Tellurium-induced neuropathy: metabolic alterations associated with demyelination and remyelination in rat sciatic nerve.

Rats fed a diet containing 1.25% elemental tellurium initiated on postnatal day 20 undergo a transient neuropathy characterized by synchronous demyelination of peripheral nerves. In sciatic nerve, the extent of demyelination was maximal after 5 days of tellurium exposure; there was a loss of 25% of the myelin, as assayed by concentration of myelin-specific P0 protein. Tellurium-induced alterations in the metabolic capacity of Schwann cells were examined by measuring the synthesis of myelin lipids in vitro in isolated sciatic nerve segments. Exposure to tellurium resulted in an early marked decrease of approximately 50% in overall incorporation of [14C]acetate into lipids, with a preferential depression in synthesis of cerebrosides, cholesterol, and ethanolamine plasmalogens (components enriched in myelin). Most dramatically, within 1 day of initiation of tellurium exposure, there was a profound increase in [14C]acetate-derived radioactivity in squalene; 23% of incorporated label was in this intermediate of cholesterol biosynthesis, compared to less than 0.5% in controls. In association with the remyelinating phase seen after 5 days of tellurium exposure, synthesis of myelin components gradually returned to normal levels. After 30 days, metabolic and morphologic alterations were no longer apparent. We suggest that the sequence of metabolic events in sciatic nerve following tellurium treatment initially involves inhibition of the conversion of squalene to 2,3-epoxysqualene, and that this block in the cholesterol biosynthesis pathway results, either directly or indirectly, in the inhibition of the synthesis of myelin components and breakdown of myelin.

Age-related changes in the mevalonate metabolism in vivo in chick kidneys.

The mevalonate incorporation in vivo into total nonsaponifiable lipids by chick kidneys drastically increased after hatching, reaching similar levels to those previously observed in liver. Cholesterol was the major sterol formed from mevalonate from 11 days onward, while a fraction of polar nonsaponifiable lipid(s) was observed as the major compound(s) synthesized at 5-8 days. Relative percentages of squalene, squalene oxide(s) and lanosterol synthesized from mevalonate also increased between 11-18 days after hatching. Results in this paper demonstrate for the first time the accumulation of a fraction of nonsaponifiable lipid(s) identified as lanosterol derivatives and cholesterol precursors formed by kidneys from [5-14C]mevalonate in experiments carried out in vivo, as well as their evolution during postnatal period.

Purification to homogeneity and some properties of squalene synthetase.

Squalene synthetase has been purified to homogeneity from yeast. It is a single polypeptide of Mr 47,000. This enzyme catalyzes the synthesis of squalene from farnesyl diphosphate via presqualene diphosphate. In the presence of reduced pyridine nucleotides, presqualene diphosphate and squalene are produced in a ratio of 6:1 from either the purified protein or the crude microsomal fraction.

Specific inhibition of fungal sterol biosynthesis by SF 86-327, a new allylamine antimycotic agent.

SF 86-327 is a new antimycotic agent of the allylamine type. Its primary action appears to be the inhibition of ergosterol biosynthesis at the point of squalene epoxidation, as was previously found with the related compound naftifine. Biosynthesis was measured by incorporation of [14C]acetate into sterols in cells of Candida albicans, Candida parapsilosis, Torulopsis glabrata, and the dermatophyte Trichophyton mentagrophytes. There was a positive correlation between the SF 86-327 concentrations needed for inhibition of growth and of sterol synthesis in these four fungi. The greater antifungal efficacy of SF 86-327 in comparison with naftifine was also reflected in the relative activities of the two compounds as sterol synthesis inhibitors. Inhibition was maximal at neutral pH. A similar degree of inhibition was found in cell-free extracts when [14C]mevalonate was used as substrate. In all cases, inhibition of sterol synthesis was accompanied by a parallel accumulation of labeled squalene. SF 86-327 and naftifine had no significant effect on initial enzymes of the ergosterol pathway, measured by incorporation of [14C]acetyl coenzyme A, or on steps distal to squalene epoxidation, measured by conversion of labeled squalene 2,3-epoxide or lanosterol. Both allylamines were highly selective for fungal, as opposed to mammalian, sterol biosynthesis. SF 86-327 caused slight inhibition of squalene epoxidation in a rat liver cell-free system, but at concentrations three to four orders of magnitude greater than those required for inhibition of the fungal pathway.

Gorgosterol biosynthesis: localization of squalene formation in the zooxanthellar component of various gorgonians.

Four species of gorgonians: three related pseudoplexaurids Pseudoplexaura porosa, P. flagellosa and P. wagenaari; and the unrelated Pseudopterogorgia americana, are sources of zooxanthellae capable, in purified broken cell preparations, of converting [14C]labeled farnesyl pyrophosphate into squalene. More extensive studies with P. porosa and P. americana zooxanthellae preparations characterize the conversion as enzymatic and demonstrate farnesyl pyrophosphate and reduced pyridine nucleotide as substrates. NADPH and NADH are essentially equivalent. Anaerobic conditions are not required. Despite numerous attempts zooxanthellae formation of sterols, including gorgosterol, from radioactive substrates was unsuccessful. By enzyme studies, we can show only the conversion of mevalonate into squalene as a zooxanthellae capability.

Squalene, lanosterol and cholesterol synthesis from acetate in neonatal chick tissues.

Acetate incorporation into squalene, lanosterol and cholesterol by liver and kidney slices and intestinal mucosa scrapes from neonatal chick was studied. Contrary to what is observed when using mevalonate as substrate, cholesterol was the main nonsaponifiable synthesized from acetate in all the conditions assayed. Low percentages of squalene and lanosterol were synthesized by liver and kidney slices, while in intestinal mucosa squalene was practically undetectable. The highest percentage of radioactivity in cholesterol was found in liver, followed by intestinal mucosa and kidney. Relative percentages of squalene, lanosterol and cholesterol were practically similar in each tissue at any incubation time and acetate concentration considered. Only in kidney these percentages, especially in the case of squalene, seemed to decrease at higher acetate concentrations (8-12 mM).

Increased 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol biosynthesis in freshly isolated hairy cell leukemia cells.

Freshly isolated hairy cells from the peripheral blood of patients with hairy cell leukemia (HCL) synthesize 3-5-fold greater amounts of cholesterol, lanosterol, and squalene from [1-14C]-acetate than do normal human peripheral blood mononuclear cells under basal conditions of culture (i.e., in the presence of low-density lipoprotein). HCL cells also exhibit an eightfold increase in the activity of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase. These changes cannot be ascribed to increased rates of cellular proliferation in the HCL cells, nor are they a consequence of an increased rate of loss of newly synthesized cholesterol into the culture medium. The increased rate of cholesterol biosynthesis in HCL cells may result in an increase in their total cellular cholesterol content, as well as in an increase in their plasma membrane cholesterol:phospholipid molar ratio. These changes, in turn, are probably responsible for some of the clinical manifestations of this disease.

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.

The in vivo incorporation of acetate into different non-saponifiable lipids by neonatal chick tissues.

The in vivo incorporation of [l-14C]acetate into non-saponifiable lipids was higher in neonatal chick liver than in intestinal mucosa, brain and kidneys, and proportional to the amount of substrate injected (2-20 mumole). 14CO2 expired in the breath was also proportional to the dose of acetate. Radioactivity from [l-14C]acetate accumulated by liver was maximal 30 min after the injection of acetate and decreased afterwards. Acetate was mainly incorporated into cholesterol by all the tissues assayed, although small percentages of lanosterol and squalene were obtained in liver. In this tissue, distribution of radioactivity was practically independent from the dose of substrate injected while in intestinal mucosa, brain and kidneys the percentage of cholesterol increased with this dose. The time course of the in vivo formation of different non-saponifiable lipids by neonatal chick tissues was also studied. More than 90% of radioactivity in this fraction obtained 15 min after the acetate injection was recovered as cholesterol in liver and kidneys, while in brain and intestinal mucosa this percentage was about 50% at this time, increasing afterwards. A high percentage of lanosterol was found in brain and intestinal mucosa 15 min after the injection of acetate.

Incorporation of mevalonate into squalene, lanosterol and cholesterol by different neonatal chick tissues.

The role of neonatal chick liver and kidneys in the incorporation of mevalonic acid into squalene, lanosterol and cholesterol was studied. Differences between the synthesizing ability of these and other tissues and the influence of the in vivo or in vitro conditions were also examined. In the in vivo experiments, distribution of radioactivity among the nonsaponifiable lipids was not dependent of the doses of mevalonic acid injected. About 80-95% of radioactivity was recovered as cholesterol in liver and brain, whereas in kidneys this percentage was only about 35%. Squalene and lanosterol were formed by kidneys in a high percentage, higher than in liver and other tissues. 12 hr after mevalonate injection, the percentage of cholesterol formed by kidneys increased until more than 50%. In the in vitro experiments carried out in the presence of 0.045-4.0 mM mevalonate, cholesterol was also the main nonsaponifiable identified, but in a lesser percentage than in vivo. In the same conditions, the incorporation of mevalonic acid by kidneys was maximal into squalene. After in vitro incubations for 2 hr, the percentage of cholesterol in kidneys also increased.

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.

Effects of unsaturated fatty acid deprivation on neutral lipid synthesis in Saccharomyces cerevisiae.

The effects of unsaturated fatty acid deprivation on lipid synthesis in Saccharomyces cerevisiae strain GL7 were determined by following the incorporation of [14C]acetate. Compared to yeast cells grown with oleic acid, unsaturated fatty acid-deprived cells contained 200 times as much 14C label in squalene, with correspondingly less label in 2,3-oxidosqualene and 2,3;22,23-dioxidosqualene. Cells deprived of either methionine or cholesterol did not accumulate squalene, demonstrating that the effect of unsaturated fatty acid starvation on squalene oxidation was not due to an inhibition of cell growth. Cells deprived of olefinic supplements displayed additional changes in lipid metabolism: (i) an increase in 14C-labeled diacylglycerides, (ii) a decrease in 14C-labeled triacylglycerides, and (iii) increased levels of 14C-labeled decanoic and dodecanoic fatty acids. The changes in squalene oxidation and acylglyceride metabolism in unsaturated fatty acid-deprived cells were readily reversed by adding oleic acid. Pulse-chase studies demonstrated that the [14C]squalene and 14C-labeled diacylglycerides which accumulated during starvation were further metabolized when cells were resupplemented with oleic acid. These results demonstrate that unsaturated fatty acids are essential for normal lipid metabolism in yeasts.

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.

[Formation of squalene and pigment 462 by the culture of Methanobacillus kuzneceovii]. / Obrazovanie skvalena i pigmenta-462 kul'turoi Methanobacillus kuzneceovii.

Using hexane extraction, squalene, its four hydroforms and an unknown orange-yellow coloured compound termed as pigment 462 (since it has a specific absorption maximum in the visible region of the spectrum) were isolated from a thermophylic methane-producing culture of Methanobacillus kuzneceovii. Pigment 462 is rapidly reduced by sodium borohydride and is readily oxidized by air oxygen; its functional group is the keto group. The physico-chemical properties of pigment 462 indicate that the pigment is one of the components of the electron transport chain of methane-producing bacteria and is localized in the membrane.

[Activities of 3-hydroxyl-3-methylglutaryl-CoA reductase and acetyl-CoA carboxylase and the rate of mevalonic acid, squalene, sterol and fatty acid biosynthesis from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in rat liver: effects of Triton WR 1339, starvation and cholesterol diet]. / Aktivnost' 3-gidroksi-3-metilglutaril-CoA-reduktazy, atsetil-CoA-karboksilazy i skorost' biosinteza mevalonovoi kisloty, skalena, sterinov i zhirnykh kislot iz [1-14C]atsetil-CoA i [2-14C]malonil-CoA v pecheni krysy: vliianie vvedeniia tritona WR 1339 golodaniia i kormleniia kholesterinom.

The effects of Triton WR 1339, starvation and cholesterol diet on the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acetyl-CoA carboxylase and on the rates of mevalonic acid (MVA) biosynthesis from acetyl-CoA and malonyl-CoA in the soluble (140 000 g) and microsomal fractions of rat liver, on the rate of incorporation of these substrates into squalene, cholesterol and lanosterol in the rat liver postmitochondrial fraction and on the rate of fatty acid biosynthesis was studied. The administration of Triton WR 1339 (200 mg per 100 g of body weight twice) stimulated the activity of HMG-CoA reductase and MVA biosynthesis from acetyl-CoA and malonyl-CoA in the intact and solubilized microsomal fractions and had no effect on these parameters in the soluble fraction. Starvation for 36 hrs did not cause inhibition of the reductase activity or MVA biosynthesis from both substrates in the soluble fraction. Alimentary cholesterol significantly increased the activity of HMG-CoA reductase, had no effect on the rate of MVA biosynthesis from acetyl-CoA and stimulated the malonyl-CoA incorporation in to MVA in the soluble fraction. Starvation an alimentary cholesterol inhibited the HMG-CoA reductase activity and MVA biosynthesis from both substrates in the solubilized microsomal fraction. Triton WR 1339 stimulated 4--19-fold the lipid formation in the total unsaponified fraction and its components i.e. squalene, lanosterol, cholesterol, from acetyl-CoA and only insignificantly (1,2--1,7-fold) increased malonyl-CoA incorporation into these compounds. Starvation and alimentary cholesterol repressed lanosterol and cholesterol biosynthesis from acetyl-CoA, decreased malonyl-CoA incorporation into these sterols and had no influence on squalene biosynthesis from the two substrates. Triton WR 1339 and starvation inhibited the acetyl-CoA carboxylase activity, unaffected by alimentary cholesterol. No significant changes in the rate of fatty acid biosynthesis from the substrates were observed. The data obtained provide evidence for the existence of autonomic pathways of MVA biosynthesis localized in the soluble and microsomal fractions of rat liver. The pathway of MVA biosynthesis in the soluble fraction is less sensitive to regulatory factors. Sterol biosynthesis from malonyl-CoA is also more resistant to regulatory effects than sterol biosynthesis from acetyl-CoA. This suggests that HMG-CoA reductase localized in the soluble fraction takes part in MVA and sterol biosynthesis from malonyl-CoA.

[Activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acetyl-CoA carboxylase and rate of biosynthesis of mevalonic acid, squalene, sterols and fatty acids from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in rat liver: changes induced by daily rhythm]. / Aktivnost' 3-gidroksi-3-metilglutaril-CoA-reduktazy, atsetil-CoA-karboksilazy i skorost' biosinteza mevalonovoi kisloty, skvalena, sterinov i zhirnykh kislot iz [I-14C]atsetil-CoA i [2-14C]malonil-CoA v pecheni krysy: izmenenie v zavisimosti ot vremeni sutok.

The activity of 3-hydrosy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and the rate of mevalonic acid (MVA) synthesis from [I-14C]acetyl-CoA and [2-14C]malonyl-CoA in the soluble (X140000 g) and microsomal fractions of rat liver and in a reconstituted system containing the soluble and microsomal fractions were studied. The changes in the activity of HMG-CoA reductase and the rate of MVA biosynthesis in the fractions at different times of the day were analyzed. The daily rhythms of the rate of acetyl-CoA and malonyl-CoA incorporation into squalene, sterols and fatty acids in the postmitochondrial fraction and the daily changes in the acetyl-CoA carboxylase activity of the soluble fraction of rat liver were compared. The incorporation of labelled acetyl-CoA and malonyl-CoA into MVA showed that the latter can be synthesized from these two substrates both in the soluble and microsomal fractions. Malonyl-CoA is a preferable substrate for MVA synthesis in the soluble fraction. MVA synthesis from acetyl-CoA proceeds fastr in the intact and solubilized microsomes than in the soluble fraction. The activity of HMG-CoA reductase was found in the soluble and microsomal fractions in practically equal amounts. The enzyme activity was increased in the microsomal fraction after its solubilization. The rate of MVA biosynthesis from acetyl-CoA and the activity of HMG-CoA reductase in the soluble fraction are practically unaffected by day-to-night changes. The activity of HMG-CoA reductase and MVA biosynthesis from acetyl-CoA in the intact and solubilized microsomal fractions reached their maximal values in the middle of the dark period. The rate of MVA biosynthesis from malonyl-CoA was decreased in the middle of the dark period in all fractions studied and reached its maximum in the middle of the light period. The daily rhythms of the acetyl-CoA carboxylase activity in the soluble fraction and the rate of MVA biosynthesis from malonyl-CoA in all fractions show a coincidence. a comparison of incorporation by the postmitochondrial fractions of acetyl-CoA and malonyl-CoA into the total non-saponified lipid fraction and its components, e. g. squalene, lanosterol and cholesterol, as well as into sterols precipitated by digitonin, showed that malonyl-CoA incorporation into the total non-saponified lipid fraction was more intensive than that of acetyl-CoA. However, acetyl-CoA was far more efficiently incorporated into sterols precipitated by digitonin or isolated by TLC than malonyl-CoA. The rate of acetyl-CoA incorporation into the total non-saponified lipid fraction and into squalene, lanosterol and cholesterol was maximal in the middle of the dark period and minimal in the middle of the light period. On the contrary, the rate of malonyl-CoA incorporation into these products was minimal in the middle of the dark period and maximal in the middle of the light period. The rate of fatty acid biosynthesis from acetyl-CoA was increased in the middle of the light and dark periods...

Squalene and sterol synthesis in isolated small-intestinal cells of the rat.

To compare the synthesis rate of cholesterol in different cells of the small intestine, isolated villous and crypt cells were incubated with a mixture of 14C-acetate and 3H-mevalonate in the presence of unlabeled carriers. The synthesis rate of squalene (includes the portion converted to sterols) from acetate was tenfold higher in the crypt than villous cells. The synthesis rate of squalene from mevalonate and the cyclization rate of squalene (the portion found in sterols) were about twofold higher in crypt than in villous cells. The conversion of acetate to squalene was correlated with that to fatty acids in the crypt cells only (r = 0.823), and the ratio of the two synthesis rates (squalene/fatty acids) was threefold higher in crypt than in villous cells. Despite the significant differences in the synthesis rates of squalene and sterols the concentrations of squalene and methyl sterols were similar in the two cell types. THe cholesterol content was, however, consistently higher in villous than in crypt cells, but the concentration was not correlated with the synthesis of squalene in the two cell types. The appearance of labeled squalene was clearly lower than that of labeled sterols in the lipoprotein-free incubation medium, but no differences were found between villous and crypt cells.