BiochimicaL et Biophysics &ta

ELSEVIER Biochimica et Biophysics Acta 1214 (1994) 79-87

Modulations in hepatic branch-point enzymes involved in isoprenoid biosynthesis upon dietary and drug treatments of rats

Magnus Andersson a,b,*, Johan Ericsson ‘, Eeva-Liisa Appelkvist ‘, Sophia Schedin ‘, Tadeusz Chojnacki d, Gustav Dallner a,c

a Clinical Research Center, Nouum, Huddinge Hospital, S-141 86 Huddinge, Sweden h Department of Neurology, Huddinge Hospital, Karolinska Institute& Huddinge, Sweden

’ Department of Biochemistry, Stockholm Unicer.Gty, Stockholm, Sweden d Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland

Received 25 January 1994; revised 18 April 1994

Abstract

Three branch-point enzymes of the mevalonate pathway, farnesyl pyrophosphate synthase, cis-prenyltransferase and squalene synthase were characterized in rat hepatic cytosol, microsomes and peroxisomes isolated from rats after treatment with peroxisome proliferators, inducers of the endoplasmic reticulum or modulators of lipid metabolism. Cholestyramine and phenobarbital induced primarily the cytosolic farnesyl pyrophosphate synthase, whereas clofibrate and phthalates elevated the corresponding peroxisomal activity. cis-Prenyltransferase activities in microsomes were induced 4-5fold after clofibrate, phthalate and phenobarbital administration, but these same treatments affected the peroxisomal activity to only a limited extent. Squalene synthase activity in microsomes was completely abolished, but the peroxisomal activity was unaffected after administra- tion of cholesterol. On the other hand, clofibrate and phthalate induced only the microsomal activities. Mevinolin treatment greatly increased peroxisomal and cytosolic farnesyl pyrophosphate synthase activities, but not the mitochondrial activity, and the cis-prenyltransferase activities were elevated in peroxisomes, but not in microsomes. These results demonstrate that the branch-point enzymes in cholesterol and dolichol biosynthesis at various cellular locations are regulated differentially and that the capacities of peroxisomes and the endoplasmic reticulum to participate in the synthesis of polyisoprenoid lipids is affected profoundly by treatment with different xenobiotics.

Key words: Microsome; Peroxisome; Farnesyl pyrophosphate synthase; cis-Prenyltransferase; Squalene synthase; Mevalonate

pathway

1. Introduction

Cholesterol, dolichol and ubiquinone share the same

initial biosynthetic pathway, i.e., the conversion of three

acetyl-CoA units into farnesyl pyrophosphate [l-3]. The key regulatory enzyme in this initial portion of the

Abbreviations: IPP, isopentenyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl pyroposphate; SCP, sterol carrier protein; HMG, 3-hydroxy-3-methylglutharyl; DEHP, di(2-eth- ylhexyljphthalate; LDL, low-density lipoprotein; ER, endoplasmic reticulum.

* Corresponding author. Fax: + 46 8 7795585.

mevalonate pathway is HMG-CoA reductase, which has been localized to both the endoplasmic reticulum [4,5] and to peroxisomes [6,7] in rat liver.

FPP synthase catalyzes the condensation of IPP (C,) units with both DMAPP (C,) and GPP (C,,) to pro- duce FPP CC,,) [8]. Most of this activity is recovered in the cytosolic fraction from liver, although relatively high activity is present in mitochondria and some activ-

ity is also found in peroxisomes, microsomes and nuclei [9-111. FPP serves as the common substrate for the first committed enzymes in the synthesis of cholesterol, dolichol and ubiquinone [12]. Furthermore, FPP is the substrate for FPP: protein transferase involved in pro- tein prenylation, as well as for all-trans GGPP syn- thase, which is also involved in protein prenylation.

0005-2760/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0005-2760(94)00075-A

Squalene synthase is the first committed enzyme in cholesterol biosynthesis and catalyzes the condensation of two FPP units to produce presqualene pyrophos- phate, which is subsequently reduced to squalene in the preserrce of reduced nucleotides. This enzyme may play a regulatory role in sterol biosynthesis, since it regulates the flow of metabolites into the sterol portion of the mevaionate pathway [13j. Squalene synthase was found to be present in the endoplasmic reticulum and peroxisomes [IO]. In immu~ohistochemic~l studies, us- ing an antisera produced against the isolated microso- ma1 squalene synthase, mostly precipitations related to the endoplasmic reticulum were observed [14]. The yeast microsomal enzyme has been purified and cloned f75]. Recently, a proteolytic fragment of the rat hepatic enzyme has been purified to apparent bomogenei~ II61.

Not only is FPP converted to squalene, whose sole fate is sterol synthesis, but this compound also serves as substrate for cis-prenyltransferase, the first commit- ted enzyme in dolichol biosynthesis. This transferase catalyzes repetitive c& additions of IPP to a FPP primer, with the resultant formation of long-chain polyprenyl pyrophosphates [2]. This enzyme activity has been observed in preparations from a number of sources, both prokaryotic [17-191 and eukaryotic [20- 241. In most eukaryotic systems investigated, c&-pre- nyltransferase activity is associated with the endoplas- mic reticulum, but recently, a high specific activity of this enzyme has been measured in rat liver peroxi- somes [lo). It has been proposed that microsomal cis- prenyltra~sferase in rat liver is dependent on SCP2 for activity f25f.

Peroxisomes are known to play an essential role in a variety of processes connected with lipid metabolism. Enzymes associated with both the synthesis and degra- dation of lipids are localized in this organelle. Peroxi- somes contribute to cellular fatty acid &oxidation, oxidizing long-chain fatty acids preferentially t261. En- zymes involved in the initial steps of plasmalogen syn- thesis are also localized in this organelle [271.

Immunolabeling and cell fractionation studies have shown that acetoacetyl-CoA thiolase, HMG-CoA re- ductase and mevalonate kinase activities are also pre- sent in peroxisomes. In addition, the Iast two common steps of the mevalonate pathway, IPP isomerase and FPP synthase, could also be demonstrated to be pre- sent in this organelle [28]. Peroxisomes supplemented with cytosol can convert mevalonic acid into choles- terol and dolichol 129-311. Four enzymes in the termi- nal portion of cholesterol synthesis, i.e., dihydrolanos~ terol oxidase, steroid-14-reductase, steroid-8-isomerase and steroid-3-ketoreductase are also found in this sub- cellular compartment [333.

In the present investigation, we have assayed certain of the branch-point enzymes of the mevafonate path-

way, i.e., FPP synthase, squalene synthase and cis-pre- nyltransferase, in rat liver cytosol, microsomes and peroxisomes. Treatment with peroxisome proliferators, inducers of the endoplasmic reticulum and modulators and inhibitors of lipid metabolism were used to demon- strate differences in the properties and regulation of these enzymes in the different subcellular compart- ments.

2. Materials and methods

2.1. Chemicals

cw-Unsaturated poI~~re~~ls, used as standards, were prepared from Sclrbus suecicu as described previously 1341. Polyprenyl pyrophosphates were prepared accord- ing to Popjak et al. 1351. Tritium-labeled IPP and FPP were prepared from the corresponding labeled alcohols as described earlier f21]. All unlabeled isoprenoid py- rophosphates were synthesized from the corresponding alcohols using the same procedure. The isopentenol, geraniol, dimethylallyl alcohol, farnesol and geranyl- geraniol isomers were generous gifts from Dr. T. Take- gawa (Kururay, Okayama, Japan). Upon TLC analysis in different solvent systems, all polyprenols and phos- phoryIated de~vatives gave single spots. Mevinolin was kindly supplied by Dr. A.W. Alberts (Merck, Sharp and Dohme, Rahway, NJ, USA).

For determination of the amounts of dolichol, dolichyl phosphate and cholesterol, the samples were mixed with 1 vol. methanol and 0.5 vol. 60% KOH and subsequently hydrolyzed for 45 min at 100°C. Chloro- form and methanol were then added to obtain a final chloroform/methanol~water ratio of 3 : 2 : 1. The up- per, aqueous phase was removed and the lower chloro- form-rich phase washed three times with Folch upper phase [36]. Dolichyl phosphate was separated from neutral lipids using a DEAE-Sephadex ion exchanger f371.

2.3. Reversed-phase HPLC

Chromatographic separations were performed on a Shimadzu LC4A system using a Waters WISP autoin- jector. The sample was injected onto a Hewlett-Packard Hypersil ODS C18,3 pm column with a size of 60 x 4.8 mm. For neutral lipids (cholesterol and dolichol) a linear gradient was used from the initial methanol/ water, 9: 1, in reservoir A to methanol/2-propanol/ hexane, 2: 1: 1, in reservoir B. The flow rate was 2 mI/min and the program time 33 min, The eluate was

M. Anderson et al. /Biochimica et Biophysics Acta 1214 (1994) 79-87 81

monitored at 210 nm. In the case of dolichyl-P, a convex gradient (setting - 1) was run from water/ methanol/ 2-propanol, 5 : 60: 40, to 40% 2- propanol/hexane, 3 : 7, both solvents containing 20 mM phosphoric acid, during a period of 17 min and at a flow rate of 2 ml/min. All the peaks were well resolved and quantitation was based on integration of peak areas, using internal standards to compensate for sam- ple losses. Recoveries were 65-85%.

2.4. Treatment of animals

Male Sprague-Dawley rats (180-200 g) were used in these experiments. The animals were fed standard lab- oratory chow (19% protein, 51% carbohydrate, 4% total fat, 0.007% cholesterol) or standard laboratory chow containing clofibrate (0.6%) or DEHP (2%) for 7 days or cholesterol (5%) or cholestyramine (5%) for 4 days before sacrifice. In some experiments, pheno- barbital (8 mg/lOO g body weight) was injected intra- peritoneally, once daily for 5 days. In the case of mevinolin treatment, the rats were fed chow mixed with mevinolin (500 mg/kg food) for 21 days.

2.5. Isolation of rat liver subfractions

Rat liver peroxisomes were isolated on a Nycodenz gradient as described earlier [33]. Isolation of the mi- crosomal fraction was also performed as described previously [33]. The microsomal pellet was suspended in 0.15 M Tris-HCl (pH S.O>, and recentrifuged at 105 000 X g for 60 min in order to remove adsorbed cytosolic proteins. The final cytosolic fraction was pre- pared by recentrifugation of the postmicrosomal super- natant at 105 000 x g for 180 min.

2.6. Incubations

cis-Prenyltransferase activity was measured in a 300 ~1 incubation medium containing 25 mM imidazole chloride (pH 7.01, 1 mM MgCl,, 0.1 mM dithiothreitol, 20 PM [3H]IPP (0.21 Ci/mmol) and 50 PM FPP. When the peroxisomal activity was measured, the medium also routinely contained 5 mM KF and the reaction was started by the addition of 100-200 pg protein. When microsomal cis-prenyltransferase activ- ity was assayed, the medium contained 100 mM KF and 1.0% Triton X-100 and the reaction was initiated by the addition of 250-500 pg protein. The reactions were allowed to proceed for 30-60 min at 37°C and thereafter terminated by the addition of 2.0 ml n- butanol, saturated with water, and the lipids were subsequently extracted, dephosphorylated and ana- lyzed by HPLC 1381.

When FPP synthase activity was assayed, the same incubation conditions were employed with certain

modifications. trans-GPP at a concentration of 50 mM was used as the allylic substrate and the reaction medium contained 5 mM KF. The reaction was started by the addition of 5-10 pg protein in the case of cytosol, while the peroxisomal assay system contained 50-100 pg protein. The reaction was allowed to con- tinue for 10 min at 37°C and then terminated by the addition of 2.0 ml n-butanol. The reaction products were subsequently extracted, dephosphorylated and analyzed by reversed-phase HPLC [381.

Squalene synthase activity was assayed under the same conditions as described above for cis-pre- nyltransferase, with the exception that the incubation medium contained 100 PM L3HlFPP (44 Ci/mol) as the only substrate. The medium also contained 1 mM NADPH, 1 mM glucose-6-phosphate and 1.2 units of glucose-6-phosphate dehydrogenase. The reaction was started by the addition of 100-200 pg protein, allowed to continue for 30 min at 37°C and then terminated by the addition of 6 ml chloroform/methanol (2:l). The reaction products were subsequently extracted and an- alyzed by HPLC.

2.7. Extraction and chromatography of lipids for assays of enzymatic activities

In all cases, except when squalene synthesis was measured, the incubation medium was extracted by extensive vortexing with 2.0 ml n-butanol saturated with water. After addition of 1.0 ml 2 M KCl, the samples were centrifuged at a low speed in order to separate the organic and aqueous phases. The butanol phases were pooled and an aliquot removed for scintil- lation counting. This procedure efficiently extracted FPP and polyprenyl pyrophosphates. Enzyme activities were expressed as the amount of radioactivity incorpo- rated into butanol-extractable products.

In order to determine absolute enzyme activities and to analyze the distribution of products, the butanol phase was evaporated under N,. The residue was re- dissolved in 200 ~1 5% n-octyl-P-glycopyranoside and dephosphorylated enzymatically according to the method of Wong and Lennarz [39]. After dephospho- rylation, the products were extracted twice with 3 ml ether/petrolium ether 1: 1 and the extract then dried under N,. The residue was redissolved in 50 ~1 metha- nol/2-propanol/hexane, 2 : 1: 1 and analyzed by re- versed-phase HPLC using a Hewlett-Packard Hypersil ODS C,, column. A convex gradient was employed from the initial methanol/water 9 : 1 in pump system A to methanol/2-propanol/hexane 2 : 1: 1 in pump sys- tem B, at a flow rate of 1.5 ml/min and with a program time of 30 min. The absorbance at 210 nm and radioac- tivity of the effluent were monitored using a UV-detec- tor and a radioactivity flow detector (Radiomatic In- struments, Tampa, FL), respectively. The individual

radioactive products were identified using unlabeled internal standards added to each run.

When squalene synthase was assayed, the lipid products were extracted with 6 ml chloroform/ methanol, 2: 1 at 37°C for 60 min. The protein was removed by centrifugation and the organic phase trans- ferred to a new tube. This organic phase was parti- tioned by the addition of 1.0 ml water and 0.67 ml methanol. The aqueous phase was discarded and the organic phase washed three times with Folch upper phase. The organic phase was then evaporated under N, and the residue redissolved in 50 ~1 methanol/2- propanol/hexane, 2 : 1: 1 and analyzed by reversed- phase HPLC as described above.

2.8. Chemical measurement

Protein was determined by the Lowry procedure using bovine serum albumin as standard [40].

3. Results

3.1. Lipid compositions of microsomes and peroxisomes

In these experiments a number of conditions and treatments known to change cellular structure in spe- cific manners were employed. It has been established that cholesterol feeding down-regulates HMG-CoA re- ductase and, thereby, decreases the biosynthesis of sterols. Cholestyramine causes sequestration of bile acids and increases the flow of metabolites through the mevalonate pathway. Clofibrate is a h~olipidemic drug, while DEHP is a plasticizer, both causing peroxi- some proliferation, i.e., increasing the numbers of this organelle and the levels of certain of its enzymatic

constituents. Phenobarbital is often employed as an effective inducer of the endoplasmic reticulum and associated hydroxylation reactions, also changing vari- ous aspects of the lipid metabolism associated with this organelle. Mevinohn is an inhibitor of HMG-CoA re- ductase used in clinical therapy.

Since isolated microsomes and peroxisomes were used here to measure various enzyme activities in- volved in dolichol and cholesterol synthesis, we ana- lyzed the lipid contents of these subfractions. Liver microsomes are known to contain similar amounts of dolichol and dolichyl-P and considerably higher con- centrations of cholesterol (Table 1). A high cholesterol diet increases microsomal dolichol content by about 20% whereas all the other treatments caused only small modifications in this parameter. The level of dolichyl-P is decreased greatly, by about 40%, in micro- somes from DEHP-treated rats and the other treat- ments resulted in only small elevations or decreases of this lipid, which is of basic importance in glycoprotein synthesis. The cholesterol content of microsomes in- creases 50% after dietary supply of cholesterol, while the other treatments had only a limited influence on cholesterol content. In comparison with microsomes, peroxisomes contain less dolichol, dolichyl-P and cholesterol. The peroxisomal dolicho1 content remains unchanged in connection with a high cholesterol diet and phenobarbital treatment. Administration of cho- lestyramine doubles this dolichol content, whereas treatment with clofibrate, DEHP and mevinolin gives a decrease of 18, 32 and 41%, respectively. Only choles- tyramine treatment is without effect on peroxisomal dolichyl-P, the level of which is elevated greatly upon administration of phenobarbital. Cholesterol diet or treatment with clofibrate, DEHP or mevinolin lower the content of this lipid in peroxisomes by 20-30%.

Table 1 Lipid compositions of rat liver microsomes and peroxisomes after various treatments

Microsomes (% of Control) Peroxisomes t% of Control)

Dolichol Dolichyl-P Cholesterol Dolichol Dolichyi-P Cholesterol

Control 1OOa 1OOb 100 c 100 d 100 r 100 f Cholesterol 119 88.2 152 98.1 82.5 95.2 Cholestyramine 10.5 90.4 102 174 104 88.5 Clofibrate 90.5 98.5 99.2 82.1 83.6 113 DEHP 108 60.4 104 68.2 74.0 124 Phcnobarbitai 93.3 96.0 105 99.8 164 106 Mevinolin 109 86.9 85.1 59.2 72.4 54.8

Rats were supplied with normal rat chow supplemented with 5% cholesterol (4 days), 5% cholestyramine (4 days), 0.6% clofibrate (7 days), 2% DEHP (7 days), or mevinolin (500 mg/kg food. 21 days). Phenobarbital (8 mg/lOO g) was injected i.p. once daily for 5 days. Isolated total microsomes and peroxisomes were analyzed. The mean values of 6 experiments are given. a 0.241 0.020 pg/mg protein; b 0.191 0.017 gg/mg protein; ’ 27.4 + 3.6 pg/mg protein; d 0.216 i 0.019 ,ug/mg protein; ’ + + 0.105 & 0.015 pg/mg protein; ’ 6.7 + 0.4 pg/mg protein. All values are means + SD.

M. Anderson et al. /Biochimica et Biophysics Acta 1214 (1994) 79-87 83

The peroxisomal content of cholesterol is increased after DEHP treatment and decreased substantially af- ter administration of mevinolin. The other drugs mod- ify peroxisomal lipid composition to only a limited extent.

3.2. FPP synthase, cis-prenyltransferase, and squalene synthase activities in isolated subcellular fractions

Peroxisomes contain FPP synthase activity, which is, however, only 2% of that found in cytosol (Table 2). The responses of the enzyme activities in these two compartments to various treatments are quite differ- ent. Cholestyramine treatment doubles the peroxiso- ma1 activity, while elevating the cytosolic activity by 13-fold. Both clofibrate and DEHP increase the perox- isomal activity several fold, but elevate the cytosolic activity only moderately. Phenobarbital treatment dou- bles only the cytosolic FPP synthase activity.

Peroxisomes demonstrate a high cis-prenyltrans- ferase activity, exceeding that found in microsomes by 3-fold on a protein basis. Both cholestyramine treat- ment and dietary cholesterol elevate this enzyme activ- ity at least 2-fold in both organelles. The peroxisome proliferators clofibrate and DEHP do not affect the peroxisomal activity, in contrast to the microsomal activity, which is elevated more than 4-fold by these compounds. The ER inducer phenobarbital increases this activity to some extent in peroxisomes, but exten- sively in microsomes.

The specific activity of squalene synthase in peroxi- somes is about the same as in microsomes. Cholestyra- mine treatment increases the peroxisomal and, in par- ticular, the microsomal activity. Both clofibrate and DEHP decrease the peroxisomal and increase the mi- crosomal activity. In contrast, a high cholesterol diet

completely abolishes microsomal squalene synthase ac- tivity whereas the peroxisomal activity is increased by 30%. Phenobarbital increases the peroxisomal activity 3-fold and the microsomal activity 4-fold.

3.3. Changes in the distribution of products formed by cis-prenyltransferase

The product distributions of the cis-prenyltrans- ferase reaction in microsomes and peroxisomes are shown in Fig. 1. The polyprenyl pyrophosphates pro- duced by the microsomal enzyme from untreated ani- mals contained 15-19 isoprene units, the two dominat- ing isoprenoids being those with 16 or 17 units (Fig. 1A). This chain-length distribution was not affected by any of the treatments employed, even though the total enzyme activity was very much increased (Fig. 1B).

In the case of untreated rats, the peroxisomal cis- prenyltransferase produced polyprenyl pyrophosphates containing 16-21 isoprene units, with the two dominat- ing species being those containing 18 or 19 units (Fig. 10. Both of the peroxisome proliferators, clofibrate and DEHP changed this distribution considerably. This effect was most pronounced in connection with clofi- brate treatment, after which the family of polyprenyl pyrophosphates produced by peroxisomes consisted of as many as 16 different derivatives (Fig. lD>. The shortest product contained 15 isoprene units, while the longest contained as many as 30 isoprene units.

3.4. Mevinolin treatment

Mevinolin, a well known inhibitor of HMG-CoA reductase, influences all of the branch-point enzymes measured to various extents (Table 3). FPP synthase activity is low in peroxisomes, but the mitochondrial

Table 2

FPP synthase, cis-prenyltransferase and squalene synthase activities in hepatic subcellular fractions isolated from rats treated with different

compounds

FPP-synthase cis-Prenyltransferase Squalene synthase

Peroxisomes Cytoso1 Peroxisomes Microsomes Peroxisomes Microsomes (o/o of control)

Control 100 a 100b 100 c 100 d 100 e 100 f Cholesterol 112 120 186 225 134 12

Cholestyramine 215 1272 226 292 168 336 Clofibrate 297 129 115 410 60 165 DEHP 521 187 94 437 79 315 Phenobarbital 128 224 181 545 268 390

The enzyme activities in microsomes, peroxisomes and cytosol were measured as described under “Materials and Methods”. In the case of

cb-prenyltransferase, the products were dephosphorylated after extraction. All products were analyzed using an HPLC system equipped with a

radioactivity flow detector. The results are the means of 5-8 experiments. a 8.22 f 0.73 pmol IPP per pg protein per h; b 432 f 46 pmol IPP per pg protein per h; ’ 380 + 32 pmol IPP per kg protein per h; d 117 k 14 pmol IPP per yg protein per h; e 380 f 36 pmol FPP per mg protein per min; f 415 k 39 pmol FPP per mg protein per min. All values are means f S.D.

84 M. Anderson et al. / Biochimica et Biophysics Acta 1214 (1994) 79-87

R~b”,iO” tima. n-0,” Relention mle. In,”

Fig. 1. Distribution of the products formed by cis-prenyltransferases in microsomes and peroxisomes. c&Prenyltransferase activities were

measured in microsomes and peroxisomes isolated from the livers of rats subjected to different treatments. The reaction products were extracted,

dephosphorylated and analyzed using an HPLC system equipped with a radioactivity flow detector. cis-Prenyltransferase activities were assayed

in (A) control microsomes; (B) microsomes from rats treated with clofibrate; (0 control peroxisomes; (D) peroxisomes from rats treated with

clofibrate. The numbers above the peaks indicate the number of isoprene units present in the individual polyisoprenoids

activity is 16% of that of the cytosol. Mevinolin treat- ma1 activity was as much as 17-fold after this treat- ment increases both the peroxisomal and cytosolic ac- tivities 4-fold but does not affect the mitochondrial activity. Microsomal cis-prenyltransferase activity re- mains unchanged upon administration of this inhibitor, but this same activity is induced greatly in peroxisomes.

ment.

4. Discussion

After the mevinolin treatment employed in our ex- Many of the enzymes of the mevalonate pathway periments, the microsomes isolated from the rats of demonstrate a pronounced multicompartment localiza- our strain exhibited a 2-fold increase in their squalene synthase activity. Significantly, the increase in peroxiso-

tion, most of them being present in two or more organelles [12]. It is of considerable intrest to establish

Table 3

Effects of mevinolin treatment on branch-point enzymes in subfractions from rat liver

Enzyme Treatment Microsomes Peroxisomes Mitochondria Cytosol

FPP-synthase a Control 9.78 f 0.6 70.8 + 0.8 444 f 28

Mevinolin 37.4 + 0.4 67.2 + 0.8 1710 + 163

cis-Prenyltransferase b Control 125 + 9.2 396 k 48

Mevinolin 114 & 10 1070 k 178

Squalene synthase ’ Control 405 f 59 383 + 39 Mevinolin 729 + 63 5040 * 685

The rats were fed normal rat chow or normal rat chow supplemented with 0.05% (w/w) mevinolin for 21 days. The radioactive products were analyzed using a reversed phase HPLC equipped with a radioactivity flow detector. The values are the means of 6 experiments. a pmol IPP/pg protein/h; b pmol IPP/mg protein/h; ’ pmol IPP/mg protein/min. All values are means i S.D.

M. Anderson et al. / Biochimica et Biophysics Acta 1214 (1994) 79-87 85

whether the enzymes present at various locations are identical proteins or are distinct isoenzymes. This lat- ter pattern is quite common, e.g., the systems for p-oxidation of fatty acids in mitochondria and peroxi- somes have similar functions, but involve different pro- teins. The multiplicity of FPP synthase in liver is indi- cated by the finding that several copies of the gene encoding FPP synthase are present in the genome, although this question is not quite settled yet 141-431. If the enzymes in different organelles are isoenzymes and functionally distinct, we may expect individual regulation.

In this study we wanted to investigate early effects of these various treatments on mevalonate pathway enzymes. As the lipid analyses demonstrated, at this time no major modifications in lipid composition of microsomes or peroxisomes had occurred. It is possible that prolonged administration and/or different doses of these drugs might elicit responses in enzyme activi- ties other than these described in the present study [44,45].

The three branch-point enzymes in the mevalonate pathway, i.e., FPP synthase, cis-prenyltransferase and squalene synthase, are all present in a number of organelles and compartments and are influenced dif- ferently, from both quantitative and qualitative points of view, by various treatments. FPP synthase is present not only in the cytosol, but also in peroxisomes and other organelles. This activity is induced in the cytosol upon treatment with cholestyramine or phenobarbital but only in peroxisomes after clofibrate administration.

Mevinolin treatment induced the peroxisomal and cytosolic but not the mitochondrial activities. The spe- cific cis-prenyltransferase activity in hepatic peroxi- somes exceeds that found in microsomes by 3-fold. The two peroxisome proliferators employed here do not influence the peroxisomal, but greatly induce the mi- crosomal enzyme. Mevinolin treatment has the oppo- site effect, inducing only the peroxisomal activity.

Squalene synthase is present in both peroxisomes and microsomes, which exhibit about the same specific activity. Similar to the other branch-point enzymes, this enzyme is also subject to very distinct and individual regulation. Clofibrate and DEHP decrease the peroxi- somal and elevate the microsomal activity.

In some cases, e.g., treatment with clofibrate or DEHP, there is a discrepancy between the lipid con- tent and branch-point enzyme activity in the same subfraction. However, there is no direct relationship between these two parameters, i.e., an increase in enzyme activity is often not associated with increased membrane lipid content. It is well established that the majority of newly synthesized lipids, both in the ER and peroxisomes, are rapidly transported away from these organelles in order to supply other membranes with lipids or for secretory purposes [31,32].

Interestingly, mevinolin treatment of rats increases all of the enzyme activities measured in peroxisomes, despite the fact that this drug is not a peroxisomal proliferator. Since we have studied isolated subfrac- tions employing an excess of substrate, the decrease in cytoplasmic substrate pool in vivo has no influence on the activities measured here. In agreement with previ- ous observations, these results indicate that mevinolin influences enzyme proteins at the translational level in addition to directly activating or inhibiting certain en- zymes [45,46].

Cholesterol is known, directly or indirectly, to down- regulate sterol biosynthesis, primarily by inhibiting HMG-CoA reductase [13]. However, it is also known that LDL, as well as cholesterol-feeding down-regulate the activity of squalene synthase both in isolated cells and in rat liver, although the effects on this enzyme are not as pronounced as those on HMG-CoA reductase [ 16,48,49].

The effect of a high-cholesterol diet on squalene synthase in our study was most remarkable. The micro- somal activity is almost completely abolished, while no inhibition is observed in the case of peroxisomes.

The question arises as to why HMG-CoA reductase demonstrates a co-regulation with microsomal, but not with peroxisomal squalene synthase. In previous stud- ies it was found that peroxisomal cholesterol synthesis, in contrast to the microsomal process, is not influenced by the level of cholesterol in the diet [29]. Hepatic peroxisomes appear to contain not only the terminal portion of the biosynthetic sequence for cholesterol, but also at least five of the initial eight enzymes in the mevalonate pathway. Consequently, it is very likely that the complete pathway for cholesterol synthesis, from acetyl-CoA, is present in peroxisomes. It is possible that a high-cholesterol diet, the classical down-regu- lator of the cytoplasmic-microsomal system, is not a regulator of the peroxisomal counterpart.

Interestingly, treatment with clofibrate or DEHP results in a broadened chain-length distribution of polyisoprenoids, with components containing 15-30 isoprene units being produced in in vitro incubations with peroxisomes. This is a remarkable finding, since in all previous investigations, even under serious patho- logical conditions, modifications in this parameter were found to be very limited [50,51]. However, following prolonged DEHP treatment of rats, the chain-lengths of hepatic dolichol derivatives are broadened, shifting to longer isoprenoids, in agreement with the observa- tions presented here [44].

As discussed above, the specific activities of the different enzymes at various locations are modified by different treatments. The situation is somewhat differ- ent when one considers changes in total activity on a cellular or tissue basis. The specific peroxisomal FPP synthase activity increases 5-fold after DEHP treat-

86 h4. Anderson et al. /Biochimica et Biophysics Acta 1214 (1994) 79-87

ment, i.e., a very pronounced effect. The increase in the total peroxisomal activity is, however, much greater, since a one-week treatment with DEHP increases the number of peroxisomes per hepatocyte by 8- to lo-fold [52]. Thus the total increase in peroxisomal FPP syn- thase activity per cell is as high as 40- to 50-fold.

It will be necessary in the future to determine the total contribution of peroxisomes and microsomes to these synthetic activities on a gram liver basis. This will, however, require considerable effort involving an- alytical subfractionation and extensive determinations of marker enzymes. We have recently performed such investigations on control liver tissue and analogous studies on liver after various treatments should be performed in the future [53].

The experiments presented in this study support the proposal that rat liver peroxisomes contain substantial levels of the branch-point enzymes cis-prenyltrans- ferase, squalene synthase and FPP synthase. Further- more, there appear to be different enzymatic systems for the biosynthesis of dolichol and cholesterol in per- oxisomes and the ER. The functional significance of this compartmentalization of the biosynthesis of these two compounds is not yet clear, but it is reasonable to expect that these lipids have different functions in the two different organelles.

Acknowledgement

This work was supported by the Swedish Medical Research Council.

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