Inhibition of rat liver sterol formation by isoprenoid and conjugated ene compounds

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Pharmacological Research Communications, Vol. 14, lVo. 10, 1982

INHIBITION OF RAT LIVER STEROL FORMATIONBY ISOPRENOID AND CONJUGATEDENE COMPOUNDS ROBERT J. MORIMand MAYASANDRAV. SRIKANTAIAH Department of Pathology, Harbor-UCLA Medical Center, Torrance, CA 90509 R e c e i v e d m f i n a l ~ r m l 3 September1982

SUMMARY. The isoprenoid compounds dolichol, c i t r a l , ~-tocopherol, ubiquinone, geraniol, arid phytol and the dierie pentachloropentadienoic acid, when introduced in sonicated liposomes with phosphatidyl serine, inhibited the conversion of squalene to sterols by the reaction mixture.

Other compounds

tested and found to have no effect on this reaction were lycopene (an isopreno~d), eleostearic acid, 4' methoxychalcone and 5(~-methyl vanillidene) rhodanine (ene compounds), Only c i t r a l , ~-tocopherol, squalene and pentachloropentadienoic acid were found to inhibit the incorporation of 3H-mevalonic acid into sterols.

INTRODUCTION. Recently our labor~tory demonstrated that squalene from squalene-phospholipid vesicles was converted into sterols by rat liver microsomes plus cytosol containing sterol carrier protein l (Morin and Srikantaiah, ]980).

Secondary regulation of cholesterol biosynthesis seems

to be occurring at this stage, where the conversion of water insoluble squalene 1:o sterols involves the participation of a cytosolic carrier protein (Srikantaiah, et a l . , 1976). Inhibitors of these reactions may be of value in the control of hypercholesterolemia, and could be effective by competing with the substrate for either the enzymes or sterol carrier protein I. is an isoprenoid compound containing six isoprene units.

Squalene

There are a large

number of naturally occurringisoprenoid compounds that are structurally 0031-6989/82/100941--07/$03.00/0

© 1982 The Italian Pharmacological Society

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Pharmacological Research Communications, Vol. 14, No. 10, 1982

similar to squalene. All of these compounds are water insoluble, as is squalene, and must be introduced into the reaction mixtures as vesicles. The present paper describes our studies of the in vitro inhibition of squalene to sterol conversion activity of rat liver microsomes and cytosol by various isoprenoid and ene type compounds.

MATERIALS AND METHODS. DL-(5-3H) mevalonic acid (25 mCi/m mol) was purchased from New England Nuclear, Boston, MA. The following compounds were purchased from Sigma Chemical Co., St. Louis, MO.: NAD, NADPH, bovine brain phosphatidyl serine, FAD, lycopene, ~-tocopherol, dolichol and ubiquinone 50.

Eleostearic

acid, 4' methoxychalcone, 5(~-methyl vanillidene) rhodanine, citral (mixture of cis and trans isomers), pentachloropentadienoic acid, geraniol and phytol were obtained from the Aldrich Chemical Co., Milwaukee, WI. 3H-squalene was prepared by incubating DL-(5-3H) mevalonic acid with a 20,O00xg rat liver supernatant as described by (Tchen, 1980); activity of the resulting 3H-squalene was 4.5 mCi/m mol. Liposomes containing 3H-squalene were prepared from purified radioactive squaiene by sonicating with phosphatidyl serine in phosphate buffer under N2 as already described (Morin and Srikantaiah, 1980).

Liposomes containing the compounds to be tested for inhibition were

also prepared in a similar manner. Microsome and cytosol fractions from rat livers were prepared as described previously (Srikantaiah, et a l . , 1976). The reaction mixture for studying the inhibition of squalene to sterol conversion consisted of l.O ml of O.02M potassium phosphate buffer, pH 7.4, with lO-4 M EDTA, containing the following reactants:

microsomes (2.0 mg protein), cytosol

protein (the 105,000xg supernatant, 3.0 mg), NADPH(l.2mM), NAD+ (3mM), FAD (O. ImM), and 3H-squalene-phospholipid liposomes, with and without liposomes containing the compounds to be tested for inhibitory effects.

The final

concentration of 3H-squalene was IOwMand that of phosphatidyl serine was O.13mM. After 2 hours incubation (the reaction is linear up to 2 hours)

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Pharmacological Research Communications, VoL 14, No. 10, 1982

the substrate and products were extracted into petroleum ether, separated by s i l i c i c acid column chromatograp|~ (Scallen, et a l . , 1968), and the radioactivity in each fraction determined by liquid scintillation counting. Activities were expressed as n moles of sterol product formed per 2 hours per 3 mg of cytosol protein. For the inhibition studies involving the conversion of mevalonic acid to sterols, rat livers were homogenized with O.02M phosphate buffer, pH 7.4, (3 ml per gm of liver) containing 30mMnicotinamide and 4mM magnesium chloride.

The homogenate was centrifuged at 20,000 g in a Sorwal high speed

ce~trifuge for lO minutes. The supernatant from this centrifugation was used for the inhibition studies.

The actual reaction mixture (2.0 ml)

contained 1.8 ml of the 20,000 g supernatant in phosphate buffer, NADPH (l.2mM), NAD+ (3mM), Mg++ (4mM) ATP (lOmM), 3H-mevalonic acid (2~Ci), and each compound under evaluation for inhibitory effects.

After 2 hours of

incubation under oxygen the reactions were stopped with an equal volume of 15% ethanolic KOH. The reaction mixture was saponified at 80°C for 30 minutes and extracted twice with 4 ml of petroleum ether each time.

The combined extracts

were evaporated to dryness and the squalene and post squalene products separated by s i l i c acid column chromatography as previously described (Scallen, et a l . , 1968). ratios of squalene to pos

Conversion a c t i v i t i e s were calculated from the squalene products (Scallen, et a l . , 1968).

RESULTS AND DISCUSSION. The results of testing for in vitro inhibition of squalene to sterol conversion by various isoprenoid and erie type compounds are indicated in Table I. concentrations,

All compounds were tested at two different

The variability in the concentrations used for different

compounds was necessitated by the differing capacities of liposomes for each of these. All compounds were added in the same volume, 3-4 experiments were done in duplicate for each compound, and the mean percentage inhibition

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Table I.

Inhibition of Squalene to Sterol Conversion by Isoprenoid and Other Ene Compounds

Inhibitor Used Lycopene

Ubiquinone

~-Tocopherol

Dolichol

Citral

Geraniol

Pentachloropentadienoic Acid

Phytol

Phosphatidyl Serine (control)

Concentration (mM)

Percent Inhibition*

± 3

0.02

20

0.08

18 + 5

O.Ol

43 + 6

0.04

66 + 6

0.01

78 + 8

0.04

90 + 7

0.01

50 + 6

0.04

80 + 8

0.02

35 + 4

0.06

76 + 6

0.02

38 + 2

0.04

72 + 5

0.005

40 + 2

0.01

70 + 7

O.ll

45 + 3

0.02

65 + 3

m

0.05

2 + 0.02

O.l

6+0.03

*Means+standard deviations . . . . . . . . . . . TEe I ml incubation mixtures consisted of microsomCs (2 mg), cytosol protein (3 mg), FAD (O.l mM), NADPH(1.2 mM), NAD* (3 mM), and ~H-squalene added as squalene-phosphatidyl serine vesicles (10~M, lO,O00 dpm), and the inhibitors prepared as inhibitor-phosphatidyl serine vesicles at the concentrations listed above. Final concentrations of inhibitors in the liposomes were determined by gas-liquid chromatography. No degradation of any of the inhibitors occurred during sonication, and concentrations were the same as added before sonication. Vesicles were prepared by mixing l.O mg of phosphatidyl serine/ml buffer with squalene or the inhibitor and sonicating for lO minutes at O°C.

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Table 2. Compounds Tested

Effect of Isoprenoid and Ene Compounds on Incorporation of Mevalonic Acid Into Sterols Concentration (mM)

Control

SquaIene(S) Mean ~S.D.

DPM Post Squalene Products (PSP) Mean ~ S.0.

S/PSP Ra~io

500 + 300

22000 + 6000

0.023

tycopene

0.08

600 ! 250

26000 ! 3000

0.023

Ubiquinone

0.04

620 ! 400

23000 ~ 5000

O.OZ7

Dolichol

0.04

490 + 300

27000 + 3000

0.018

Citral

0.02

200 ÷ 50

14000 + ] 8 0 0 "

0.014

0.06

160 + 32*

6000 + 800*

0.026

0.005

180 + 30

9000 + I000"

0.020

Pentachloropentadienoic acid ~-Tocopherol

Eleostearic acid

Squalene

0.01

60 + 25*

3900 + 200*

0.015

O.Ol

400 ~_ lO0

160DO +_ 2000

0.025

0.04

240 + 60

O.Ol

700 ~ 80

32000 ~ 4000

0.02]

0.02

go0 + go

41000 + 4000*

0.022

O.Ol

900 ~ I00

8000 ~ 700*

0.112

0.02

1400 + 70"

3000 + 600*

0.466

8000 + 3000*

0.030

l~wo-ml Of each reaction mixture Conta#ned 1.8 }M-of rat-1~ver~O,OOOxg supernatant (45-56 mg protein), NADPH(l.2mH), ~IAD+ (3mr4), Hg+ (4raM), AIP (lOmI4), buffer and the above compounds added as liposomes. Incubations were conducted for 2 hours at 37°C under 02 . *Indicates results significantly different from controls at p< O.Ol.

calculated for each. Under the experimental conditions stated in Table ], the conversion activity without any !nhibitors was 4.2 n moles of stero] products per 3 mg of cytosol protein per 2 hours. Amongthe isoprenoid compounds tested, all the compounds except lycopene had significant inhibitory activity.

Control incubations using liposomes of phosphatidyl

serine alone showed no inhibition.

Other compounds without effect (not

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shown in the Table) were eleostearic acid, 4'-methoxychalcone and 5(m-methyl vanillidene) rhodanine. The same compounds were tested for inhibition of 3H-mevalonic acid incorporation into sterols (Table 2).

The method employed gives only the

radioactivity in the squalene fraction and in a second fraction consisting of all the post-squalene intermediates.

Any inhibition of the conversion

of squalene to product will result in an accumulation of squalene. Citral, pentachloropentadienoic acid, and m-tocopherol, all of which inhibited the incorporation of mevalonic acid to sterols, did not result in accumulation of squalene, but rather inhibited the formation of squalene i t s e l f (the ratio of squalene to post squalene intermediates was unchanged). Since many squalene precursors are also isoprenoid compounds, the specific stage at which this inhibition may occur is presently unknown. Unlabeled squalene did inhibit the incorporation of mevalonic acid into sterols, with an accumulation of squalene due to the competing effect of the non-radioactive squalene with the endogenous labeled squalene produced. Eleostearic acid was the only compound which stimulated the incorporation of mevalonic acid into squalene and other products. The inhibition of squalene to sterol conversion by the above isoprenoid and ene compounds appears to be specific, since all of the similar compounds tested did not inhibit this reaction.

Since the phospholipids themselves did

not inhibit the reaction, an inhibition due to nonspecific competition For binding between the liposomes containing the substrate and liposomes containing the inhibitors can be ruled out.

Not all the compounds that inhibited the

conversion of squalene to sterols inhibited the incorporation of mevalonic acid into sterols.

The compounds that did inhibit the latter acted at an earlier

step than conversion of squalene to sterols.

The specific step between

isopentenyl pyrophosphate and squalene at which inhibition may occur is presently unknown. Somemono and bicyclic terpenes have been shown to inhibit the

PharmacologicalResearch Communications, Vol. 14, No. 10, 1982

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reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonic acid (Clegg, et al., 1980).

Inhibition of cholesterol biosynthesis at a step other than the

traditional 3-hydroxy-3-methylglutaryl CoA reductase offers a unique approach, and since most of the inhibitors used in this study are dietary constituents, they have potential clinical value.

A number of other isoprenoid compounds

are being screened ~or inhibitory activity, and those that are most effective will be subsequently tested for in vivo hypocholesterolemic effects.

ACKNOWLEDGEMENTS. This work was supported by Research Award #697G2-2 from the American Heart Association, Greater Los Angeles A f f i l i a t e and by the N.I.Ho Biomedical Research Support Grant to Harbor-UCLA Medical Center. REFERENCES. Clegg, R.J., Middleton, B., Bell, G.D. and White, D.A. (1980) Biochem. Pharm., 29, 2125-2127. Morin,~R.J. and Srikantaiah, M.V. (1980) J. Lipid. Res., 21, I143-I146. Scallen, T.J., Dean, W.J. and Schuster~ M.W. (1968) J. Bio-l. Chem., 243, 5202-5206. Srikantaiah, M.V., Hansbury, E., Loughran, E.D. and Scallen, T.J. (1976) J. Biol. Chem., 251, 5496-5504. Srikantaiah, M.V., Lew, D.W. and Morin, R.J. (1980) L i p i d , L5, 555~5. Tchen, T.T. (1963) Methods Enzymol., 6, 509-512.