- Email: [email protected]
Studies on estrogen regulation of cholesterol biosynthesis in rat liver microsomes
357
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 55499
STUDIES
ON ESTROGEN
BIOSYNTHESIS
REGULATION
IN RAT LIVER
OF CHOLESTEROL
MICROSOMES
S.MUKHERJEEANDA.BHOSE Laboratories of Lipid Research, Department of Applied Chemistry, University of Calcutta, Calcutta (India)
(Received June Ioth,1968)
SUMMARY
An inhibition of cholesterol biosynthesis from acetate has been demonstrated in the liver of rats previously administered IOO pg (per IOO g rat) of I7b-estradiol for periods varying from 4 to 56 days. The site of estrogen control of hepatic sterol synthesis has been investigated in microsomal preparations. The inhibitory effect of exogenous estrogen is observed mostly at premevalonate stages of biosynthesis; the conversion of mevalonate to squalene or of squalene to cholesterol is not significantly altered by hormonal administration. The first estrogen-sensitive step in the pathway of biosynthesis appears to be the condensation of acetoacetyl-CoA and acetyl-CoA, since markedly reduced activity of the hydroxymethylglutaryl-CoA-condensing enzyme has been demonstrated in liver microsomes isolated from treated animals. A decrease in the microsomal hydroxymethylglutaryl-CoA reductase activity also results from the hormone treatment. The overall depression in the rate of cholesterol synthesis is determined by the hormonal inhibition of mevalonate synthesis in microsomes. Estrogen has very little influence on hydroxymethylglutaryl-CoA-cleavage enzyme activity.
INTRODUCTION
The effects of dietary cholesterol and fasting have predominated most studies attempting to elucidate the physiological mechanism controlling hepatic synthesis of cholesterol from acetate. GOULD AND POPJAK' provided evidence that exogenous cholesterol inhibits cholesterol synthesis by controlling steps between acetate and mevalonate. BUCHER, OVERATH AND LYNEN~ found that fasting greatly reduced the incorporation of acetate into cholesterol in rat liver by suppressing the activity of hydroxymethylglutaryl-CoA reductase. The effect of starvation on the microsomal reductase has since been confirmed by WIELAND et a1.3. SIPERSTEIN AND FAGAN%& made strikingly similar observations of the control of hydroxymethylglutaryl-CoA reductase activity in their studies on the mechanism of feed-back inhibition of cholesterol synthesis by dietary cholesterol. On the other hand, evidence of a metabolic Biochim. Biophys. Acta, 164 (1968) 357-368
S. MUKHERJEE, A. BHOSE
358
block between mevalonate and squalene in livers of fasted rats was suggested by SCAIFE AND MIGICOVSKY~. This implied that regulation of hepatic biosynthesis of cholesterol might involve more than one enzymic site. Further support for this hypothesis was obtained from the more recent studies of GOULD AND SWYRYD’ who demonstrated that prolonged hydroxymethylglutaryl-CoA
cholesterol feeding not only depressed the activity of reductase but almost completely inhibited the conver-
sion of mevalonic acid into squalene by affecting two other enzymes in the post-mevalonate stages of biosynthesis. The incorporation of acetate into cholesterol in rat liver is greatly influenced by endogenous as well as exogenous hormones. Evidences
of decreased sterol syn-
thesis in rat liver by estrogen administration have been presented in many experimental studies-lo, but the precise biochemical site for estrogen inhibition of cholesterol synthesis from acetate has not been established. While BOYD’~ suggested that endogenous estrogen influenced cholesterol synthesis at a point between acetate and mevalonate, MEROLA AND ARNOLD~~ demonstrated that oral administration of this hormone decreased the rate of decarboxylation of mevalonate by liver homogenate. In the present investigation
an attempt was made to locate the enzymic site
or sites of estrogen control of the hepatic synthesis of cholesterol from acetate. Because no hormone has yet been shown to influence sterol synthesis when added in vitro, the effects of estrogen were determined in microsomes isolated from rats pretreated with the hormone. The rates of conversion of acetate to mevalonate, mevalonate to squalene and squalene to cholesterol were compared in microsomal preparations from control and hormone-treated
rats. The results indicate that the hormone
markedly influences the biosynthesis at the pre-mevalonate stage; furthermore, the depression of hydroxymethylglutaryl-CoA synthesis appears to be of great significance in the estrogen regulation of the overall synthesis of cholesterol.
MATERIALS AND METHODS
Animals Young adult male albino rats of CDRI (Lucknow) strain were placed on stock rations and paired according to identical weight and age. The pairs were then separated; one of each pair was subjected to hormone treatment, while the other served as the control.
Hormone treatment ~@Estradiol, dissolved in propylene glycol, was administered intramuscularly each day while the control animal received an equal volume of the solvent. The dose of hormone was selected on the basis of preliminary short-term experiments. At lower doses, ranging from 5 to 50 pg/Ioo g rat weight, estrogen did not significantly influence the synthesis in vitro of cholesterol from radioacetate. Upon increasing the dosage from 50 to 150 ,ug based on IOO g animal weight, marked and consistent depression of sterol synthesis could be demonstrated when the level of hormone administered was IOO pg/day per IOO g rat weight. This dosage was selected for studying the effect of estrogen on enzymes of cholesterol biosynthesis from acetate in the liver. Biochim. Biophys. Ada,
164 (1968) 357-368
REGULATIONOF CHOLESTEROLBIOSYNTHESIS
359
Materials
All biochemicals used in this investigation were purchased from Sigma Chemical Co., U.S.A., and all reagent-grade chemicals used were purchased from E. Merck and Co., Germany. [I-l*C]Acetate, [z-%]mevalonic acid lactone and [I-Xlmevalonate were obtained from the Radiochemical Centre, England. [14C]Squalene was prepared in the laboratory from [I-%]acetate according to LANGDONAND BLOCH’~. [r,3,5-14C]hydroxymethylglutaric acid was obtained by direct enzymatic synthesis recommended by LYNEN~~.Both radioactive and unlabelled hydroxymethylglutaric acid were converted to their coenzyme A ester via anhydride by the method of HILZ et aLIs. Hydroxymethylglutaric acid CoA-condensing enzyme, used in the preparation of labelled hydroxymethylglutaric acid, was isolated from baker’s yeast according to LYNEN et al.16. Mevalonic acid kinase, used for the assay of mevalonic acid, was prepared according to TCHEN”. Preparation
of liver
Freshly excised rat livers cooled to o” were homogenized in a loose-fitting allglass homogenizer in Bucher’s medium, using z ml of buffer per g liver. The homogenates were centrifuged for IO min to obtain a 500 xg supernatant fraction employed in part of the study. Washed microsomes and dialyzed soluble enzymes were prepared from this supernatant fluid according to POPJAK’*. Microsomal extracts used for studying hydroxymethylglutaryl-CoA synthesis were obtained by the procedure of RUDNEYlD. Microsomes were directly sedimented from a 5000 xg supernatant fraction by ultracentrifugation at IOOOOO >cg for 30 min, the microsomal pellet homogenized in 2 vol. of 10-3 M versene, allowed to stand at o” for 5 min and recentrifuged at IOOOOO xg for 30 min. The resulting supernatant solution, containing the microsomal hydroxymethylglutaryl -CoA-condensing enzyme, was used for studying the rates of microsomal synthesis of hydroxymethylglutarylCoA. A soluble enzyme preparation from liver microsomes, used for studying the conversion of hydroxymethylglutaryl-CoA to mevalonic acid, was prepared by the method of BRODIE~~.Microsomes, sedimented from a IO ooo xg supernatant fraction, were washed and resuspended in potassium phosphate-bicarbonate buffer (0.1 M phosphate and 0.07 M bicarbonate), (pH 7.0) ; the protein concentration was adjusted to 30 mg per ml and the suspension stirred at o” for 6 h. The clear supernatant fluid was decanted and used for assaying hydroxymethylglutaryl-CoA-reductase activity. Incubation,
isolation ad
assay
The incubations were carried out as described for each experiment in the accompanying tables. Cholesterol was isolated for counting by saponification and digitonin precipitation, while the procedure outlined by LANGDONAND BLOCH’~for its preparation was employed for isolating squalene. Estimation of acetoacetate in tissue preparations. After incubation with [I-~~C]acetate, the mixture was chilled, protein precipitated by addition of trichloroacetic acid and the mixture centrifuged. The radioactive acetoacetate was decarboxylated by incubating it with aniline hydrochloride in the presence of 0.1 M potassium acetate buffer (pH 5.8) ; the acetone formed was converted to z,4-dinitrophenylhydrazone and Biochim. Biophys. Acta, 164 (1968) 357-368
S. MUKHERJEE, A. BHOSE
360
extracted with carbon tetrachloride. The extract was washed with potassium acetate, potassium bicarbonate and water and then concentrated in vacua. An aliquot was plated for the assay of radioactivity. For assay of hydroxymethylgluta~l-Co~4-condensing enzyme, the method of RUDNEY*~was employed. After incubation the flasks were heated to boiling, carrier hydroxymethylglutaric acid (0.1 M) was added and the acyl derivative hydrolyzed with 6 M KOH. After adding celite (z g/ml) directly to the mixture, [l*C!hydroxymethylglutaric acid was recovered by continuous overnight extraction with ether. The ether was removed under reduced pressure, the extract acidified with 0.5 M H&SO, and 50 mg of charcoal added. The suspension was filtered and the clear filtrate applied onto ~owex-~-(formate) columns, from which hydrox~~ethylglutaric acid was isolated by eluting with formic acid. Mevalonic acid, synthesized in microsomal extracts and prepared according to BRODIE~~,was converted to phosphomevalonic acid for assay according to BWCHER, OVERATH AKD LUNEN~. After incubation, the reaction was stopped by heating for 2 min on a boiling-water bath and extracted overnight with ether. The extract was made alkaline with 6 M KOH and the ether removed under reduced pressure. The residue containing the potassium salt of mevalonic acid was neutralized to pH 7.0 and re-incubated in the presence of 0.02 M solution of ATP, MgF, and potassium phosphate buffer (pH 7.0), serum albumin (3 mg/ml) and an excess of yeast mevalonic acid kinase. After a z-h incubation, Dowex-50 (H+ form) was added in excess and the mixture re-extracted overnight with ether. The radioactivity of the aqueous phase containing the phosphomevalonic acid was assayed. Hydroxymethylglutaryl-CoA-cleavage enzyme activity of unfractionated liver homogenates was assayed in the presence of excess hydroxymethylglutaryl-CoA-condensing enzyme, as recommended by BUCHER, OVERATHAND LYNF,N%,with slight modification. At the end of a 3o-min incubation, the [lPC]acetoacetate formed from [*4C]hydroxymethylglutaryl-CoA was estimated as described earlier. The radioactivity was assayed in a Tracerlab Superscaler gas-flow counter and self-absorption correction made whenever necessary. RESULTS The conversion of [r-Xlacetate to cholesterol in livers of control and estrogeninjected rats was measured over a period of eight weeks. The biosynthesis of cholesterol, studied in the 500 xg supernatant fraction of liver homogenate, is represented in Fig. I. Cholesterol synthesis was depressed to a significant extent within the first week of hormonal treatment, and by eight weeks, synthesis decreased to nearly 56% of the control value Marked inhibition of sterol biosynthesis was also observed in microsomes obtained from livers of estrogen-administered animals (Table I). The average fall in synthesis was nearly 43.5% in livers of rats given estrogen for eight days. However, when hormonal treatment was continued for eight weeks, there was no significant change from the values obtained after 8 days of hormonal administration. When microsomes from normal livers were incubated with soluble enzyme preparations from rats pretreated with estrogen, no depression of sterol synthesis resulted (Table II). Moreover, when microsomal preparations from normal and hormoneBiochim.
Biophys.
Acta,
164
(1968)
357-368
REGULATION OI; CHOLESTEROL BIOSYNTHESIS
4
6
I4 DAYS
26 OF TREATMENT
361
56
Fig. I. Inhibition of cholesterol synthesis in 500 x g supernatant fraction of liver homogenates of estrogen-administered rats. The rate of synthesis in estrogen-injected rats is expressedaspercentage of the rate for normal rat liver in the corresponding supernatant fraction. Each point represents the average of 6-8 experiments performed in duplicate. I ml of I : 4 homogenate in Bucher’s medium was incubated in air at 37” for 2 h in a medium containing 30 pmoles of GSH, I pmole of NADH, 0.5 ,umole of NADPH, 0.5 pmole of CoASH, 5 pmoles of ATP, 7 pmoles of MgCl, and 20 pmoles of [r-Klacetate (9. roB counts/min). TABLE
I
ESTROGEN
ADMINISTRATION
OF SYNTHESIS
OF CHOLESTEROL
FROM
ACETATE
IN RAT
LIVER
MICROSOMES
Washed microsomes (20-30 mg) were incubated in air at 37” for 3 h in presence of soluble enzymes in a medium containing 300 pmoles of potassium phosphate buffer (pH 7.0), IOO ,umoles nicotinamide, 30 pmoles GSH, 5 pmoles ATP, 7 pmoles M&l,, 1.5 pmoles CoASH, 1.0 ,umole NADH, 2.5 ,umoles NADPH and 200 ,umoles [I-%]acetate (9.10~ counts/min) in a total volume of 2.1 ml. Results expressed as pmoles acetate converted to cholesterol per mg of microsomal protein in I h. Expt. No.
Microsomes
Duration of treatment 8 weeks
8 days Cholesterol (countslmin)
Acetate + Cholesterol (,umoleslh)
Cholesterol (counts/m&)
Acetate + Cholesterol (pmoleslh)
Normal Estrogen-treated*
3375 2 160
0.037 0.024
2740 1500
0.030 0.017
2
Normal Estrogen-treated*
3870 2340
0.043 0.026
3010 I 760
0.034 0.020
3
Normal Estrogen-treated*
3150 2070
0.035 0.023
2560 1410
0.028 0.016
Normal Estrogen-treated*
3600 2345
0.040 0.026
3570 2070
0.039 0.023
I
4
* roe pg of estradiol per roe g weight of animal per day.
treated rats were combined and conversion of acetate to cholesterol was studied in the presence of normal supernatant fraction, the synthesis was merely additive. These experiments clearly exclude the possibility of formation of sterol-synthesis inhibitors following estrogen administration. The location
of site of inhibition
In order to locate the possible site of estrogen control of the enzymic site of cholesterol synthesis from acetate in liver, the microsomal rates of formation of mevalonic acid, squalene and cholesterol were measured using labelled acetate, Biochim. Biophys. Acta, 164 (1968) 357-368
S. MUKHERJEE, A. BHOSE
362 TABLE
II
CONVERSION
OF ACETATE
TO CHOLESTEROL
IN
MIXED
CELLULAR
FRACTIONS
Conditions and additions the same as those for Table I. Estrogen treatment continued for 8 weeks. Cellular fvactioxs Soluble fraction
Microsomes
Normal Normal Normal Hormone-treated Normal + hormone-treated TABLE SYNTHESIS TREATED
(I
Normal Hormone-treated Hormone-treated Hormone-treated Normal
: I)
Acetate + Cholesterol (countslminpev mg micvosomepvotein pev h) x IO-~ Expt. I
Expt.
2.64 2.58 2.58 I.55 2.21
2.56 2.56 2.57 1.50 2.22
2
III OF MEVALONIC
ACID
FROM
ACETATE
IN
LIVER
MICROSOME
OF NORMAL
AND
ESTROGEN-
RATS
Incubations carried out as described in Table I. The mixture contains in addition IOO pmoles of mevalonic acid. The mevalonate radioactivity was assayed by convelting it to phosphate as described in text. Results expressed as pmoles of mevalonate formed per h per mg of microsomal protein. Microsomes
Mevalonate (counts/m&) x Ioe3
Acetate + Mevalonic acid (~moles/h per mg)
Normal Estrogen-treated*
1.76
0.039
I.01
0.022
2
Normal Estrogen-treated*
I
.67 0.98
0.036
3
Normal Estrogen-treated**
I.89 0.95
0.042
4
Normal Estrogen-treated**
1.81
0.045 0.025
Expt. No. I
1.11
0.022
0.021
* Hormone treatment for 8 days. ** Hormone treatment continued for 8 weeks.
mevalonate and squalene as precursors for the above three stages of biosynthesis. The incorporation of [I-Wlacetate into mevalonic acid using mevalonate trap is shown in Table III. After 8 days of estrogen administration, a marked decrease in mevalonic acid synthesis was obtained. When estrogen treatment was continued for eight weeks, there was, however, no further decrease in the rates of conversion of acetate to mevalonic acid. Estrogelz
e$ect on steps after mevalonate
The transformation of [z-Xlmevalonate to squalene in microsomes of normal and estrogen-treated rat liver is represented in Table IV. The results indicate that estrogen treatment does not influence the conversion of mevalonic acid to subsequent intermediates leading to squalene synthesis in the microsomes. This fails to support the earlier finding in that mevalonic acid clecarboxylation occurs considerably less often in liver homogenates of rats pretreated with estrogenla. The microsomal rates of synthesis of cholesterol from squalene, represented in Table IV, were also unaffected by estrogen administration. Even when the duration of hormonal treatment was extended to a period of eight weeks, it did not influence the Biochim. Biophys. Ada,
164 (1968) 357-368
REGULATION TABLE EFFECT
OFCHOLESTEROL
BIOSYNTHESIS
363
IV OF ESTROGEN
TREATMENT
ON CONVERSION
OF MEVALONATE
TO SQUALENE
AND OF SQUALENE
TO CHOLESTEROL
Washed microsomes (21~35 mg) were incubated in a medium containing soluble enzymes and 2oo pmoles potassium phosphate buffer (pH 7.4), 15 pmoles ATP, 7 pmoles MgCI,, 8 pmoles NAD+, IOO pmoles nicotinamide, 30 pmoles GSH and 2 pmoles [z-‘*C]mevalonic acid (0.1 ,uC). Incubation carried out in N, gas phase for 3 h at 37“. For experiments on cholesterol synthesis from squalene incubation was carried out in air. The medium contained in addition I pmole NADH, I pmole NADP+, 2.5 ,umoles NADPH and 0.5 pmole of [%]squalene (9800 counts/min) suspension prepared according to TCHEN~~. Micvosomes
Mevalonate -+ Squalene (counts/min per mg microsomal protein) X xo-3
Sqwlene + Cholesterol (countslmin per mg microsomal protein) x 10-l
Normal Estrogen-treated*
3.67 3.51
975 912
2
Normal Estrogen-treated*
3.20 3.22
890 902
3
Normal Estrogen-treated*
3.5’ 3.47
845 960
Ex$t. No.
I
* Duration of hormone treatment
8 days.
biosynthesis of cholesterol when squalene was used as the precursor for its synthesis. In view of this apparent discrepancy between the present observation and that of MEROLA AND ARNOLD~~, mevalonic acid decarboxylation was re-investigated, using unfractionated liver homogenates in place of microsomes, to find out whether cellular fractions other than microsomes were responsible for the reported decrease in mevalonic acid decarboxylation. From the results presented in Table V, it would appear that pretreatment of rats with estrogen did not alter the rate of decarboxylation of [I-14C]mevalonic acid in liver. Additional evidence supporting this finding was obtained from rates of conversion of [z-W]mevalonic acid to cholesterol in portions of liver homogenates used for studying its decarboxylation. No significant change in the TABLE EFFECT INTO
V OF ESTROGEN
CHOLESTEROL
TREATMENT BY
RAT LIVER
ON DECARBOXYLATION
OF MEVALONATE
AND
ITS INCORPORATION
HOMOGENATE
The composition of the incubation mixture was essentially the same as in Table I. For decarboxylation or incorporation experiment the mixture contained I PC of either [I-‘“Clmevalonic acid or [a-%]mevalonic acid and 0.5 ml of 1:2 homogenate in potassium phosphate buffer (pH 7.0). Incubation was for I h at 37” in air. Ex+t. No.
Homogenate
I
Total counts in ‘4C02
counts/min per mg liver
[&*C]Mevalonicacid + Cholesterol (countslmin) x ro-4
Normal Estrogen-treated*
21200 21800
790 780
I.51 1.50
2
Normal Estrogen-treated*
16750 22 030
770 762
1.62 1.70
3
Normal Estrogen-treated*
25 890 27260
810 740
r.gr I.90
4
Normal Estrogen-treated*
23350 24000
760 770
I.33 1.50
* Hormone treatment
Decarboxylation
continued
of [I-r%]mevalonic
acid
for 8 days. Biochim. Biophys. Acta, 164 (1968) 357-368
S. MUKHERJEE, A. RHOSE
364 TABLE SYNTHESIS
VI OF
FROM
ACETOACETATE
ESTROGEN-TREATED
[I-I%]ACETATE
IN LIVER
HOMOGENATES
OF NORMAL
AND
RATS
The incubation mixture contained I ml of homogenate of liver in z vol. of 0.05 M potassium phosphate buffer (pH 7.4), 2opmoles of [I-%]acetate (12.2 mC/mmole), I pmole of semicarbazide, 7 pmoles M&l,, 30 pmoles GSH, r pmole CoASH. Incubation carried out for 30 min at 37”. I ml aliquot of incubation mixture deproteinized with IO yb trichloroacetic acid, incubated with 4 ml of aniline hydrochloride and I ml of I M acetate buffer (pH 5.6) in glass-stoppered tube for 90 min. The [“Clacetone converted to z,4-dinitrophenylhydrazone, extracted with carbon tetrachloride, washed with sodium acetate, sodium bicarbonate and water and radioactivity of hydrazone determined. E@pt. No. I
Homogenate
Acetoacetate (countslmin pev ml) x IO-~
Normal
22.1 21.0
Estrogen-treated* 2
25.4 26.0
Normal
Estrogen-treated* * Hormone treatment
for 8 days.
incorporation of [G4C]mevalonic acid into squalene could be demonstrated following hormonal administration for 8 days (Table V). The foregoing experiments thus strongly favour the hypothesis that estrogen inhibition of hepatic synthesis of cholesterol from acetate is due to hormonal influences on enzymes of mevalonic acid biosynthesis and steps subsequent to mevalonic acid are relatively independent of estrogen control. Eflect
of estrogen
on steps prior
to mevalonate
To obtain information on the site of estrogen inhibition of mevalonic acid synthesis from acetate, rates of formation of acetoacetate, hydroxymethylglutaric acid and mevalonic acid were compared in livers of normal and estrogen-treated rats. The acetoacetate production was studied in unfractionated liver homogenates in 0.05 M phosphate buffer (pH 7.4) and from results in Table VI it is evident that estrogen had no influence on the synthesis of acetoacetate. This indicated that the site TABLE
VII
EFFECT OF ESTROGEN TREATMENT ON THE INCORPORATION METHYLGLUTARIC ACID BY RAT LIVER MICROSOMAL EXTRACT
OF
[I-I'CIACETATE INTO HYDROXY-
0.5 mlof microsomalextract(200-250pg protein) incubatedin air at37'in presence of 200 pmoles potassium phosphate buffer (pH 7.4), 15 pmoles ATP, 7 pmoles MgCl,, 1.5 pmole CoASH, loo ,umoles hydroxymethylglutaric acid and 200 pmoles [I-%]acetate (9.10~ counts/min). Hydroxymethylgluteric acid isolated as described in text.
Radioactivity in hydvoxymethylglutaric acid
Acetate --f H~~droxymethylglutaric acid (pmoles/h per mg protein) x IO-~
Expt. No.
Micvosomal extract
I
Normal Estrogen-treated*
25.4 3.6
0.56 0.08
Normal Estrogen-treated*
24.1
0.53
8.0
0.18
Normal Estrogen-treated*
15.7 4.5
0.35 0.10
(countslmin)
2
3
x Io@
* Hormone treatment for 8 days. Biochim. Biophys. Acta, 164 (1968) 357-368
3%
REGULATION OF CHOLESTEROL BIOSYNTHESIS
of estrogen inhibition of sterol synthesis must be between acetoacetyl-CoA and mevalonic acid. To determine the effect of the hormone on the condensation step between acetyl-CoA and acetoacetyl-CoA, the hepatic activities of hydroxymethylglutaryl-CoA-condensing enzyme in control and estrogen-treated rats were compared in versene extracts of microsomes by the procedure of RUDNEY~~. The results of this study, shown in Table VII, clearly indicated that estrogen administration & vivo markedly depressed the synthesis of hydroxymethylglutaryl-CoA; the condensing enzyme activity in livers of these animals was only 30% of the control value. The microsomal extract used in this experiment contained most of the hepatic-condensing enzyme because the microsomes were directly sedimented from the 3000 xg supernatant fractions of liver homogenates. The effect of estrogen, however, appears to be considerably exaggerated probably due to the high activity of the versene-disrupted microsomal preparations since the actual inhibition of sterol synthesis in livers of estrogen-treated rats was only 45% of the control value. The marked depressionof hydroxymethylglutaric acid synthesis from radioacetate may, however, reflect a predominant estrogen control of the hydroxymethylglutaryl-CoA-condensing enzyme activity. To obtain unequivocal evidence of estrogen inhibition of condensing enzyme activity, it became imperative to study the hormonal influences on ancilliary enzymes, which may affect the concentration of hydroxymethylglutaryl-CoA in the liver of these animals. A comparison of hydroxymethylglutaryl-CoA-cleavage enzyme in liver homogenates of control and estrogen-treated rats was carried out using labelled hydroxymethylglutaryl-CoA. To provide for approximately similar rates for the condensation reaction, an excess of condensing enzyme from yeast was added to the assay mixture to overcome any deficiency of this enzyme in the liver of estrogentreated rats. The results of this assay, given in Table VIII, showed that no appreciable alteration in the cleavage enzyme activity occurred from hormonal administration. Because the depression of hydroxymethylglutaric acid synthesis under the experimental conditions employed cannot arise from increased deacylase action, the results can be interpreted as being due to estrogen control of the hepatic activity of the condensing enzyme. The evidences, therefore, imply that estrogen treatment TABLE
VIII
CLEAVAGEOF TREATED
HYDROXYMETHYLGLUTARYL-cd
BY LIVER
HOMOGENATES
OF NORMAL
AND
ESTROGEN-
RATS
0.05 ml of homogenate incubated with 1.0 pmole of [W]hydroxymethylglutaryl-CoA (5800 counts/min) in a medium containing 2oo pmoles Tris buffer (pH 74, 7 pmoles Na,S, 0.3 pmole CoASH, 14 ,umoles cysteine, 4 pmoles MgCl,, I unit of yeast hydroxymethylglutaryl-CoA-condensing enzyme and 0.3 mg serum albumin. Incubation at 37” for 30 min. Acetoacetate decarboxylated and isolated as described in text for counting. Results expressed as pmoles acetoacetate formed per mg protein in homogenate per h.
Expt. NO.
Microsomal
extract
Acetoacetate (,umoleslmg
Control Estrogen-treated*
0.96
2
Control Estrogen-treated*
I.02
0.97.
3
Control Estrogen-treated*
0.88 0.92
I
* Hormone
formed protein
per h per ml)
1.10
treatment for 8 days. Biochim.
Biophys.
Acta,
164 (1968) 357-368
366 TABLE EFFECT
S. MUKHERJEE, A. BHOSE IX OF
ESTROGEN
MEVALONATE
IN
ADMINISTRATION
MICROSOMAL
EXTRACTS
ON OF
CONVERSION RAT
OF
HYDROXYMETHYLGLUTARYL-cob.
TO
LIVER
The incubation mixture contained IOO ,umoles Tris buffer (pH 7.8), 30 pmoles GSH, 8 pmoles potassium EDTA, I ,umole NADH, I ,umole NADPH, 0.5 pmole CoASH, IO pmoles [‘“Clhydroxymethylglutaryl-Coil (5800 counts/min per pmole), 0.5 ml microsomal extract (zoo ,ug protein), 0.5 mg serum albumin in a total volume of 0.95 ml. Expt. No.
Micvosomal extract
I
Normal Estrogen-treated*
5oo 235
0.86 0.40
2
Normal Estrogen-treated*
305 185
0.52 0.32
3
Normal Estrogen-treated*
501 340
0.86 0.58
* Hormone
treatment
Hydroxymethylglutavyl CoA --f Mevalonic acid _ (counts/min per mg proteinjx IO+ (pmoleslh per mgprotein) x
IO-~
for 8 days.
causes a lowering of hydroxymethylglutaryl-CoA concentration for mevalonate synthesis in the livers of rats pretreated with the hormone. The microsomal rates of conversion of [14C]hydroxymethylglutaryl-CoA to mevalonic acid are represented in Table IX. The activity of hydroxymethylglutarylCoA reductase was studied in partially concentrated extracts of microsomes and the results showed that the hormone also caused a significant lowering of mevalonic acid synthesis in microsomes. The microsomal-reductase activity was approx. 50% lower for the treated animals as compared to controls, suggesting the existence of a second hormone-sensitive step in the biosynthetic pathway. DISCUSSION
From the evidences presented above, it is clear that estrogen inhibits cholesterol biosynthesis in rat liver by depressing the activity of enzymes responsible for the formation of hydroxymethylglutaryl-CoA and mevalonic acid. Although there are other pathways for its formation a2--24,hydroxymethylglutaryl-CoA is synthesized in liver mainly from acetyl-CoA and acetoacetyl-Cob by the action of the condensing enzyme. There is little evidence to suggest that reduced rates of hydroxymethylglutaryl-CoA formation in livers of estrogen-treated rats are due to any influence of the hormone on ancilliary enzymes, such as hydroxymethylglutaryl-CoA-cleavage enzyme, or hydroxymethylglutaryl-CoA deacylase or due to a decrease in acetoacetate formation. The comparative data on synthesis rates of compounds hydrolyzable to hydroxymethylglutaric acid in microsomal extracts of control and estrogen-treated rats, lead to the conclusion that inhibition of hepatic synthesis of cholesterol by estrogen is primarily due to the hormonal control of the condensation step. The diminished rate of microsomal synthesis of mevalonic acid from hydroxymethylglutarylCoA observed in rats pretreated with estrogen may presumably arise from an adaptive change in the hepatic concentration of the microsomal reductase resulting from a shortage of supply of its substrate or may represent a partial inhibition of the reductase activity by estrogen. A decrease in NADPH concentration in the liver of Biochim. Biophyys. Ada,
164 (1968) 357-368
367
REGULATION OF CHOLESTEROL BIOSYNTHESIS
estrogen-treated rats may also limit the conversion of hydroxymethylglutaryl-CoA by the NADPH-dependent microsomal reductase, but under the experimental conditions employed, the diminished rate of synthesis of mevalonic acid cannot be attributed to a change in the hepatic level of the nucleotide by the action of estrogensensitive transhydrogenate in the liverzs. A comparison of the microsomal rates of formation of mevalonic acid and cholesterol from acetate further indicates that estrogen inhibits both these reactions to nearly the same extent. Thus the net inhibition of cholesterol biosynthesis results from the hormonal control of the two key enzymes in the pre-mevalonate stages and is of quantitative significance in determining the overall change in synthesis of cholesterol from acetate in the liver of estrogen-administered rats. This conclusion seems justified because only minor effects have been obtained in the rates of incorporation of mevalonic acid to squalene or cholesterol following estrogen treatment. It thus appears that the most distinguishing feature of the estrogen control of hepatic synthesis of cholesterol from acetate is its inhibitory effect on the hydroxymethylglutaryl-CoA-condensing enzyme, while the natural inhibition caused by starvation3 or by cholesterol feeding496 concerns the activity of hydroxymethylglutaryl-CoA reductase. Although it is difficult to predict the exact mechanism of estrogen regulation of cholesterol biosynthesis from acetate, it seems possible that by inhibiting the activity of the condensing enzyme, estrogen may divert a part of the acetoacetyl-Cob to other metabolic pathways, thereby restricting its utilization for sterol synthesis. ACKNOWLEDGEMENTS
This investigation was supported by the U.S. Department of Agriculture Grant (FG-In-I&) under PL-480. Partial financial assistance during the initial stages of the investigation was received from the Council of Scientific and Industrial Research. The authors wish to acknowledge the technical assistance of Miss M. BOSE and MISS S. GUPTA in this investigation. The authors are indebted to Prof. J. W. PORTER for his assistance in the preparation
of hydroxymethylglutaryl-CoA
reductase
employed
in this study.
REFERENCES R. G. GOULD AND G. POPJAK. Biochem. J., 66 (1957) 51. N. L. R. BUCHER, P. OVERATH AND F. LYNEN, Biochim. Biophys. Acta, 40 (1960) 491. 0. WIELAND, G. LOFFLER, L. WEISS AND I. NENFELDT, Biochem. Z., 333 (1960) IO. M. D. SIPERSTEIN AND V. M. FAGAN, in G. WEBER, Advances in Enzyme Regulation, Vol. 2, Pergamon Press, New York,
1964. p. 249.
5 M. D. SIPERSTEIN AND V. M. FAGAN. 1. Biol. Chem.. UI 110661 602. 6 J. F. SCAIFE AND B. B. MIGICOVSKY; tan. J. Biochek,’ 3; (&5’7) 615. R. G. GOULD AND E. A. SWYRYD, J. Lipid Res., 7 (1966) 698. 87L. C. FILLIOS, R. KAPLAN, R. S. MARTIN AND F. STARE, Am. J. Physiol., 193 (1958) 47. 9 G. S. BOYD, Federation Proc., 21 (1962) Suppl. No. II, p. 86. 10 S. MUKHERJEE, S. GUPTA AND A. BHOSE, J. Atherosclerosis Res., 7 (1967) 435. Metabolism. Academic Press. New York. II G. S. BOYD, in J. K. GRANT, The Control of_ Li+id .
1963, p.
79.
-
12 A. J. MEROLA AND A. ARNOLD, Science, 144 (1964) 3616. I3 R. G. LANGDON AND K. BLOCH, J. Biol. Chem., zoo (1953) 135. 14 F. LYNEN, in G. E. W. WOLSTENHOLME AND M. O’CONNOR, CibaFound. Steroids, (1959) 95.
Symp. Biosyn. Terpenes
Biochim. Biophys. Acta, 164 (1968) 357-368
S. MUKHERJEE,
368
A. BHOSE
H. HILZ, J. KNAPPE, E. RINGELMANN AND F. LYNEN, Biochem. Z., 329 (1958) 476. F. LYNEN, H. EGGERER, U. HENNING AND I. KASSEL, Angew. Chem., 70 (1958) 739. T. T. TCHEN, J. Biol. Chem., 233 (1958) 1100. G. POPJAK, in G. PINCUS, Hormones and Athevosclerosis, Academic Press, New York, 1959, p. 7. H. RUDNEY, J. Biol. Chsm., 227 (1957) 363. J. BRODIE, Ph.D. Dissertation, University of Wisconsin, 1962. T. T. TCHEN, in S. P. COLOWICK AND N. 0. KAPLAN, Methods in Enzymology, Vol. V, Academic Press, New York, 1963. p. 505. 22 A. DEL CAMPILLO-CAMPBELL,E. K. DEKKER AND M. J. COON, Biochim. Biophys. Acta, 31 (1959)
15 16 17 18 Ig 20 21
481. 23 F. LYNEN, 481.
J. KNAPPE, E. LORCH, G. JOTTING AND E. RINGELMANN,
24 B. K. BACHHAWAT AND M. J. COON, J. Biol. Chem., 231 (1958) 625. 25 L. A. PESCH, Biochim. Biophys. Acta, 81 (1964) 229. Biochim.
Biophys.
Acta, 164 (1968) 357-368
Anger.
Chem., 71 (1959)
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 55499
STUDIES
ON ESTROGEN
BIOSYNTHESIS
REGULATION
IN RAT LIVER
OF CHOLESTEROL
MICROSOMES
S.MUKHERJEEANDA.BHOSE Laboratories of Lipid Research, Department of Applied Chemistry, University of Calcutta, Calcutta (India)
(Received June Ioth,1968)
SUMMARY
An inhibition of cholesterol biosynthesis from acetate has been demonstrated in the liver of rats previously administered IOO pg (per IOO g rat) of I7b-estradiol for periods varying from 4 to 56 days. The site of estrogen control of hepatic sterol synthesis has been investigated in microsomal preparations. The inhibitory effect of exogenous estrogen is observed mostly at premevalonate stages of biosynthesis; the conversion of mevalonate to squalene or of squalene to cholesterol is not significantly altered by hormonal administration. The first estrogen-sensitive step in the pathway of biosynthesis appears to be the condensation of acetoacetyl-CoA and acetyl-CoA, since markedly reduced activity of the hydroxymethylglutaryl-CoA-condensing enzyme has been demonstrated in liver microsomes isolated from treated animals. A decrease in the microsomal hydroxymethylglutaryl-CoA reductase activity also results from the hormone treatment. The overall depression in the rate of cholesterol synthesis is determined by the hormonal inhibition of mevalonate synthesis in microsomes. Estrogen has very little influence on hydroxymethylglutaryl-CoA-cleavage enzyme activity.
INTRODUCTION
The effects of dietary cholesterol and fasting have predominated most studies attempting to elucidate the physiological mechanism controlling hepatic synthesis of cholesterol from acetate. GOULD AND POPJAK' provided evidence that exogenous cholesterol inhibits cholesterol synthesis by controlling steps between acetate and mevalonate. BUCHER, OVERATH AND LYNEN~ found that fasting greatly reduced the incorporation of acetate into cholesterol in rat liver by suppressing the activity of hydroxymethylglutaryl-CoA reductase. The effect of starvation on the microsomal reductase has since been confirmed by WIELAND et a1.3. SIPERSTEIN AND FAGAN%& made strikingly similar observations of the control of hydroxymethylglutaryl-CoA reductase activity in their studies on the mechanism of feed-back inhibition of cholesterol synthesis by dietary cholesterol. On the other hand, evidence of a metabolic Biochim. Biophys. Acta, 164 (1968) 357-368
S. MUKHERJEE, A. BHOSE
358
block between mevalonate and squalene in livers of fasted rats was suggested by SCAIFE AND MIGICOVSKY~. This implied that regulation of hepatic biosynthesis of cholesterol might involve more than one enzymic site. Further support for this hypothesis was obtained from the more recent studies of GOULD AND SWYRYD’ who demonstrated that prolonged hydroxymethylglutaryl-CoA
cholesterol feeding not only depressed the activity of reductase but almost completely inhibited the conver-
sion of mevalonic acid into squalene by affecting two other enzymes in the post-mevalonate stages of biosynthesis. The incorporation of acetate into cholesterol in rat liver is greatly influenced by endogenous as well as exogenous hormones. Evidences
of decreased sterol syn-
thesis in rat liver by estrogen administration have been presented in many experimental studies-lo, but the precise biochemical site for estrogen inhibition of cholesterol synthesis from acetate has not been established. While BOYD’~ suggested that endogenous estrogen influenced cholesterol synthesis at a point between acetate and mevalonate, MEROLA AND ARNOLD~~ demonstrated that oral administration of this hormone decreased the rate of decarboxylation of mevalonate by liver homogenate. In the present investigation
an attempt was made to locate the enzymic site
or sites of estrogen control of the hepatic synthesis of cholesterol from acetate. Because no hormone has yet been shown to influence sterol synthesis when added in vitro, the effects of estrogen were determined in microsomes isolated from rats pretreated with the hormone. The rates of conversion of acetate to mevalonate, mevalonate to squalene and squalene to cholesterol were compared in microsomal preparations from control and hormone-treated
rats. The results indicate that the hormone
markedly influences the biosynthesis at the pre-mevalonate stage; furthermore, the depression of hydroxymethylglutaryl-CoA synthesis appears to be of great significance in the estrogen regulation of the overall synthesis of cholesterol.
MATERIALS AND METHODS
Animals Young adult male albino rats of CDRI (Lucknow) strain were placed on stock rations and paired according to identical weight and age. The pairs were then separated; one of each pair was subjected to hormone treatment, while the other served as the control.
Hormone treatment ~@Estradiol, dissolved in propylene glycol, was administered intramuscularly each day while the control animal received an equal volume of the solvent. The dose of hormone was selected on the basis of preliminary short-term experiments. At lower doses, ranging from 5 to 50 pg/Ioo g rat weight, estrogen did not significantly influence the synthesis in vitro of cholesterol from radioacetate. Upon increasing the dosage from 50 to 150 ,ug based on IOO g animal weight, marked and consistent depression of sterol synthesis could be demonstrated when the level of hormone administered was IOO pg/day per IOO g rat weight. This dosage was selected for studying the effect of estrogen on enzymes of cholesterol biosynthesis from acetate in the liver. Biochim. Biophys. Ada,
164 (1968) 357-368
REGULATIONOF CHOLESTEROLBIOSYNTHESIS
359
Materials
All biochemicals used in this investigation were purchased from Sigma Chemical Co., U.S.A., and all reagent-grade chemicals used were purchased from E. Merck and Co., Germany. [I-l*C]Acetate, [z-%]mevalonic acid lactone and [I-Xlmevalonate were obtained from the Radiochemical Centre, England. [14C]Squalene was prepared in the laboratory from [I-%]acetate according to LANGDONAND BLOCH’~. [r,3,5-14C]hydroxymethylglutaric acid was obtained by direct enzymatic synthesis recommended by LYNEN~~.Both radioactive and unlabelled hydroxymethylglutaric acid were converted to their coenzyme A ester via anhydride by the method of HILZ et aLIs. Hydroxymethylglutaric acid CoA-condensing enzyme, used in the preparation of labelled hydroxymethylglutaric acid, was isolated from baker’s yeast according to LYNEN et al.16. Mevalonic acid kinase, used for the assay of mevalonic acid, was prepared according to TCHEN”. Preparation
of liver
Freshly excised rat livers cooled to o” were homogenized in a loose-fitting allglass homogenizer in Bucher’s medium, using z ml of buffer per g liver. The homogenates were centrifuged for IO min to obtain a 500 xg supernatant fraction employed in part of the study. Washed microsomes and dialyzed soluble enzymes were prepared from this supernatant fluid according to POPJAK’*. Microsomal extracts used for studying hydroxymethylglutaryl-CoA synthesis were obtained by the procedure of RUDNEYlD. Microsomes were directly sedimented from a 5000 xg supernatant fraction by ultracentrifugation at IOOOOO >cg for 30 min, the microsomal pellet homogenized in 2 vol. of 10-3 M versene, allowed to stand at o” for 5 min and recentrifuged at IOOOOO xg for 30 min. The resulting supernatant solution, containing the microsomal hydroxymethylglutaryl -CoA-condensing enzyme, was used for studying the rates of microsomal synthesis of hydroxymethylglutarylCoA. A soluble enzyme preparation from liver microsomes, used for studying the conversion of hydroxymethylglutaryl-CoA to mevalonic acid, was prepared by the method of BRODIE~~.Microsomes, sedimented from a IO ooo xg supernatant fraction, were washed and resuspended in potassium phosphate-bicarbonate buffer (0.1 M phosphate and 0.07 M bicarbonate), (pH 7.0) ; the protein concentration was adjusted to 30 mg per ml and the suspension stirred at o” for 6 h. The clear supernatant fluid was decanted and used for assaying hydroxymethylglutaryl-CoA-reductase activity. Incubation,
isolation ad
assay
The incubations were carried out as described for each experiment in the accompanying tables. Cholesterol was isolated for counting by saponification and digitonin precipitation, while the procedure outlined by LANGDONAND BLOCH’~for its preparation was employed for isolating squalene. Estimation of acetoacetate in tissue preparations. After incubation with [I-~~C]acetate, the mixture was chilled, protein precipitated by addition of trichloroacetic acid and the mixture centrifuged. The radioactive acetoacetate was decarboxylated by incubating it with aniline hydrochloride in the presence of 0.1 M potassium acetate buffer (pH 5.8) ; the acetone formed was converted to z,4-dinitrophenylhydrazone and Biochim. Biophys. Acta, 164 (1968) 357-368
S. MUKHERJEE, A. BHOSE
360
extracted with carbon tetrachloride. The extract was washed with potassium acetate, potassium bicarbonate and water and then concentrated in vacua. An aliquot was plated for the assay of radioactivity. For assay of hydroxymethylgluta~l-Co~4-condensing enzyme, the method of RUDNEY*~was employed. After incubation the flasks were heated to boiling, carrier hydroxymethylglutaric acid (0.1 M) was added and the acyl derivative hydrolyzed with 6 M KOH. After adding celite (z g/ml) directly to the mixture, [l*C!hydroxymethylglutaric acid was recovered by continuous overnight extraction with ether. The ether was removed under reduced pressure, the extract acidified with 0.5 M H&SO, and 50 mg of charcoal added. The suspension was filtered and the clear filtrate applied onto ~owex-~-(formate) columns, from which hydrox~~ethylglutaric acid was isolated by eluting with formic acid. Mevalonic acid, synthesized in microsomal extracts and prepared according to BRODIE~~,was converted to phosphomevalonic acid for assay according to BWCHER, OVERATH AKD LUNEN~. After incubation, the reaction was stopped by heating for 2 min on a boiling-water bath and extracted overnight with ether. The extract was made alkaline with 6 M KOH and the ether removed under reduced pressure. The residue containing the potassium salt of mevalonic acid was neutralized to pH 7.0 and re-incubated in the presence of 0.02 M solution of ATP, MgF, and potassium phosphate buffer (pH 7.0), serum albumin (3 mg/ml) and an excess of yeast mevalonic acid kinase. After a z-h incubation, Dowex-50 (H+ form) was added in excess and the mixture re-extracted overnight with ether. The radioactivity of the aqueous phase containing the phosphomevalonic acid was assayed. Hydroxymethylglutaryl-CoA-cleavage enzyme activity of unfractionated liver homogenates was assayed in the presence of excess hydroxymethylglutaryl-CoA-condensing enzyme, as recommended by BUCHER, OVERATHAND LYNF,N%,with slight modification. At the end of a 3o-min incubation, the [lPC]acetoacetate formed from [*4C]hydroxymethylglutaryl-CoA was estimated as described earlier. The radioactivity was assayed in a Tracerlab Superscaler gas-flow counter and self-absorption correction made whenever necessary. RESULTS The conversion of [r-Xlacetate to cholesterol in livers of control and estrogeninjected rats was measured over a period of eight weeks. The biosynthesis of cholesterol, studied in the 500 xg supernatant fraction of liver homogenate, is represented in Fig. I. Cholesterol synthesis was depressed to a significant extent within the first week of hormonal treatment, and by eight weeks, synthesis decreased to nearly 56% of the control value Marked inhibition of sterol biosynthesis was also observed in microsomes obtained from livers of estrogen-administered animals (Table I). The average fall in synthesis was nearly 43.5% in livers of rats given estrogen for eight days. However, when hormonal treatment was continued for eight weeks, there was no significant change from the values obtained after 8 days of hormonal administration. When microsomes from normal livers were incubated with soluble enzyme preparations from rats pretreated with estrogen, no depression of sterol synthesis resulted (Table II). Moreover, when microsomal preparations from normal and hormoneBiochim.
Biophys.
Acta,
164
(1968)
357-368
REGULATION OI; CHOLESTEROL BIOSYNTHESIS
4
6
I4 DAYS
26 OF TREATMENT
361
56
Fig. I. Inhibition of cholesterol synthesis in 500 x g supernatant fraction of liver homogenates of estrogen-administered rats. The rate of synthesis in estrogen-injected rats is expressedaspercentage of the rate for normal rat liver in the corresponding supernatant fraction. Each point represents the average of 6-8 experiments performed in duplicate. I ml of I : 4 homogenate in Bucher’s medium was incubated in air at 37” for 2 h in a medium containing 30 pmoles of GSH, I pmole of NADH, 0.5 ,umole of NADPH, 0.5 pmole of CoASH, 5 pmoles of ATP, 7 pmoles of MgCl, and 20 pmoles of [r-Klacetate (9. roB counts/min). TABLE
I
ESTROGEN
ADMINISTRATION
OF SYNTHESIS
OF CHOLESTEROL
FROM
ACETATE
IN RAT
LIVER
MICROSOMES
Washed microsomes (20-30 mg) were incubated in air at 37” for 3 h in presence of soluble enzymes in a medium containing 300 pmoles of potassium phosphate buffer (pH 7.0), IOO ,umoles nicotinamide, 30 pmoles GSH, 5 pmoles ATP, 7 pmoles M&l,, 1.5 pmoles CoASH, 1.0 ,umole NADH, 2.5 ,umoles NADPH and 200 ,umoles [I-%]acetate (9.10~ counts/min) in a total volume of 2.1 ml. Results expressed as pmoles acetate converted to cholesterol per mg of microsomal protein in I h. Expt. No.
Microsomes
Duration of treatment 8 weeks
8 days Cholesterol (countslmin)
Acetate + Cholesterol (,umoleslh)
Cholesterol (counts/m&)
Acetate + Cholesterol (pmoleslh)
Normal Estrogen-treated*
3375 2 160
0.037 0.024
2740 1500
0.030 0.017
2
Normal Estrogen-treated*
3870 2340
0.043 0.026
3010 I 760
0.034 0.020
3
Normal Estrogen-treated*
3150 2070
0.035 0.023
2560 1410
0.028 0.016
Normal Estrogen-treated*
3600 2345
0.040 0.026
3570 2070
0.039 0.023
I
4
* roe pg of estradiol per roe g weight of animal per day.
treated rats were combined and conversion of acetate to cholesterol was studied in the presence of normal supernatant fraction, the synthesis was merely additive. These experiments clearly exclude the possibility of formation of sterol-synthesis inhibitors following estrogen administration. The location
of site of inhibition
In order to locate the possible site of estrogen control of the enzymic site of cholesterol synthesis from acetate in liver, the microsomal rates of formation of mevalonic acid, squalene and cholesterol were measured using labelled acetate, Biochim. Biophys. Acta, 164 (1968) 357-368
S. MUKHERJEE, A. BHOSE
362 TABLE
II
CONVERSION
OF ACETATE
TO CHOLESTEROL
IN
MIXED
CELLULAR
FRACTIONS
Conditions and additions the same as those for Table I. Estrogen treatment continued for 8 weeks. Cellular fvactioxs Soluble fraction
Microsomes
Normal Normal Normal Hormone-treated Normal + hormone-treated TABLE SYNTHESIS TREATED
(I
Normal Hormone-treated Hormone-treated Hormone-treated Normal
: I)
Acetate + Cholesterol (countslminpev mg micvosomepvotein pev h) x IO-~ Expt. I
Expt.
2.64 2.58 2.58 I.55 2.21
2.56 2.56 2.57 1.50 2.22
2
III OF MEVALONIC
ACID
FROM
ACETATE
IN
LIVER
MICROSOME
OF NORMAL
AND
ESTROGEN-
RATS
Incubations carried out as described in Table I. The mixture contains in addition IOO pmoles of mevalonic acid. The mevalonate radioactivity was assayed by convelting it to phosphate as described in text. Results expressed as pmoles of mevalonate formed per h per mg of microsomal protein. Microsomes
Mevalonate (counts/m&) x Ioe3
Acetate + Mevalonic acid (~moles/h per mg)
Normal Estrogen-treated*
1.76
0.039
I.01
0.022
2
Normal Estrogen-treated*
I
.67 0.98
0.036
3
Normal Estrogen-treated**
I.89 0.95
0.042
4
Normal Estrogen-treated**
1.81
0.045 0.025
Expt. No. I
1.11
0.022
0.021
* Hormone treatment for 8 days. ** Hormone treatment continued for 8 weeks.
mevalonate and squalene as precursors for the above three stages of biosynthesis. The incorporation of [I-Wlacetate into mevalonic acid using mevalonate trap is shown in Table III. After 8 days of estrogen administration, a marked decrease in mevalonic acid synthesis was obtained. When estrogen treatment was continued for eight weeks, there was, however, no further decrease in the rates of conversion of acetate to mevalonic acid. Estrogelz
e$ect on steps after mevalonate
The transformation of [z-Xlmevalonate to squalene in microsomes of normal and estrogen-treated rat liver is represented in Table IV. The results indicate that estrogen treatment does not influence the conversion of mevalonic acid to subsequent intermediates leading to squalene synthesis in the microsomes. This fails to support the earlier finding in that mevalonic acid clecarboxylation occurs considerably less often in liver homogenates of rats pretreated with estrogenla. The microsomal rates of synthesis of cholesterol from squalene, represented in Table IV, were also unaffected by estrogen administration. Even when the duration of hormonal treatment was extended to a period of eight weeks, it did not influence the Biochim. Biophys. Ada,
164 (1968) 357-368
REGULATION TABLE EFFECT
OFCHOLESTEROL
BIOSYNTHESIS
363
IV OF ESTROGEN
TREATMENT
ON CONVERSION
OF MEVALONATE
TO SQUALENE
AND OF SQUALENE
TO CHOLESTEROL
Washed microsomes (21~35 mg) were incubated in a medium containing soluble enzymes and 2oo pmoles potassium phosphate buffer (pH 7.4), 15 pmoles ATP, 7 pmoles MgCI,, 8 pmoles NAD+, IOO pmoles nicotinamide, 30 pmoles GSH and 2 pmoles [z-‘*C]mevalonic acid (0.1 ,uC). Incubation carried out in N, gas phase for 3 h at 37“. For experiments on cholesterol synthesis from squalene incubation was carried out in air. The medium contained in addition I pmole NADH, I pmole NADP+, 2.5 ,umoles NADPH and 0.5 pmole of [%]squalene (9800 counts/min) suspension prepared according to TCHEN~~. Micvosomes
Mevalonate -+ Squalene (counts/min per mg microsomal protein) X xo-3
Sqwlene + Cholesterol (countslmin per mg microsomal protein) x 10-l
Normal Estrogen-treated*
3.67 3.51
975 912
2
Normal Estrogen-treated*
3.20 3.22
890 902
3
Normal Estrogen-treated*
3.5’ 3.47
845 960
Ex$t. No.
I
* Duration of hormone treatment
8 days.
biosynthesis of cholesterol when squalene was used as the precursor for its synthesis. In view of this apparent discrepancy between the present observation and that of MEROLA AND ARNOLD~~, mevalonic acid decarboxylation was re-investigated, using unfractionated liver homogenates in place of microsomes, to find out whether cellular fractions other than microsomes were responsible for the reported decrease in mevalonic acid decarboxylation. From the results presented in Table V, it would appear that pretreatment of rats with estrogen did not alter the rate of decarboxylation of [I-14C]mevalonic acid in liver. Additional evidence supporting this finding was obtained from rates of conversion of [z-W]mevalonic acid to cholesterol in portions of liver homogenates used for studying its decarboxylation. No significant change in the TABLE EFFECT INTO
V OF ESTROGEN
CHOLESTEROL
TREATMENT BY
RAT LIVER
ON DECARBOXYLATION
OF MEVALONATE
AND
ITS INCORPORATION
HOMOGENATE
The composition of the incubation mixture was essentially the same as in Table I. For decarboxylation or incorporation experiment the mixture contained I PC of either [I-‘“Clmevalonic acid or [a-%]mevalonic acid and 0.5 ml of 1:2 homogenate in potassium phosphate buffer (pH 7.0). Incubation was for I h at 37” in air. Ex+t. No.
Homogenate
I
Total counts in ‘4C02
counts/min per mg liver
[&*C]Mevalonicacid + Cholesterol (countslmin) x ro-4
Normal Estrogen-treated*
21200 21800
790 780
I.51 1.50
2
Normal Estrogen-treated*
16750 22 030
770 762
1.62 1.70
3
Normal Estrogen-treated*
25 890 27260
810 740
r.gr I.90
4
Normal Estrogen-treated*
23350 24000
760 770
I.33 1.50
* Hormone treatment
Decarboxylation
continued
of [I-r%]mevalonic
acid
for 8 days. Biochim. Biophys. Acta, 164 (1968) 357-368
S. MUKHERJEE, A. RHOSE
364 TABLE SYNTHESIS
VI OF
FROM
ACETOACETATE
ESTROGEN-TREATED
[I-I%]ACETATE
IN LIVER
HOMOGENATES
OF NORMAL
AND
RATS
The incubation mixture contained I ml of homogenate of liver in z vol. of 0.05 M potassium phosphate buffer (pH 7.4), 2opmoles of [I-%]acetate (12.2 mC/mmole), I pmole of semicarbazide, 7 pmoles M&l,, 30 pmoles GSH, r pmole CoASH. Incubation carried out for 30 min at 37”. I ml aliquot of incubation mixture deproteinized with IO yb trichloroacetic acid, incubated with 4 ml of aniline hydrochloride and I ml of I M acetate buffer (pH 5.6) in glass-stoppered tube for 90 min. The [“Clacetone converted to z,4-dinitrophenylhydrazone, extracted with carbon tetrachloride, washed with sodium acetate, sodium bicarbonate and water and radioactivity of hydrazone determined. E@pt. No. I
Homogenate
Acetoacetate (countslmin pev ml) x IO-~
Normal
22.1 21.0
Estrogen-treated* 2
25.4 26.0
Normal
Estrogen-treated* * Hormone treatment
for 8 days.
incorporation of [G4C]mevalonic acid into squalene could be demonstrated following hormonal administration for 8 days (Table V). The foregoing experiments thus strongly favour the hypothesis that estrogen inhibition of hepatic synthesis of cholesterol from acetate is due to hormonal influences on enzymes of mevalonic acid biosynthesis and steps subsequent to mevalonic acid are relatively independent of estrogen control. Eflect
of estrogen
on steps prior
to mevalonate
To obtain information on the site of estrogen inhibition of mevalonic acid synthesis from acetate, rates of formation of acetoacetate, hydroxymethylglutaric acid and mevalonic acid were compared in livers of normal and estrogen-treated rats. The acetoacetate production was studied in unfractionated liver homogenates in 0.05 M phosphate buffer (pH 7.4) and from results in Table VI it is evident that estrogen had no influence on the synthesis of acetoacetate. This indicated that the site TABLE
VII
EFFECT OF ESTROGEN TREATMENT ON THE INCORPORATION METHYLGLUTARIC ACID BY RAT LIVER MICROSOMAL EXTRACT
OF
[I-I'CIACETATE INTO HYDROXY-
0.5 mlof microsomalextract(200-250pg protein) incubatedin air at37'in presence of 200 pmoles potassium phosphate buffer (pH 7.4), 15 pmoles ATP, 7 pmoles MgCl,, 1.5 pmole CoASH, loo ,umoles hydroxymethylglutaric acid and 200 pmoles [I-%]acetate (9.10~ counts/min). Hydroxymethylgluteric acid isolated as described in text.
Radioactivity in hydvoxymethylglutaric acid
Acetate --f H~~droxymethylglutaric acid (pmoles/h per mg protein) x IO-~
Expt. No.
Micvosomal extract
I
Normal Estrogen-treated*
25.4 3.6
0.56 0.08
Normal Estrogen-treated*
24.1
0.53
8.0
0.18
Normal Estrogen-treated*
15.7 4.5
0.35 0.10
(countslmin)
2
3
x Io@
* Hormone treatment for 8 days. Biochim. Biophys. Acta, 164 (1968) 357-368
3%
REGULATION OF CHOLESTEROL BIOSYNTHESIS
of estrogen inhibition of sterol synthesis must be between acetoacetyl-CoA and mevalonic acid. To determine the effect of the hormone on the condensation step between acetyl-CoA and acetoacetyl-CoA, the hepatic activities of hydroxymethylglutaryl-CoA-condensing enzyme in control and estrogen-treated rats were compared in versene extracts of microsomes by the procedure of RUDNEY~~. The results of this study, shown in Table VII, clearly indicated that estrogen administration & vivo markedly depressed the synthesis of hydroxymethylglutaryl-CoA; the condensing enzyme activity in livers of these animals was only 30% of the control value. The microsomal extract used in this experiment contained most of the hepatic-condensing enzyme because the microsomes were directly sedimented from the 3000 xg supernatant fractions of liver homogenates. The effect of estrogen, however, appears to be considerably exaggerated probably due to the high activity of the versene-disrupted microsomal preparations since the actual inhibition of sterol synthesis in livers of estrogen-treated rats was only 45% of the control value. The marked depressionof hydroxymethylglutaric acid synthesis from radioacetate may, however, reflect a predominant estrogen control of the hydroxymethylglutaryl-CoA-condensing enzyme activity. To obtain unequivocal evidence of estrogen inhibition of condensing enzyme activity, it became imperative to study the hormonal influences on ancilliary enzymes, which may affect the concentration of hydroxymethylglutaryl-CoA in the liver of these animals. A comparison of hydroxymethylglutaryl-CoA-cleavage enzyme in liver homogenates of control and estrogen-treated rats was carried out using labelled hydroxymethylglutaryl-CoA. To provide for approximately similar rates for the condensation reaction, an excess of condensing enzyme from yeast was added to the assay mixture to overcome any deficiency of this enzyme in the liver of estrogentreated rats. The results of this assay, given in Table VIII, showed that no appreciable alteration in the cleavage enzyme activity occurred from hormonal administration. Because the depression of hydroxymethylglutaric acid synthesis under the experimental conditions employed cannot arise from increased deacylase action, the results can be interpreted as being due to estrogen control of the hepatic activity of the condensing enzyme. The evidences, therefore, imply that estrogen treatment TABLE
VIII
CLEAVAGEOF TREATED
HYDROXYMETHYLGLUTARYL-cd
BY LIVER
HOMOGENATES
OF NORMAL
AND
ESTROGEN-
RATS
0.05 ml of homogenate incubated with 1.0 pmole of [W]hydroxymethylglutaryl-CoA (5800 counts/min) in a medium containing 2oo pmoles Tris buffer (pH 74, 7 pmoles Na,S, 0.3 pmole CoASH, 14 ,umoles cysteine, 4 pmoles MgCl,, I unit of yeast hydroxymethylglutaryl-CoA-condensing enzyme and 0.3 mg serum albumin. Incubation at 37” for 30 min. Acetoacetate decarboxylated and isolated as described in text for counting. Results expressed as pmoles acetoacetate formed per mg protein in homogenate per h.
Expt. NO.
Microsomal
extract
Acetoacetate (,umoleslmg
Control Estrogen-treated*
0.96
2
Control Estrogen-treated*
I.02
0.97.
3
Control Estrogen-treated*
0.88 0.92
I
* Hormone
formed protein
per h per ml)
1.10
treatment for 8 days. Biochim.
Biophys.
Acta,
164 (1968) 357-368
366 TABLE EFFECT
S. MUKHERJEE, A. BHOSE IX OF
ESTROGEN
MEVALONATE
IN
ADMINISTRATION
MICROSOMAL
EXTRACTS
ON OF
CONVERSION RAT
OF
HYDROXYMETHYLGLUTARYL-cob.
TO
LIVER
The incubation mixture contained IOO ,umoles Tris buffer (pH 7.8), 30 pmoles GSH, 8 pmoles potassium EDTA, I ,umole NADH, I ,umole NADPH, 0.5 pmole CoASH, IO pmoles [‘“Clhydroxymethylglutaryl-Coil (5800 counts/min per pmole), 0.5 ml microsomal extract (zoo ,ug protein), 0.5 mg serum albumin in a total volume of 0.95 ml. Expt. No.
Micvosomal extract
I
Normal Estrogen-treated*
5oo 235
0.86 0.40
2
Normal Estrogen-treated*
305 185
0.52 0.32
3
Normal Estrogen-treated*
501 340
0.86 0.58
* Hormone
treatment
Hydroxymethylglutavyl CoA --f Mevalonic acid _ (counts/min per mg proteinjx IO+ (pmoleslh per mgprotein) x
IO-~
for 8 days.
causes a lowering of hydroxymethylglutaryl-CoA concentration for mevalonate synthesis in the livers of rats pretreated with the hormone. The microsomal rates of conversion of [14C]hydroxymethylglutaryl-CoA to mevalonic acid are represented in Table IX. The activity of hydroxymethylglutarylCoA reductase was studied in partially concentrated extracts of microsomes and the results showed that the hormone also caused a significant lowering of mevalonic acid synthesis in microsomes. The microsomal-reductase activity was approx. 50% lower for the treated animals as compared to controls, suggesting the existence of a second hormone-sensitive step in the biosynthetic pathway. DISCUSSION
From the evidences presented above, it is clear that estrogen inhibits cholesterol biosynthesis in rat liver by depressing the activity of enzymes responsible for the formation of hydroxymethylglutaryl-CoA and mevalonic acid. Although there are other pathways for its formation a2--24,hydroxymethylglutaryl-CoA is synthesized in liver mainly from acetyl-CoA and acetoacetyl-Cob by the action of the condensing enzyme. There is little evidence to suggest that reduced rates of hydroxymethylglutaryl-CoA formation in livers of estrogen-treated rats are due to any influence of the hormone on ancilliary enzymes, such as hydroxymethylglutaryl-CoA-cleavage enzyme, or hydroxymethylglutaryl-CoA deacylase or due to a decrease in acetoacetate formation. The comparative data on synthesis rates of compounds hydrolyzable to hydroxymethylglutaric acid in microsomal extracts of control and estrogen-treated rats, lead to the conclusion that inhibition of hepatic synthesis of cholesterol by estrogen is primarily due to the hormonal control of the condensation step. The diminished rate of microsomal synthesis of mevalonic acid from hydroxymethylglutarylCoA observed in rats pretreated with estrogen may presumably arise from an adaptive change in the hepatic concentration of the microsomal reductase resulting from a shortage of supply of its substrate or may represent a partial inhibition of the reductase activity by estrogen. A decrease in NADPH concentration in the liver of Biochim. Biophyys. Ada,
164 (1968) 357-368
367
REGULATION OF CHOLESTEROL BIOSYNTHESIS
estrogen-treated rats may also limit the conversion of hydroxymethylglutaryl-CoA by the NADPH-dependent microsomal reductase, but under the experimental conditions employed, the diminished rate of synthesis of mevalonic acid cannot be attributed to a change in the hepatic level of the nucleotide by the action of estrogensensitive transhydrogenate in the liverzs. A comparison of the microsomal rates of formation of mevalonic acid and cholesterol from acetate further indicates that estrogen inhibits both these reactions to nearly the same extent. Thus the net inhibition of cholesterol biosynthesis results from the hormonal control of the two key enzymes in the pre-mevalonate stages and is of quantitative significance in determining the overall change in synthesis of cholesterol from acetate in the liver of estrogen-administered rats. This conclusion seems justified because only minor effects have been obtained in the rates of incorporation of mevalonic acid to squalene or cholesterol following estrogen treatment. It thus appears that the most distinguishing feature of the estrogen control of hepatic synthesis of cholesterol from acetate is its inhibitory effect on the hydroxymethylglutaryl-CoA-condensing enzyme, while the natural inhibition caused by starvation3 or by cholesterol feeding496 concerns the activity of hydroxymethylglutaryl-CoA reductase. Although it is difficult to predict the exact mechanism of estrogen regulation of cholesterol biosynthesis from acetate, it seems possible that by inhibiting the activity of the condensing enzyme, estrogen may divert a part of the acetoacetyl-Cob to other metabolic pathways, thereby restricting its utilization for sterol synthesis. ACKNOWLEDGEMENTS
This investigation was supported by the U.S. Department of Agriculture Grant (FG-In-I&) under PL-480. Partial financial assistance during the initial stages of the investigation was received from the Council of Scientific and Industrial Research. The authors wish to acknowledge the technical assistance of Miss M. BOSE and MISS S. GUPTA in this investigation. The authors are indebted to Prof. J. W. PORTER for his assistance in the preparation
of hydroxymethylglutaryl-CoA
reductase
employed
in this study.
REFERENCES R. G. GOULD AND G. POPJAK. Biochem. J., 66 (1957) 51. N. L. R. BUCHER, P. OVERATH AND F. LYNEN, Biochim. Biophys. Acta, 40 (1960) 491. 0. WIELAND, G. LOFFLER, L. WEISS AND I. NENFELDT, Biochem. Z., 333 (1960) IO. M. D. SIPERSTEIN AND V. M. FAGAN, in G. WEBER, Advances in Enzyme Regulation, Vol. 2, Pergamon Press, New York,
1964. p. 249.
5 M. D. SIPERSTEIN AND V. M. FAGAN. 1. Biol. Chem.. UI 110661 602. 6 J. F. SCAIFE AND B. B. MIGICOVSKY; tan. J. Biochek,’ 3; (&5’7) 615. R. G. GOULD AND E. A. SWYRYD, J. Lipid Res., 7 (1966) 698. 87L. C. FILLIOS, R. KAPLAN, R. S. MARTIN AND F. STARE, Am. J. Physiol., 193 (1958) 47. 9 G. S. BOYD, Federation Proc., 21 (1962) Suppl. No. II, p. 86. 10 S. MUKHERJEE, S. GUPTA AND A. BHOSE, J. Atherosclerosis Res., 7 (1967) 435. Metabolism. Academic Press. New York. II G. S. BOYD, in J. K. GRANT, The Control of_ Li+id .
1963, p.
79.
-
12 A. J. MEROLA AND A. ARNOLD, Science, 144 (1964) 3616. I3 R. G. LANGDON AND K. BLOCH, J. Biol. Chem., zoo (1953) 135. 14 F. LYNEN, in G. E. W. WOLSTENHOLME AND M. O’CONNOR, CibaFound. Steroids, (1959) 95.
Symp. Biosyn. Terpenes
Biochim. Biophys. Acta, 164 (1968) 357-368
S. MUKHERJEE,
368
A. BHOSE
H. HILZ, J. KNAPPE, E. RINGELMANN AND F. LYNEN, Biochem. Z., 329 (1958) 476. F. LYNEN, H. EGGERER, U. HENNING AND I. KASSEL, Angew. Chem., 70 (1958) 739. T. T. TCHEN, J. Biol. Chem., 233 (1958) 1100. G. POPJAK, in G. PINCUS, Hormones and Athevosclerosis, Academic Press, New York, 1959, p. 7. H. RUDNEY, J. Biol. Chsm., 227 (1957) 363. J. BRODIE, Ph.D. Dissertation, University of Wisconsin, 1962. T. T. TCHEN, in S. P. COLOWICK AND N. 0. KAPLAN, Methods in Enzymology, Vol. V, Academic Press, New York, 1963. p. 505. 22 A. DEL CAMPILLO-CAMPBELL,E. K. DEKKER AND M. J. COON, Biochim. Biophys. Acta, 31 (1959)
15 16 17 18 Ig 20 21
481. 23 F. LYNEN, 481.
J. KNAPPE, E. LORCH, G. JOTTING AND E. RINGELMANN,
24 B. K. BACHHAWAT AND M. J. COON, J. Biol. Chem., 231 (1958) 625. 25 L. A. PESCH, Biochim. Biophys. Acta, 81 (1964) 229. Biochim.
Biophys.
Acta, 164 (1968) 357-368
Anger.
Chem., 71 (1959)