Inhibition of Hepatic Cholesterol Synthesis

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 inhibition 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-phosphate buffer with the aid of Tween 80 at a Tween 80-to-squa- phatase assay (21) was performed. Glucose 6-phosphate hydrol- lene ratio of 80:1 (wt/wt) before addition to the incubation ysis was calculated as the difference between the amount of mixture. All samples were incubated in a water bath shaker at glucose present in incubated samples and the amount in boiled- 37°C for 2 hr with shaking at medium intensity. The incubation enzyme blanks. Results are expressed as ,umol of glucose pro- was stopped by pouring the mixture into 10 ml of 10% (wt/wt) duced per mg ofprotein. Protein in the microsomal suspensions potassium hydroxide in methanol. was determined according to the method of Lowry et aL (22). Isolation and Quantitation of Labeled Sterols. Three milli- grams each ofcarrier cholesterol and "lanosterol" was added to RESULTS each sample. The samples were then saponified and extracted with diethyl ether. The resulting neutral lipid fraction (NLF) Dietary Standards. A dose-response relationship was estab- was chromatographed on thin-layer plates ofsilica gel 60 G de- lished between dietary cholesterol and the incorporation ofla- veloped in benzene/diethyl ether, 9: 1 (vol/vol). Bands with belfrom [1-14C]acetate into sterols by mouse liverhomogenates the same rate of movement as cholesterol (4-desmethylsterol) (Table 1). The conversion ofacetate into sterols declined sharply and "lanosterol" (4,4,14-trimethylsterol) were made visible with as the amount ofcholesterol in the diet was increased from 0% iodine vapor. These regions together with the one in between (control, taken as 100%) to 0.5% (18% of control), after which (4-monomethylsterols) were scraped from the plate and eluted no furtherchange was observed. The commercial mixture called directly into toluene/Omnifluor. Radioactivity was determined "lanosterol" was administered as a negative control. Our results with a 720 series Nuclear Chicago liquid scintillation spectrom- agreed with those of Langdon and Bloch (23), who reported that eter. Results given are the total of the radioactivity in the des- dietary "lanosterol" did not depress sterol synthesis from acetate in rat liver preparations. The data listed in Table 1 show that cholesterol in the diet (0.25%) did not depress the incor- H H R ., poration of acetate into fatty acids significantly below control values. Furthermore, liver homogenates from cholesterol-fed mice had the same capacity as homogenates from control mice to incorporate label from [2-"'C]mevalonic acid into sterols, and CHOLESTEROL 20-EPICHOLESTEROL (E)-20(22)-DEHYDRO- there was only a slight depression in the ability of the homog- CHOLESTEROL enates to permit label from [11-'4C]squalene to pass to sterols. oR R The observed effects ofcholesterol feeding are in general agree- ment with values given in the literature for rats (24-26). Dietary Sterols with Altered Stereochemistry in the Side Chain. Table 1 shows that liver homogenates prepared from PREGN-5-EN-3 -OL (E)-17(20)-DEHYDRO- (Z)-17(20)-DEHYDRO- mice fed (E)-17(20)-dehydrocholesterol (Fig. 1) displayed ap- CHOLESTEROL CHOLESTEROL proximately the same extent of depression in sterol synthesis from acetate as did homogenates from mice fed cholesterol. As =R= with cholesterol, (E)-17(20)-dehydrocholes'terol did not bring about a significant depression in the conversion of mevalonic acid to sterols. Similarly, the incorporation ofacetate into sterols was strongly depressed in liver preparations from mice fed the FIG. 1. Partial structures of sterols used in this work. The re- sterol with unsaturation in the 20(22) position (Fig. 1). At 0.5% mainder of the structures is in all cases the same and is that found in ofthe diet, (E)-20(22)-dehydrocholesterol actually appeared to cholesterol. be somewhat more effective than cholesterol. However, the Downloaded by guest on September 25, 2021 Biochemistry: Erickson. and Nes Proc. NatL Acad. Sci. USA 79 (1982) 4875 Table 1. Effect of dietary test sterols on the hepatic conversion of labeled substrates to sterols and fatty acids [14C]Mevalonate [14CI~cetate converted to ['4C]Squalene Food [ CIAcetate converted to converted to converted to % of diet eaten, Sterols, Fatty acids, sterols, sterols, Sterol in diet (wt/wt) g/mouse % of control % of control % of control % of control None - 4.2 ± 0:2 100 (24) 100 (4) 100 (4) 100 (6) Cholesterol 0.125 4.5 ± 0.2 57.5 ± 6.7 (8) 0.25 4.3 ± 0.3 27.5 ± 4.8 (8) 93.1 + 11.8 (6) 101.7 + 4.3 (4) 84.6 ± 5.1 (6) 0.50 3.9 ± 0.2 17.6 ± 2.4 (7) 1.0 4.1 ± 0.2 16.6 ± 4.1 (6) 2.0 3.6 ± 0.1 18.3 ± 2.9 (7) 5.0 4.0 ± 0.3 16.2 ± 1.9 (3) - Lanosterol 0.50 4.4 ± 0.2 97.0 ± 7.6 (5) Pregn-5-en-30-ol 0.25 3.8 ± 0.3 27.4 ± 3.1 (4) 99.7 (2) 99.5 ± 1.3 (4) 0.50 3.2 ± 0.1 20.2 (2) 89.3 (2) - (E)-17(20)-Dehydro- cholesterol 0.25 3.4 ± 0.1 25.0 ± 3.4 (8) 99.4 ± 7.3 (8) 91.3 ± 2.9 (6) (E)-20(22)-Dehydro- 0.25 4.6 ± 0.2 17.7 ± 2.5 (6) 100.3 ± 1.5 (4) cholesterol 0.50 3.7 ± 0.3 10.7 ± 1.1 (6) (Z)-17(20)-Dehydro- 0.25 3.5 ± 0.1 7.6 ± 1.0 (12) 92.6 ± 5.6 (6) 38.3 ± 2.0 (11) 31.4 + 2.9 (5) cholesterol 0.50 3.2 ± 0.3 4.8 ± 0.8 (5) 20-Epicholesterol 0.25 4.1 ± 0.4 25.3 ± 2.8 (9) 106.7 ± 13.5-(8) 91.2 ± 1.8 (5) 0.50 4.3 ± 0.1 35.4 ± 4.1 (6) Mice were fed the sterol-enriched diets for 26 hr. Results are expressed as percent of control (no dietary sterol). A set of controls was run with each sterol tested. Values represent the mean ± SEM. Numbers in parenthesis are the numbers of mice used. observed differences in potency between cholesterol and the as at a site (or sites) subsequent to mevalonate in the pathway. A2'(2)-sterol are not highly significant as determined by a one- The data listed in Table 2 reflect the fate ofmevalonate in liver sided t test (0.25% = 0.05 < P < 0.10; 0.5% = 0.02 < P < 0.05). homogenates from control and (Z)-17(20)-dehydrocholesterol- The results listed in Table 1 also show that liver homogenates fed mice. In homogenates from mice fed the (Z)-sterol, the con- from mice fed the Aww-sterol had the same capacity to convert version ofmevalonate into substances found in the entire NLF mevalonate into sterols as did control mice. Neither (E)-17(20)- after saponification was depressed to41% ofcontrol. In the same nor (E)-20(22)-dehydrocholesterol depressed the incorporation experiments, mevalonate metabolism to sterols was depressed of acetate into fatty acids. to 36% of control. Because mevalonate conversion into the (Z)-17(Z0)-Dehydrocholesterol (Fig. 1) also reduced the con- whole NLF was reduced by approximately the same extent as version of acetate into sterols by liver homogenates. At equiv- conversion to sterols, (Z)-17(20)-dehydrocholesterol apparently alent dietary amounts, the (Z)-A'7(0)-sterol actually depressed did not cause a major buildup oflabel in squalene or squalene hepatic synthesis more effectively than did cholesterol. The oxide. Thus, an important postmevalonate site (or sites) of in- observed difference was found to be highly significant (one- hibition must lie at, or prior to, the synthesis of squalene. Ex- sided t test, P << 0.001 at each of two dose levels). Whether amination of the NLFs by thin-layer chromatography verified this increased activity is the result of enhanced absorption, al- that there was no substantial accumulation oflabel in fractions tered metabolism, or greater intrinsic activity ofthe sterol itself that cochromatographed with squalene in samples prepared in the liver remains to be elucidated. However, as described from (Z)-17(20)-dehydrocholesterol-fed mice. in the following sections, we did examine other aspects of the The recovery of label in the isopentenoid pyrophosphate activity of this sterol. fiction (Table 2) indicates a rate of conversion of mevalonate- The conversion of acetate into sterols by homogenates pre- to these compounds that was similar in control (0.16 limol/g pared from mice fed 20-epicholesterol (Fig. 1) at 0.25% of the ofliver per 2 hr) and in (Z)-17(20)-dehydrocholesterol-fed mice diet was 25% ofcontrol. When the sterol was 0.5% ofthe diet, (0.22 Aumol/g of liver per 2 hr). The major effect that the (Z)- a depression in sterol synthesis from acetate was still observed sterol feeding had on the sterol biosynthetic enzymes that lie but itwas less pronounced (35.4% ofcontrol) than that observed distal to mevalonate on the pathway can be seen by a consid- at-the lower dose. This trend is somewhat inconsistent, because eration of the total flux of label from [14C]mevalonate into two greater depression would have been expected. However, the fractions: the NLF and the isopentenoid pyrophosphates, the results were reproducible. sum ofwhich includes all metabolites past mevalonic acid py- Regulatory Sites Affected by Dietary (Z)-17(20)-Dehydro- rophosphate on the sterol pathway. The results from control cholesterol. Results obtained by incubating liver homogenates mice showed a total of 1.09 ;Lmol ofmevalonate converted per from (Z)-17(20)-dehydrocholesterol-fed mice with various sub- g ofliver. For (Z)-17(20)-dehydrocholesterol-fed mice, the total strates, [14C]acetate, [14C]mevalonic acid, and ['4C]squalene was 0.60 imol of mevalonate converted per g of liver. Thus, (Table 1) indicate that the (Z)-sterol depressed sterol synthesis liver homogenates from (Z)-17(20)-dehydrocholesterol-fed mice at a number ofenzymatic sites in the pathway. The conversion had only a 55% capacity (as compared to controls) to convert ofboth mevalonate and squalene to sterols was reduced to about mevalonate into the total measured metabolites-i.e., products a third (38 and 31%, respectively) of control values, and the that lie after mevalonic acid pyrophosphate in the pathway. The conversion of acetate was almost completely depressed (8% of fate ofthe excess mevalonate is not known. However, it is prob- control). Because the depression in sterol synthesis from mev- able that the total utilization ofmevalonate is depressed due to alonate is insufficient to account for the total inhibition ob- an effect on one or more ofthe enzymes that convert it to the served when acetate was the substrate, the (Z)-sterol apparently isopentenoid pyrophosphates. The suggestion that one or more acted at a site (or sites) between acetate and mevalonate as well ofthese enzymes participates in the overall regulation ofsterol Downloaded by guest on September 25, 2021 4876 Biochemistry: Erickson and Nes Proc. Natl. Acad. Sci., USA 79 (1982) Table 2. Mevalonate metabolism by liver homogenates from (Z)-17(20)-dehydrocholesterol-fed mice Total flux of 1'4C]Mevalonate totr"~ecaovaere [14C]Mevalonate to sterols [14C]Mevalonate to NLF isopentenoid pyrophosphates metabolites, tumol/g liver % of Iumol/g liver % of ,umol/g liver % of pmol/g liver Sterol in diet per 2 hr control per 2 hr control per 2 hr control per 2 hr None 0.64 ± 0;03 (4) - 0.93 ± 0.03 (4) 0.16 ± 0.03 (4) 1.09 (Z)-17(20)-Dehydro- cholesterol (0;25%) .0.23 ± 0.02 (5) 35.9 0.38 ± 0.03 (5) 40.9 0.22 ± 0.02 (5) 137.5 0.60 Mice were fed control diet or 0.25% (Z)-17(20)-dehydrocholesterol-enriched diet for 26 hr. Data are presented as the mean ± SEM with values in parenthesis indicating the numbers of -mice used.

biosynthesis is not without precedent in the literature (24, 27, ative retention-time as authentic (Z)-sterol mixed with a larger 28). amount of -cholesterol, amounted to 5% of.the second peak, It should be noted that, in some experiments, the (Z)-17(20)- which was derived from cholesterol. In the absence of the (Z)- dehydrocholesterol-fed mice ate approximately 35% less food sterol in the diet, the first peak was missing. These results sug- than control mice did. Fasting (24, 25, 29) and reduced food gest that the dietary (Z)-sterol is absorbed from the intestine and consumption (30) are known to depress the activities of many reaches the liver, but further work is required to define this of the hepatic enzymes involved in sterol synthesis. However, point. when the effect ofa 35% reduction in food intake was examined Dietary Sterol with a Shortened Side Chain. As shown in ,alone-i.e., in the absence of sterol from the diet-the incor- Table 1, liver homogenates from mice fed the C21 sterol pregn- poration ofmevalonate and squalene into sterols was depressed 5-en-39-ol converted acetate into sterols at approximately the by only 12.8% and 18.7%, respectively, compared to control same rate as did homogenates from cholesterol-fed mice. At mice fed ad lib. 0.25% ofthe diet, cholesterol and the C21 sterol depressed he- Glucose-6-Phosphatase Determination. The microsomal en- patic sterol synthesis from acetate to approximately 28% and zyme glucose-6-phosphatase was assayed in preparations from 27% ofcontrol, respectively. As with cholesterol, the C21 sterol control and (Z)-17(20)-dehydrocholesterol-fed mice. The results did not depress the conversion of mevalonate into sterols. are listed in Table 3. At 0.25% ofthe diet, the (Z)-sterol did not depress the activity ofglucose-6-phosphatase below control values. When the amount of the (Z)-sterol was increased to 0.5% DISCUSSION some reduction in activity (23%) was detected, which was sta- The work reported here implies that, unlike what has been tistically significant (0.002 < P < 0.005) and may reflect a slight found for a variety of other biological functions of sterols (13, perturbation of the enzyme. Nevertheless, this reduction in 31-37), recognition of the side chain is minimal in the various activity was small compared to the effect that the (Z)-sterol had processes (absorption, transport, etc.) that govern feedback on sterol synthesis from acetate (greater than 90% depression). inhibition. No decrease in glucose-6-phosphatase activity was observed The entire isohexyl portion of the side chain could be re- when cholesterol was fed as 0.5% of the diet. moved with retention of the same kind of activity, both quali- Presence of (Z)-17(20)-Dehydrocholesterol in Liver Prepa- tatively and quantitatively, as found with cholesterol. The fail- rations. After the feeding of(Z)-17(20)-dehydrocholesterol, this ure of the isohexyl part of the side chain to be recognized was sterol was identified by gas/liquid chromatography in the livers paralleled by our experiments with stereochemical phenomena. of the mice. The NLF of livers from mice fed the (Z)-sterol The ability to depress hepatic sterol synthesis was retained showed two sterol peaks at retention times relative to choles- without regard to whether C-22 was fixed to the right in (E)- terol of 0.92 and 1.00. The first peak, which had the same rel- 17(20)-dehydrocholesterol or to the left in (Z)-17(20)-dehydrocholesterol. Activity was also retained when C-23 was fixed rig- Table 3. Hepatic glucose-6-phosphatase activity after feeding of idly away from the nucleus in (E)-20(22)-dehydrocholesterol as (Z)-17(20)-dehydrocholesterol well as when the configuration at C-20 was inverted in 20-epicholesterol. If recognition of the side chain were crucial, one Glucose-6-Pase activity, would have expected a much higher degree ofstereospecificity Sterol in diet A.mol glucose/mg protein than the results imply. Interestingly, the lack of feedback None 4.41 ± 0.20 (5) repression to require the presence ofa sterol side chain is par- (Z)-17(20)-Dehydro- alleled by the structural correlates (side chain unnecessary) for cholesterol (0.25%) 4.36 ± 0.12 (5) formation ofphospholipid vesicles (refs. 13 and 38 and unpub- None 4.02 ± 0,06 (5) lished data),. suggesting a similar structure-activity correlation (Z)-17(20)-Dehydro- in the interaction ofsterols with lipids in the chylomicrons that cholesterol (0.5%) 3.08 ± 0.22 (5) carry sterol to the liver (39, 40). None 5.46 ± 0.21 (5) Pregn-5-en-3,-ol, (E)-17(20)-dehydrocholesterol, (E)-20(22)- Cholesterol (0.5%) 5.41 ± 0.17 (5) dehydrocholesterol, and 20-epicholesterol each acted quanti- Mice were fed control or stero-enriched diet as indicated for 26 hr. tatively in the same manner as did cholesterol, and each only The microsomal fraction of livers was isolated by centrifugation at depressed the synthesis of sterols at a point (presumably in- 106,000 x g for 1 hr. Microsomes (approximately 1 mg protein) were volving hydroxymethylglutaryl-CoA in the incubatedfor 10 min at 370C with 60 ,umol of cacodylate buffer, pH 6.5, reductase) pathway 30 ,umol of glucose 6-phosphate, and distilled water to a volume of 1.5 prior to mevalonate. However, one sterol studied, (Z)-17(20)- ml. Enzyme activity was determined by quantitation of the glucose dehydrocholesterol, was found to have unusual activity. Not liberated in the reaction. Data are listed as the mean ± SEM with only was it more effective than cholesterol in depressinghepatic values in parenthesis indicating the numbers of mice used. sterol synthesis from acetate, but also it behaved differently Downloaded by guest on September 25, 2021 Biochemistry: Erickson and Nes Proc. Natl. Acad. Sci. USA 79 (1982) 4877 from cholesterol in a qualitative sense. Although short-term (ca. 12. Dugan, R. E. (1981) in Biosynthesis of Isoprenoid Compounds, 24-hr) feedingofeither cholesterol (24-26) oroxygenated sterols eds. Porter, J. W. & Spurgeon, L. (Wiley, New York), pp. 125- (41-43) does not affect sites after mevalonic acid on the sterol 126. pathway, the (Z)-sterol depressed the conversion ofboth mev- 13. Nes, W. R., Adler, J. H., Billheimer, J. T., Erickson, K. A., Jo- alonate and squalene into sterols. This could not be correlated seph, J. M., Landrey, J. R., Marcaccio-Joseph, R., Ritter, K. S. & Conner, R. L. (1982) Lipids 17, 257-262. with an alteration in the activity of a typical enzyme (glucose- 14. Ruzicka, L., Rey, E. & Muhr, A. C. (1944) Helv. Chim. Acta 27, 6-phosphatase) ofthe endoplasmic reticulum. Furthermore, we 472-489. could find no change in the ability of the liver preparations to 15. Huang-Minlon (1949) J. Am. Chem. Soc. 71, 3301-3303. cOnvert acetate to fatty acids in the case either ofthe (Z)-sterol 16. Nes, W. R., Varkey, T. E., Grump, D. R. & Gut, M. (1976)J. or of the three other sterols in which this facet was examined. Org. Chem. 41, 3429-3433. Thus (Z)-17(20)-dehydrocholesterol appears to bring about a 17. Nes, W. R., Varkey, T. E. & Krevitz, K. (1977)J. Am. Chem. Soc. decrease in activity of a number of the enzymes involved in 99, 260-262. sterol biosynthesis but does not appear to impair overall me- 18. Kandutsch, A. A. & Packie, R. M. (1970) Arch. Biochem. Bio- tabolism or cause gross perturbation in microsomal membrane phys. 140, 122-130. 19. Bucher, N. L. R. & McGarrahan, K. (1956) J. Biol Chem. 222, characteristics. 1-15. The ability of 20-epicholesterol, pregnenol, the two (E)-di- 20. Goodman, D. S. & Popjak, G. (1960)J. Lipid Res. 1, 286-300. enols, and the (Z)-dienol to inhibit hepatic sterol synthesis 21. Nordlie, R. C. & Arion, W. J. (1966) Methods EnzymoL 9, strongly suggests the compounds are absorbed. The identifi- 619-625. cation of (Z)-17(20)-dehydrocholesterol in the liver by gas/liq- 22. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. uid chromatography supports this expectation, although more (1951)1. BioL Chem. 193, 265-275. definitive work on this point is necessary. It is perhaps worth 23. Langdon, R. G. & Bloch, K. (1953)J. BioL Chem. 202, 77-81. emphasizing that, although a quantitative comparison of the 24. Gould, R. G. & Swyryd, E. A. (1966) J. Lipid Res. 7, 698-707. actual hepatic action of the various sterols must await further 25. Bucher, N. L. R., McGarrahan, K., Gould, E. & Loud, A. V. workon absorption, transport, and metabolism (43), the positive (1959)J. BioL Chem. 234, 262-267. 26. Siperstein, M. D. & Fagan, V. M. (1966) J. BioL Chem. 241, results obtained cannot be explained, as the inactivity of lan- 602-609. osterol might be, by lack ofabsorption. Moreover, ifthe preg- 27. Brown, M. S. & Goldstein, J. L. (1980)J. Lipid Res. 21, 505-517. nenol, for instance, were absorbed less well than is cholesterol, 28. Chang, T.-Y. & Limanek, J. S. (1980)J. BioL Chem. 255, 7787- this would only mean the C21 sterol is actually more potent than 7795. cholesterol as a feedback agent. The converse, better absorp- 29. Slakey, L. L., Craig, M. C., Beytia, E., Briedis, A., Feldbrueg- tion, might explain the higher and more extensive activity of ger, D. H., Dugan, R. E., Queshi, A. A., Subbaryan, C. & Porter, the (Z)-sterol. J. W. (1972)J. BioL Chem. 247, 3014-3022. 30. Kandutsch, A. A., Heiniger, H.-J. & Chen, H. W. (1977) We thank Dr. John M. Joseph for the synthesis of some ofthe com- Biochim. Biophys. Acta 486, 260-272. pounds. This investigation, which constituted partial fulfillment of the 31. Nes, W. R., Joseph, J. M., Landrey, J. R. & Conner, R. L. (1980) requirements for the Ph.D. degree of K.A.E., was supported by Grant J. BioL Chem. 255, 11815-11821. AM-12172 from the National Institutes of Health. 32. Norman, A. W., Johnson, R. L., Corradino, R. & Okamura, W. H. (1979)J. BioL Chem. 254, 11445-11449. 1. Nes, W. R., Sekula, B. C., Nes, W. D. & Adler, J. H. (1978) J. 33. Arthur, J. R., Blair, H. A. F., Boyd, G. S., Hattersley, N. G. & % BioL Chem. 253, 6218-6225. Suckling, K. E. (1975) Biochem. Soc. Trans. 3, 963-966. 2. Nes, W. D., Patterson, G. W. & Bean, G. A. (1980) Plant Physiol 34. Tavani, D. M., Nes, W. R. & Billheimer, J. T. (1982) J. Lipid 66, 1008-1011. Res. 23, 774-781. 3. Nes, W. D., Saunders, G. A. & Heftmann, E. (1982) Lipids 17, 35. Ritter, K. S. & Nes, W. R. (1981)J. 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