Isoprenoid biosynthesis in a cell-free system from pea shoots.

A cell-free system consisting of a soluble fraction and plastid fragments from pea shoots incorporates 2-(14)C-mevalonate very actively into farnesol, squalene, geranylgeraniol, and other isoprenoids of different carbon-chain lengths. The products have different cofactor requirements, which makes it possible to channel the pathway into different products by varying the incubation mixture.

Mevalonate metabolism: role of kidneys.

More than one-half of the amount of mevalonate that is metabolized by pathways not leading to sterols is accounted for by the action of the kidneys. Conversion of mevalonate in vivo to squalene and sterols in the kidneys is confined almost entirely to the proximal and distal convoluted tubules in the cortex. More sterol than squalene is synthesized from mevalonate not only in the liver but also in the kidney.

Sterol synthesis in the human arterial intima.

Intimal sterol synthesis was examined in isolated human arterial segments obtained at surgery or at postmortem examination. The tissues were incubated with acetate-1-(14)C and mevalonate-2-(14)C and the incorporation of these precursors into sterols was determined. Intimal sterols were isolated by multiple chromatographic techniques and purified by bromination and oxidation procedures. The results indicate that the arterial intima can incorporate acetate and mevalonate into cholesterol, cholestanol, and squalene. Cholestanol was the major sterol synthesized by the arterial wall, but cholesterol production was also consistently observed. The findings suggest that local synthesis is a potential source of sterol accumulation within the arterial wall. The conversion of cholesterol to other sterols was also studied in terminally ill patients receiving labeled cholesterol before death. Tissue analyses revealed the presence of labeled cholestanol as well as cholesterol in the tissue 5-104 days after labeled cholesterol administration. The results demonstrate the conversion of cholesterol to cholestanol in man and suggest that the exchange of cholestanol between the blood and tissues is similar to that of cholesterol.

Polyisoprenoid amphiphilic compounds as inhibitors of squalene synthesis and other microsomal enzymes.

Analogues of farnesyl pyrophosphate containing a farnesyl moiety and a variety of amine residues replacing the pyrophosphate have been synthesized. Most of these compounds were effective inhibitors of the synthesis of squalene and presqualene pyrophosphate from farnesyl pyrophosphate. 50% inhibition was obtained at concentrations between 50 and 100 micron. These analogues also inhibited other microsomal enzymes so they apparently function as general inhibitors of microsomal enzymes.

Suppression of sebum secretion with 13-cis-retinoic acid: effect on individual skin surface lipids and implications for their anatomic origin.

The contribution of the keratinizing epidermis to the human skin surface lipid film has been difficult to ascertain because, after its release from the epidermal cells, epidermally derived lipid inevitably becomes mixed with sebum. In the present study, the sustainable rates of production of the 5 neutral lipid classes found on the skin surface (triglycerides + free fatty acids, wax esters, cholesterol, cholesterol esters, and squalene) were measured on the foreheads of acne patients before, during, and following treatment with 13-cis-retinoic acid, a drug which suppresses sebum production profoundly. Since sebum production was high in the patients before treatment and was suppressed to a few percent of the pretreatment level in some of the patients during treatment, data covering a wide range of sebum production rates were obtained. By using squalene as a measure of sebum production and plotting the rates of production of the other lipid classes vs the rate of production of squalene, it was possible through extrapolation to estimate the residual (i.e., epidermal) rate of production of each lipid class at zero sebum production. The results indicated that epidermis releases triglycerides + free fatty acids and cholesterol to the skin surface. The cholesterol esters in freshly secreted skin surface lipids appeared to be almost entirely sebaceous in origin. Measurements were also made of the percentages of cholesterol esters in lipid collected from the scalp after several days' accumulation and were compared to corresponding values for the forehead lipid. The percentages of cholesterol esters in scalp lipid tended to rise when sebum production was suppressed by the drug, rather than remaining relatively constant as occurred in the freshly secreted forehead lipid. This result indicated that epidermis may contribute to skin surface cholesterol esters, probably through skin surface esterification of epidermal cholesterol.

Effect of temperature on ergosterol biosynthesis in yeast.

Anaerobically grown Saccharomyces cerevisiae was aerated for 7 hr at 20 degrees, 30 degrees, or 40 degrees, in a phosphate buffer containing 2% glucose. At elevated temperature (40 degrees), de novo synthesis of squalene and sterois in the aerated yeast was only 32-35% of that at lower temperature (20 degrees or 30 degrees), and this decrease was attributed to the repression of the enzymes involved in the synthesis of mevalonate from acetyl-COA. In addition, at elevated temperature, the metabolic flux from squalene to ergosterol was blocked at squalene epoxidation, lanosterol demethylation, and ergosta-5, 7, 22, 24(28)-tetraene-3beta-ol reduction.

Formation of dehydrosqualene catalyzed by squalene synthetase in Saccharomyces cerevisiae.

When microsomal fraction of Saccharomyces cerevisiae was incubated with farnesyl pyrophosphate or presqualene pyrophosphate in the presence of Mn2+, 12,13-cis-dehydrosqualene (DeH2Sq) and some related compounds were found to be formed. Incubation in the presence of NADPH gave rise to only squalene. By heat treatment of the microsomal fraction, the DeH2Sq- and squalene-forming activities were inactivated at approximately the same rate. The elution patterns of both activities upon Sephacryl S-200 chromatography of the enzyme solubilized from the microsomal fraction with taurodeoxycholate coincided completely. These results indicate that DeH2Sq formation in yeast is catalyzed by squalene synthetase. Divalent cation was essential for this reaction and Mn2+ was six times more effective than Mg2+. DeH2Sq formation was also observed when microsomes of pig liver were used instead of yeast microsomal fraction, suggesting that this reaction is a ubiquitous one among the eucaryotes which are capable of synthesizing sterols. Based on these observations, the mechanisms of DeH2Sq and squalene formation are discussed.

Lipids in plant tissue cultures. V. Effect of environmental conditions on the lipids of Glycine soja and Brassica napus cultures.

The environmental conditions prevailing during growth of plant tissue cultures affect the concentration of certain lipid classes and the fatty acid patterns of the total lipids. Irrespective of whether the cultures are grown under continuous illumination or in the dark, aerated cultures contain larger proportions of sterols, steryl esters, steryl glycosides and various sterylglycolipids and lower concentrations of squalene than non-aerated cultures. The lipids of the latter contain larger proportions of saturated very long-chain fatty acids than those of the former cultures.

Inhibition of hepatic sterol and squalene biosynthesis in rats fed di-i-ethylhexyl phthalate.

Di-2-ethylhexyl phthalate (DEHP), a commonly used plasticizer, was found to be an inhibitor of the biosynthesis of hepatic nonsaponifiable lipids in the art. The addition of DEHP at levels of 0.5% or 1.0% to a stock diet of rats resulted in a decreased conversion of acetate-1-14C and mevalonate-5-3H into squalene, C30 sterols, and C27 sterols by liver minces or slices, in vitro. In studies conducted with 0.5% DEHP feeding from 2 to 11 days, the degree of inhibition was found to increase with the duration of DEHP feeding; the inhibition of 3H-mevalonate conversion to squalene and sterols developed more slowly, being reduced to ca. 70% control values in 11 days, whereas 14C-acetate conversion was reduced to ca. 35% of control values during the same period. 3H-mevalonate conversion to sterols and squalene was, however, found to be suppressable to the same extent as 14C-acetate conversion when diets containing 1.0% DEHP were fed for 18 days. The inhibitory effect of dietary DEHP on sterol and squalene biosynthesis from 14C-acetate and 3H-mevalonate by rat liver preparations is unlikely to be accounted for by the negative feedback of cholesterol secondary to hepatic sterol accumulation since, in these studies, hepatic total lipid and hepatic total sterol levels were similar in control and DEHP-fed rats.

Stimulation of hepatic squalene and triglyceride synthesis by dimethylsulfoxide, in vitro.

The incorporation of [14C]mevalonate and [14C]acetate into squalene by rat liver slices was increased over 7-fold by the presence of 5% dimethylsulfoxide (DMSO) in the incubation medium. The stimulation of squalene synthesis was dose-related over the concentration range of 1-5% DMSO and did not affect the incorporation of [14C]mevalonate into the C27-sterol fraction (cholesterol) but did increase (about 50%) incorporation into C30-sterol (lanosterol) at a level of 5% DMSO. The stimulation of squalene synthesis was observed under both anaerobic (N2 atmosphere) and aerobic (ambient air or 95% O2/5% CO2) conditions and may represent a direct effect of DMSO on squalene synthetase. At a level of 5%, DMSO also stimulated 7-fold the incorporation of [14C]acetate into triglycerides by liver slices; this occurred without changes in incorporation into the phospholipid or free fatty acid fractions. The disproportionate increase in lipid labeling from [14C]acetate suggests that the effects of DMSO are not simply a matter of increasing [14C]acetate entry into the tissue.

[In vitro biosynthesis of cholesterol and its precursors in the osseous tissue of rats]. / Issledovanie biosinteza kholesterina i ego predshestvennikov v kostnoi tkani krys in vitro

By thin-layer chromatography compounds of sterol origin were isolated from the nonsaponified fraction of bone tissue of rats. The occurrence of squalene, lanosterol, cholesterol and incompletely identified Cdelta2427-sterol in the organic matrix of the bone was demonstrated by means of UV- and IR-spectrometry. Bone tissue incubation with 1,2-C14-aceticacid yielded C14-labeled lanosterol, C28, 29-sterols, cholesterol and squalene. The rate of cholesterol synthesis in the bone tissue was lower than that in the liver and intestine of rats.

[Effect of triterpene glycosides on the biosynthesis of sterols and fatty acids by the yeast Saccharomyces carlsbergensis]. / Bliianie nekotorykh triterpenovykh glikozidov na biosintez sterinov i zhirnykh kislot drozhzhei Saccharmyces carlsvergensis.

The effect of cauloside C from Caulophyllum robustum M., theasaponine from Thea sinensis L. and stichoposide A from Stichopus japonicus S. on the biosynthesis of sterols and fatty acids by the yeast Saccharomyces carlsbergensis was studied. In in vivo and in vitro experiments cauloside C and theasaponine inhibited ergosterol biosynthesis at the stage of squalene-to-lanosterol transformation. In in vivo experiments stichoposide A inhibited squalene biosynthesis. The triterpene glycosides tested showed a typical feature: in in vivo and in vitro experiments they blocked the biosynthesis of fatty acids.