction appears stereospecific for the 2β-epimer. The 2α-hydroxyl group on ring A seems to favor the reduction of the 3-oxo group and it does this stereospecifically to the 3β-structure. The epimerization of the substrate, which is most likely enzymatically induced, is the first example of steroid epimerization reported in the adrenal. This is a practical preparative method for synthesizing a variety of steroids hydroxylated at C-2 from a single substrate and could be adjusted to the production of important quantities of 2-hydroxylated metabolites of corticosteroids.

Peroxisome proliferator-activated receptor α (PPARα) activators induce hepatic farnesyl diphosphate synthase gene expression in rodents

Abstract Fibrates are hypolipidemic drugs that exert multiple effects on lipid metabolism by activating peroxisome proliferator-activated receptor alpha (PPARα) and modulating the expression of many target genes. In order to investigate the link between PPARα and cholesterol synthesis, we analysed the effect of fibrates on expression of the farnesyl diphosphate synthase (FPP synthase) gene, known to be regulated by sterol regulatory element-binding proteins (SREBPs), in conjunction with HMG-CoA reductase. In wild-type mice, both fenofibrate and WY 14,643 induced FPP synthase gene expression, an effect impaired in PPARα-null mice. A three-fold induction was observed in ciprofibrate-treated rat hepatocytes, in primary culture. This effect was decreased in presence of 5,6-dichlorobenzimidazole riboside (DRB) and cycloheximide (CHX), transcription and translation inhibitors, respectively. Acyl-CoA oxidase (AOX), a bona fide PPARα target gene, was induced by ciprofibrate but slower and more strongly than FPP synthase. In addition, induction of FPP synthase gene expression was abolished in the presence of 25-hydroxycholesterol (25-OH Chol). Thus, activation of PPARα by fibrates induced FPP synthase gene expression in both hepatocytes in culture and in mouse liver. This effect is likely to be dependent on cellular sterol level, possibly through SREBP-mediated transcriptional activation.

18-Hydroxylase activity in the Y1 adrenal cell line

18-Hydroxylase activity, reported here for the first time in the mouse adrenal tumor cell line (Y1), was expressed in the metabolism of 11-deoxycorticosterone (DOC) and corticosterone (B). Detected after 24 h of incubation, it was more evident after 48 h and produced mostly 18-hydroxy-20 alpha-DHB from these exogenous substrates. However, 18-hydroxylation was quantitatively less significant than the metabolism of 20 alpha-reduction and 11 beta-hydroxylation (of DOC). The latter is also the predominant metabolism of progesterone in this cell line, during the conversion of cholesterol from the serum-supplemented culture media. The cytochrome P-450 11 beta activity of the Y1 cells is similar to that of the mouse in vivo which catalyzes the production of an 11 beta 18-dihydroxylated metabolite as the principal 18-hydroxylated steroid. It is different from that of other species, such as the rat and the bovine, both in terms of the ratio of 11 beta- to 18-hydroxylated metabolites and of the structure of these metabolites.

Partial characterization of unusual polar steroids in the urine of a child with low renin hypertension

Analysis of urinary steroids excreted by a 7-year old girl with low renin hypertension following ACTH treatment revealed several unknown steroids, which have been analysed by gas chromatography-mass spectrometry. It is proposed that these steroids are monohydroxylated derivatives of cortisol, cortisone, either or both tetrahydro and allo-tetrahydrocortisol and either or both tetrahydro and allo-tetrahydro-11-deoxycortisol. Further analysis indicated that there are two likely positions for the additional hydroxyl group, either on the A or B ring.

Synthesis and mass spectrometric analysis of hexahydrogenated products of aldosterone, 18-hydroxycorticosterone and 18-hydroxy-11-deoxycorticosterone

Abstract Stereospecific or nonstereospecific reductions of corticosteroids with an 18-hydroxyl group or an 18-oxo group were carried out either with sodium borohydride or 20β-hydroxysteroid dehydrogenase of Streptomyces hydrogenous to unambiguously characterize the two possible 20α- and 20β-epimers. The resulting products were analyzed as methyloxime trimethylsilyl ethers by gas chromatography-mass spectrometry. Characteristic ions are described and a mechanism of a specific fragmentation of the 18,20,21-trihydroxystcroids is proposed.

18-Hydroxylation in the Y-1 adrenal cell line: Response to acth and to culture conditions

The 18-hydroxylation of deoxycorticosterone in the Y-1 adrenal cell line was studied under various incubation and cell culture conditions and compared to 11 beta-hydroxylation. Repeated incubation of the substrate increased both 18- and 11 beta-hydroxylation in the Y-1 cells. Furthermore, both 18- and 11 beta-hydroxylation were increased with increased serum concentration and prolonged incubation time. While the increase in 11 beta-hydroxylation seemed to be independent of the type of serum, 18-hydroxylation was much more important in cells cultured in fetal or newborn calf serum supplemented medium than in those cultured in horse serum supplemented medium. As expected, ACTH treatment increased 11 beta-hydroxylation; however, it decreased 18-hydroxylation. The different regulation of these two hydroxylating pathways by ACTH, point to a heterogeneity of the cytochrome P-450(11) beta of the Y-1 cell line.

Evidence of formation of isomeric methoximes from 20-oxosteroids

The formation and gas chromatographic behavior of syn- and anti-isomers in position 20 of the methoxime-trimethylsilyl (MO-TMS) derivatives of many 20-oxo and 3,20-dioxo-21-hydroxysteroids is reported. The existence of such isomers was established from the gas chromatographic (GC) and mass spectrometric analysis of the MO-TMS derivatives of 3 alpha,21-dihydroxy-5 beta-pregnan-20-one and its 17 alpha-epimer. The degree of separation during GC analysis of the syn- and anti-isomers in position 20, as well as those in position 3, is associated to the position of additional hydroxyl groups on the steroid ring. These data are very important for the location of oxygenated substituents such as 2 alpha/2 beta, 6 alpha/6 beta, 11 beta, 16 alpha, 17 alpha, 18, 19 or 21-hydroxyl groups during structural studies of 20-oxo and 3,20-dioxosteroids.