Squalene, cholesterol metabolism

Y Hidaka, H Hotta, Y Nagata, Y Iwasawa, M Horie, T Kamei. Effect of a novel squalene epoxidase inhibitor, NB-598, on the regulation of cholesterol metabolism in Hep G2 cells. J Biol Chem 266 13171-13177, 1991. 

Insect steroid metabolism has two biochemically distinctive components dealkylation of phytosterols to cholesterol and polyhydroxylation of cholesterol to ecdysone. We will focus on the first of these. Lacking the ability to synthesize sterols de novo, insects instead have evolved a dealkylation pathway to convert plant sterols to cholesterol(7-10). The dealkylation pathways are apparently absent in most other higher and lower organisms, which can convert mevalonate to squalene and thence into sterols( ). Specific insecticides are possible based on these biochemical differences. 

From activated isoprene, the metabolic pathway leads via dimerization to activated geraniol (1 = 2) and then to activated farnesol = 3). At this point, the pathway divides into two. Further extension of farnesol leads to chains with increasing numbers of isoprene units—e.g., phytol (1 = 4), dolichol (1 = 14-24), and rubber = 700-5000). The other pathway involves a head-to-head linkage between two farnesol residues, giving rise to squalene (1 = 6), which, in turn, is converted to cholesterol (1 = 6) and the other steroids. 

Squalene takes part in metabolism as precursor for synthesis of steroids and structurally quite similar to (3-carotene, coenzyme qlO, vitamins Ki, E, and D. The squalene in skin and fat tissue comes from endogenous cholesterol synthesis as well as dietary resources in people who consume high amounts of olive and fish oil especially shark liver (Gershbein and Singh, 1969). Squalene is synthesized by squalene synthase which converts two units of farnesyl pyrophosphate, direct precursor for terpenes and steroids, into squalene. As a secosteroid, vitamin D biosynthesis is also regulated by squalene. Moreover, being precursor for each steroid family makes squalene a crucial component of the body. 

Although sterols like cholesterol are not synthesized de novo by parasitic flatworms, they do possess an active mevalonate pathway (Fig. 20.3) (reviewed in Coppens and Courtoy, 1996). This pathway has been studied in 5. mansoni, and all available evidence indicates that it is similar to the lipid metabolism seen in F. hepatica. The mevalonate pathway was shown to be used by 5. mansoni for the synthesis of dolichols for protein glycosylation, of quinones as electron transporters in the respiratory chain and of farnesyl and geranylgeranyl pyrophosphates as substrates for the isopreny-lation of proteins (Chen and Bennett, 1993 Foster et a/., 1993). A key enzyme in the mevalonate pathway is 3-hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) and it was shown that the schistosomal enzyme differs from the mammalian type, both structurally and in its regulatory properties (Rajkovic et ai, 1989 Chen et at., 1991). Farnesyl pyrophosphate plays a key role in the mevalonate pathway as it is the last common substrate for the synthesis of all end products (Fig. 20.3). As mentioned already, the branch leading from farnesyl pyrophosphate via squalene to cholesterol is not operative in parasitic flatworms, whereas the other branches are active, at least in S. mansoni and probably also in F. hepatica and FI. diminuta. 

Further work in the phylum Echinodermata shows a variable ability to biosynthesize steroids. In the class Holothuroidea and Echinoidea, the representatives examined could synthesize squalene but not triterpenoids or sterols from acetate. However, several examples from the class Asteroidea were able to synthesize squalene, lanosterol, and other steroids. In the later stages of steroid metabolism it was shown that cholesterol was converted into cholest-7-enol via cholestanol. 

Variation within the phylum Mollusca has been noted before. More species from the order Mesogastropoda again show that squalene and sterols can be synthesized. Although previous work with the class Bivalvia was unable to demonstrate the ability to synthesize steroids, androstane derivatives were metabolized.Detailed studies with the slug Ariolimax (order Stylommato-phora) showed how cholesterol was metabolized to pregnenolone, 17a-hydroxy-pregnenolone, and thus to androstane derivatives. 

Relatively little is known about plant sterols. (Most of the research effort in steroid metabolism has been expended in the investigation of steroid-related human diseases.) It appears, however, that the initial phase of plant sterol synthesis is very similar to that of cholesterol synthesis with the following exception. In plants and algae the cyclization of squalene-2,3-epoxide leads to the synthesis of cycloartenol (Figure 12.30) instead of lanosterol. Many subsequent reactions in plant sterol pathways involve SAM-mediated methylation reactions. There appear to be two separate isoprenoid biosynthetic pathways in plant cells the ER/cyto-plasm pathway and a separate chloroplast pathway. The roles of these pathways in plant isoprenoid metabolism are still unclear. 

A group of fungicides that inhibit squalene epoxidation has been developed primarily for use against pathogenic fungi in medicine. Epoxidation of squalene is catalyzed by squalene epoxidase (a flavoprotein) that starts the complicated cyclization of squalene. The squalene-2,3-epoxide formed by this enzyme is further metabolized to a protosterol cation intermediate, which is transformed to either cycloartenol in plants (cycloartenol synthase) or lanosterol (lanosterol synthase). Cycloartenol is the precursor to plant sterols, whereas lanosterol is the precursor of cholesterol and the other sterols in animals, and to ergosterol in plants. 

Squalene. Squalene is a highly unsaturated aliphatic hydrocarbon (C30H50) with important biological properties. It is a metabolic precursor of cholesterol. A rich source of squalene is shark liver oil. Squalene is also present in human tissue in small amounts and in some vegetable oils. 

The resolution of synthetic presqualene and prephytoene alcohols via their etienic acid derivatives has been reported. This work confirmed that the active (-f-)-enantiomers in both series have the same absolute configuration [(li , 2/ , 3/ )]. It has been established, by use of Hn.m.r., that the proton (deuteron) introduced at C-3 during the cyclization of squalene to tetrahymanol by Tetrahymena pyriformis has the 3/8 configuration. Both antipodes of the trimethyldecalol (13) have been shown to be effective inhibitors of cholesterol biosynthesis in rat liver enzyme preparations and cultured mammalian cells. The accumulation of squalene 2,3-oxide and squalene 2,3 22,23-dioxide in the treated systems indicates that inhibition occurs at the cyclization stage. The inhibitor is metabolized to the diol (14). The results of other sterol inhibition... 

Miettinen, T.A. and Vanhanen, H., Serum concentration and metabolism of cholesterol during rapeseed oil and squalene feeding. Am. J. Clin. Nutr, 59, 356-363, 1994. 

Nicholas (1961) has shown, however, that suitably fortified minced rat brain preparations from 1-year-old animals will utilize 2-G -acetate and mevalonate to form labeled nonsaponifiable material. After incubation G -squalene and G -cholesterol were isolated from the preparations, by precipitation as digitonide and conversion to the dibromide, and thus a definite conversion by adult rat brain was established. As expected, liver was much more effective in synthesizing cholesterol, but both adult brain and liver slices utilized mevalonate more efiSciently than acetate. This therefore indicates an interesting difference in the cerebral metabolism of... 

It is interesting that H and C are incorporated from L-[methyl- H, C]-methionine into cholesterol and 5a-cholest-7-en-3P-ol in normal and tumorous rats. The exact mechanism of this incorporation is obscure at present. There has been an increased interest in the biosynthesis and metabolism of cholesterol in brain tissue. This area has also been reviewed recently. The primary pathway of sterol biosynthesis in adult rat brain seems to be via A -intermediates. It is interesting that the conversion of squalene into sterols by microsomal fractions from brains of immature rats requires the lOOCKX) X g supernatant fraction from liver, the corresponding supernatant fraction from brain being inactive. ... 

Metabolism of Sterols. It is interesting that a heat-stable sterol carrier protein (SCP) has been detected in the protozoan Tetrahymena pyriformis. This protozoan-SCP (P-SCP) was required, in addition to oxygen and pyridine nucleotides, for conversion of cholesterol into cholesta-5,7,22-trien-3p-ol by the protozoan microsomal enzymes (A - and A -dehydrogenase). It is interresting that both protozoan-SCP and liver-SCP are interchangeable in cholesterol biosynthesis by liver enzymes and the oxidation of cholesterol to the triene by protozoan enzymes. The effect of numerous hypocholesteraemic compounds on the cyclization of squalene to the pentacyclic triterpenoid tetrahymanol has also been studied in Tetrahymena. ... 

By incubation or perfusion of placental tissue Math acetate, this precursor is transformed into squalene, lanosterol, and cholesterol (Levitz et al., 1962, 1964 Van Leusden and Villee, 1965 C. A. Villee, 1967, 1969). On the other hand, placental perfusion with mevalonate results in the formation of squalene and lanosterol, but not of cholesterol (Tjcvitz et al., 1962). However, the in vi(7 o conversion of both acetate and mevalonate to cholesterol was found by Zelen-ski and Villee (1966) using a preparation of minced human term placenta. Tliese authors suggest that the formation of cholesterol from these precursors is through different metabolic pathways. 

A second important anabolic pathway of acetyl-CoA produces the isoprenoid lipids, especially the steroids. Three molecules of acetyl-CoA condense at first to form a branched-chain compound, hydroxymethylglutaryl-CoA. With a superabundance of acetyl-CoA, such as occurs in some pathologic metabolic conditions (like diabetes, cf. Chapt. XX-10), acetoacetate can be formed from hydroxymethyl-glutaryl-CoA (ketogenesis). But normally, the reduction of the thioester group of that compound yields mevalonate which is then converted to isopentenyl pyrophosphate with an expenditure of 3 moles of ATP. The subsequent synthesis of squalene and cholesterol does not require any further energy supply. 

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