Inhibition of Hepatic Cholesterol Synthesis
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Proc. Nati Acad. Sci. USA Vol. 79, pp. 4873-4877, August 1982 Biochemistry Inhibition of hepatic cholesterol synthesis- in mice by sterols with shortened and stereochemically varied side chains [feedback regulation/3-hydroxy-3-methylglutaryl-coenzyme A reductase/mevalonic acid/(E)- and (Z)-17(20)-dehydrocholesterol/ pregn-5-en-3.&ol] KATHERINE A. ERICKSON* AND WILLIAM R. NESt Department ofBiological Sciences, Drexel University, Philadelphia, Pennsylvania 19104 Communicated by E. H. Ahrens, Jr., May 5, 1982 ABSTRACT Micewere fedcholesterol orvarious other sterols chain found in cholesterol. The literature provided no guidance for 26 hr, after which the amount ofhepatic cholesterol synthesis on the effect that this changewould have. The only steroids with was measured in a cell-free system. The following sterols were as shortened side chains previously examined (hormones and bile effective as cholesterol itself in, depressing the conversion of ace- acids) have had oxygen atoms on or in place of the side chain tate into sterol: pregn-5.en-3.&ol, which lacks an isohexyl group (10-12). Polar groups, ofcourse, would be expected to have a on C-20; (E)-17(2G)-dehydrocholesterol, in which the isohexyl marked effect in their own-right. In addition, many ofthe com- group is fixed to the right; (E)-20(22)-dehydrocholesterol, in which pounds-e.g., bile acids and testosterone-also had- changes C-23 is oriented away from the nucleus; and 20-epicholesterol. in the In order to make an exact comparison with cho- Moreover, when the isohexyl group was fixed to the left in (Z)- nucleus. 17(20)-dehydrocholesterol, this dietary sterol, identified in the lesterol we used the 3,(3hydroxy-A5-sterols (androst-5-en-3/-ol liver, causednotonly a depression in the conversion ofacetate into and pregn-5-en-3(-ol), which had no other polar groups and sterols but also-a depression in the conversion of both mevalonate zeroandtwocarbon atoms on C-17, respectively. Both inhibited and squalene into sterols. The incorporation of acetate into fatty hepatic sterol synthesis. Details for the pregnenol are given acids was not depressed, nor did the (Z)-sterol appear to have a here, and the results with the androstenol are given elsewhere generalized effect on membranous enzymes,, because the activity (13). of glucose-6-phosphatase was unaffected. Thus, feedback inhibi- tion was retained when .the stereochemistry of cholesterol's side MATERIALS AND METHODS chain was drastically changed and even after the nearly complete Chemicals. [1-4C]Acetic acid (sodium salt, 45-60 mCi/ removal of the side chain. This implies that the side chain is only mmol) and DL-[2-14C]mevalonic acid (dibenzylethylenedi- minimally recognized by the mechanisms involved in. feedback amine salt, 47 mCi/mmol) were obtained from New England inhibition. Nuclear and [11J4C]squalene (50-55 mCi/mmol) was from Research Products International (Mt. Prospect, IL) (1 Ci = 3.7 The influence of the size, shape, and polarity of sterols on the x 10.1 becquerels). The disodium salt ofglucose 6-phosphate, biological function ofthese compounds has been the subject of NADPH, NAD, nicotinamide, glutathione, and sodium caco- many investigations. Among them have been studies with fungi dylate were from Sigma. The glucose oxidase enzyme prepa- and protozoa on the significance ofchirality at C-20 and of the ration was from Worthington Diagnostics. Cholesterol was ob- spatial orientation of C-22 on the ability of sterols to promote tained from Baker, and a mixture called "lanosterol" from Mann growth of anaerobic Saccharomyces cerevisiae (1), to induce Research Laboratories (New York). The "lanosterol" was com- oospore formation in Phytophthora cactorum (2, 3), and to be posed- of roughly equal amounts of lanosterol and 24-dihydro- dehydrogenated by Tetrahymena pyriformis (4, 5). In all three lanosterol asjudged by gas/liquid chromatography; presumably of these cases the natural (20R) configuration (6-9) was found there was also a few percent each ofagnosterol [lanost-7,9(11), to be essential. Similarly obligatory was the ability ofthe sterol, 24-trien-3,(3ol] and 24-dihydroagnosterol (14). Pregn-5-en-3f- to have C-22 oriented to the right in the usual view of the mol- ol was prepared synthetically in this laboratory from pregnen- ecule (C-18 and C-19 toward the observer and ring Ato the left) olone by John M. Joseph by a modification (reduction of the and simultaneously to have no bulk larger than a H atom in tosylhydrazone with sodium borohydride) of the reported pro- front.- Orientation of C-22 to the right is known to occur in the cedure (15). (Z)-17(20)-, (E)-17(20)-, and (E)-20(22)-dehydro- crystalline state (6, 7). A reason why this should be the more cholesterol and 20-epicholesterol (also called 20-isocholesterol) stable form has been suggested (4) and correlated (4, 5) with the were prepared as described in the literature (16, 17). Their biological properties (1-5). structures are shown in Fig. 1. In the present investigation we have examined this stereo- Animals. Male C57BL/6J mice (Jackson Laboratory) 8 weeks chemical problem in mammals. The functional parameter used old at delivery were used at ages 11-15 weeks. Maintained on for quantitation was inhibition of hepatic cholesterol synthesis an automatically timed light schedule (lights on, 0600; off, 1800) brought about by short-term feedback from sterol in the diet they were allowed food and water ad lib. The diet was pelleted of mice. Because, unlike the results with the single-celled or- cholesterol-free test diet from United States Biochemical ganisms, we found biological activity required neither the (20R) (Cleveland, OH) supplemented with 5% safflower oil. This diet configuration nor the ability of C-22 to lie on the right, it ap- was composed of 6% cellulose flour, 4% salt mixture, 65% su- peared that the receptors involved in feedback did not recognize crose, 25% alcohol-extracted casein, and vitamin supplemen- the cholesterol side chain. In order to explore this further, we tation. The diet also contained a sterol with the same retention extended our studies to sterols with side chains smaller than the Abbreviation: NLF, neutral lipid fraction after saponification. The publication costs ofthis article were defrayed in part bypage charge * Present address: Dept. of Medical Technology, Thomas Jefferson payment. This article must therefore be hereby marked "advertise- University, Philadelphia, PA 19107. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 4873 Downloaded by guest on September 25, 2021 4874 -Biochemistry: Erickson and Nes Proc. Nad Acad. Sci. USA 79 (1982) time in gas liquid chromatography as cholesterol at a concen- methyl-, monomethyl-, and dimethylsterol fractions expressed tration ofonly 12.0 ,g ofsterol per g offood. One week before as percent ofcontrol. Typically, homogenates from control mice mice were used in experiments, the food pellets were replaced converted labeled substrates as follows (mean ± SEM): in nine by the same diet in powdered form. Sterols were-added to the experiments, [1-14C]acetate into sterols, 22.1 ± 1.5 nmol/g of powdered diet in a large volume of diethyl ether, and the sol- liver per 2hr; in six experiments, [1-'4C]acetate into fatty acids, vent was removed by stirring at room temperature. Each animal 69.9 ± 9.3 nmol/g of liver per 2 hr; in six experiments, [1- was allowed access to the sterol-enriched diet for 26 hr before 14C]mevalonate into sterols, 853.2 ± 0.1 nmol/g of liver per sacrifice. All mice received the same size portion offood (usual- 2 hr; and in six experiments, [11-14C]squalene into sterols, 44.8 ly 5 g), and the amount offood consumed during the 26-hr pe- + 3.0 nmol/g of liver per 2 hr. riod was estimated by weighing that which remained (Table 1). FattyAcids. The incorporation of ['4C]acetate intofatty acids Preparation of Homogenates and Incubation with Labeled was estimated by the method described by Kandutsch and Substrates. Mice were sacrificed between 1130 and 1200 be- Packie (18). cause Kandutsch and Packie (18) reported the rate ofsterol syn- Isopentenoid Pyrophosphates. This fraction contained the thesis in C57BL/6J mice to be high in the morning hours and A2- and A3-isopentenyl, geranyl, and farnesyl pyrophosphates. relatively constant from early morning until approximately Liver homogenates were incubated with [2- 4C]mevalonic acid 2000. Liver homogenates were prepared as described by as described. After extraction ofnonsaponifiable lipids, isopen- Bucher and McGarrahan (19). Homogenates were centrifuged tenoid pyrophosphates in the aqueous phase were converted at 800 x g for 10 min at 0-5°C. Each incubation flask contained to free alcohols by acid hydrolysis as described by Goodman and 3 ml of the liver homogenate supernatant derived from 1.4 g Popjak (20). In the presence ofcarrier geraniol and farnesol (4 of liver, 1.6 mM NAD, 16 mM glucose 6-phosphate, 1 mM mg), the alcohols (pH 10) were extracted into ether. The ether NADPH, and labeled substrate: [1-'4C]acetate (12 ,uCi, 7 extracts were evaporated to a measured volume and radioactiv- emol), DL-[2-'4C]mevalonate (1.0 ,uCi, 7.5 ,umol), or [11- ity was quantitated by liquid scintillation counting. 4C]squalene (0.7 ,uCi, 0.3 ,umol). Cofactors and water-soluble Glucose-6-Phosphatase Assay. The microsomal fractions of substrates were added to the liver homogenate supernatant in liver homogenates were suspended in 0.1 M sodium cacodylate 0.5 ml of Bucher's medium. Squalene was suspended in phos- buffer (10 ,ul/mg ofmicrosomes, pH 6.5) and a glucose-6-phos- phate buffer with the aid of Tween 80 at a Tween 80-to-squa- phatase assay (21) was performed.