Molcculnr and Cellular Endocrinology 4 ( 1976) 107 - 114

@North-Holland Publishing Company

EFFECTS OF 25HYDROXYCHOLESTEROL AND AMINOGLUTETHIMIDE

IN ISOLATED RAT ADRENAL CELLS. A MODEL FOR CONGENITAL

LIPOID ADRENAL HYPERPLASIA? *

H.E. FALKE, H.J. DEGENHART, G.J.A. ABELN and H.K.A. VISSER

Department o,rPediatrics, Erasmus iJniversit.,s and Academic Hospital Kotterdam and Sophia Children’s Hospital and Neonatal Unit, Gordelwcg 160, Kotterdam. The Netherlands

Received 9 July 197.5; accepted 9 Septcmbcr 1975

The production of corticostcrone from 25-hydroxycholesterol by isolated rat adrenal cells

is inhibited by aminoglutethimide phosphate (AGI); half-maximal inhibition is obtained at ca.

10 SM. AGI also inhibits ACTH-stimulated steroid production from endogeneous substrates; here ~~alf-~~~a~i~~al inl~ibjtion is obtained with ca. 40 MM AGI. in the presence of ACTH + AGI,

25-11ydroxycholesterol causes additive inhibition. This effect of 2Shydroxycholesterol is dosc-

dependent. ACTIi-stimulated steroid production from endogencous substrates is partially in-

hibited by S-cholene3p,24-diol.

These results may just reflect substrate competition for the side-chain cleaving system or

may be due to sonic secondary toxic effect on the cells.

Keywords: adrenal cells; 2.5hyclroxycholesterol; ACTH; aminoglutethimidc; congenital

lipoid adrenal hyperplasia.

Aminoglutethijl~ide phosphate (AGI) is a well known inhibitor of cholesterol side-chain cleavage (Cower, 1974). Cholesterol side-chain cleavage involves several steps. In the recent model of Kraaipoel et al. (1975) the first step is the conversion of cholesterol into 5,20(22)-cholestadien-30-01. This step is inhibited by AC1 (Degcnhart et al., 1974). Inhibition of cholesterol side-chain cleavage by AC1 is non-competitive (Bell and Harding, 1974).

The effect of AGI on the adrenal is comparable to congenital lipoid adrenal hyperplasia (CLAH) (Goldman, 1970), a rare but almost always fatal human inborn

error due to a deficiency of one or more of the components of the cholesterol side-

* This work was supported by a grant from the Foundation for Medical Research, Sophia

Children’s ttospital and Neonatal Unit, Rottcrdam.

107

chain cleaving system (Prader and Siebenman, 1957; Degenhart et al., 1972; Kirk- land et al., 1973). The analog of cholesterol, S-cholestene-3@,25-dial (25-hydroxy- cholesterol) is a good exogenous substrate for the cholesterol side-chain cleaving system in isolated rat adrenal cells (Falke et al., 1975b). Both in isolated rat adrenal cells and in bovine adrenal mitochondria conversion of 25-hydroxycholesterol into steroids is inhibited by AGI (Degenhart et al., 1974; Falke et al., 1975b). The pre- sent paper describes the effect of AC1 on the production of corticosterone by iso- lated rat adrenal cells incubated with ACTH and/or 25hydroxycholesterol. The possibility exists that C-24 sterols can function as intermediates in the metabolism of 25hydroxycholesteroI (Degenhart et al., 1974). Therefore we also tested 5- ch~~lelle-3~,24-diol in our incubation system.

MATERIALS AND METHODS

Materials Adrenocorticotrophin (ACTH) was a purified preparation of porcine ACTH ob-

tained from Organon, Oss (Cortrophine@). Albumin was obtained from Sigma Chemical Co. (Bovine Serum Albumin, Cohn fraction V) and HEPES from Calbio- them. AGI was a generous gift from Ciba Co., Arnhem; 25hydroxycholesterol was obtained from Steraloids; 5-cholene-3@,24-dial was synthesized in our laboratory by LiAlH~-reduction of the methyl ester of 3~-hydroxy-S-cholenic acid (Schwarz Mann). All glass surfaces in contact with the adrenal cells or ACTH solutions were siliconized (Siliclad, Clay Adams) and sterilized. Buffers were sterilized by filtration through Millipore filters (HAWP 02.500, pore size 0.45 PM).

Cells were incubated in Krebs-Ringer (pH 7.4) buffers containing 3% (W/V) al- bumin and 0.2% (w/v) glucose. These buffers were prepared with 21 mM bicarbon- ate (KRBAG) or HEPES (KRHAG). Incubations were carried out in round-bot- tomed glass centrifuge tubes under 02--CO2 (955%) for KRBAG or in air for KRHAG. The total incubation volume was 1 ml. ACTH and AC1 were added in 0.1 ml buffer, the sterols in 10 1.11 ethanol. This low concer~tration of ethanol (1%) does not influence steroidogenesis. After 2 h incubatiot~ at 37°C in a Dubnoff incubator shaking at 100 rev./min the tubes were tightly capped, frozen rapidly and stored at -20°C. The number of cells per incubation was 1-2 X 105.

Suspensions of isolated cells were prepared as described (Falke et al., 1975a). Adrenals of male Sprague-Dawley rats (200--250 g) were used. Cell yield was 5 X lo’-1 X IO6 cells per adrenal. Steroid production by cells prepared in KRBAG or KRHAG showed the same response to ACTH or 25hydroxycholesterol.

Corticosterone was measured by fluorimetry as previously described (Falke et al., 1975a). The values obtained were corrected for the small amount of fluores- cence caused by 25hydroxycholesterol (Falke et al., 1975b).

RESULTS

The conversion of 25hydroxycholestero1 (25 pg/mI) into corticosterone was completely inhibited by 70 PM AGI (fig. 1, curve C). Complete inhibition of the ef- fect of ACTH (1 ~nU~nl1) required 280 @vl AC1 (fig. 1, curve A). 50% inhibition of the effects of 2S-hydroxychoiesterol and ACTH was obtained with approx~rnately 10 and 40 PM AGI, respectively. Fig. I also shows the effect of AGI in the presence

% STEROIDOGENIC

~:OSPoNSE 1

9 120 ,

AMINOGLUTETHIMIDE PHOSPHATE pM

I:ig. 1. Effect of AGI on corticosterone production by isolated rat adrenal cells. Cells were in-

cubated with I mU ACTH/ml (curve A); 1 mlJ ACTH/ml + 5 p,e 25-hydroxycholesteruI/ml

(curve R) and 25 fig 25-hydrosycholesterol/ml (curve C). Results arc expressed in percentagcs

of corticostcronc production in the presence of 1 mU ACTH/ml without AC1 (curves A and B)

or 25 fig X-hydroxycholestcrol/ml without AGI (Curve C). Duplicate incubations arc plotted.

110 2S-ffydPo~.~cholestcrof nnd at~l~no~lutethi~lide

CORTICOSTERONE

)lg12x105cells12hrs 1.75 1

1.50

1.25

1.00

0.75

0.50

0.25

a

------___

I 1 I I

5 10 15 20 25 25-HYDROXYCHOLESTEROL p@Jl

l?g. 2. ~(~rticostcr~)ne prod~lction by isolated rat adrenal cells in the presence of 25hydrosy-

cholesterol f*-----a), 25-hydroxycholestcrol + ACT11 (1 ~~U~I~~I) (o----i~), 25-hydroxy-

cholesterol + AGI (70 &I) f=- - - - - -*) or 25-tlydroxycholesterol + ACTS (I mU/ml) + AGI

(70 PM) (o- - - - - -cl). Duplicate incubations arc plotted.

of both ACTH (1 mU/mI) and 2ShydroxycholesteroI (5 pg/ml) (curve B). In the presence of ACTH alone, 2Shydroxycholesterol induced a further increase in corti- costcronc production. With 5 pg 25hydroxycholesterol/ml the increase varied be- tween lo--25%, but was always present. At higher concentrations of the sterol more pronounced differences were observed (fig. 2). However, in the presence of both ACTH and AGI, addition of 25-hydroxycholcsterol resulted in an additive de- crease of corticosterone production. This effect of 25-hydroxycholesterol was ob- served at all AGI concentrations tested. The stimulation of corticosterone produc-

HE. Falke ct af. 111

CORTICOSTERONE

0.25

) j___________--____# _-

I I I I I 0 0.5 1.0 1.5 2.0 2.5

S-CHOLENE-3P,24-DIOL pg/ml

I:&. 3. Effect of 5-cholene-3&24-dial on steroid production by isolated rat adrenal cells incu- bated without further additions (B- - - - - -9) or with ACTff (1 mU/nll) (o o). Duplicate incubations are plotted.

tion by 25-hydroxycholesterol was dose-dependent, whether ACTH was present or not (fig. 2). It should be mentioned that the ACTH concentration used (I mU/ml) did not cause maximal stimulation of steroid production. Maximal stimulation in this series of experiments was usually found with IO-100 mU ACTH/ml. The extra stimulation of steroid production by 25hydroxycholesterol did not occur with maximal stimulating ACTH concentrations (Falke et al., 1975b). AC1 (70 MM) al- most completely inhibited steroid production at all 25hydroxycholesterol concen- trations tested. The ACTH-stimulated corticosterone production from endogenous precursors was only partially inhibited by 70 PM AGI. Again, addition of 25.hy- droxycholesterol resulted in a further inhibition of the corticosterone production. This inhibition increased at higher concentrations of the sterol.

Fig. 3 shows the effect of S-cholene-3~,24-diol on the corticosterone production by isolated rat adrenal cells. This C-24 sterol had no effect on the basal steroid pro- duction, but partially inhibited the effect of ACTH.

DISCUSSION

Both ACTH and 25hydroxycholesterol stimulate corticosterone production by isolated rat adrenal cells. In both cases corticosterone production is inhibited by AGI. Concerning its effect on the conversion of 25-hydroxycholesterol into corti-

112 25.Ilydroxycholesteroi and aminoglutethimide

costerone, AC1 concentrations 4 times higher are required for a similar degree of inhibition of the ACTH-stimulated steroid production.

The effect of AC1 on the ACTH-stimulated steroid production can be described to inhibition of the side-chain cleavage of the endogenous substrate cholesterol. There is ample evidence that ACTH stimulates the steroid production by increasing the cholesterol supply to the cholesterol side-chain cleaving system (Garren et al.,

1971; Brownie et al., 1973; Trzeciak and Boyd, 1973; Bell and Harding, 1974). AGI causes accumulation of free cholesterol in ACTH-stimulated rat adrenals (Dex- ter et al., 1967), especially in the mitochondria (Mahaffee et al., 1974). Therefore it is improbable that inhibition of the ACTH-stimulated steroid production by AGI is

due to inhibition of an ACTH-dependent process prior to cholesterol side-chain cltiavage. On the other hand ACTH appears to have no effect on the cholesterol side-chain cleaving system itself, as discussed in a previous paper (Falke et al., 1975b). In the presence of AGI, addition of 25hydroxycholesterol causes additive inhibition of ACTH-stimulated corticosterone production. We offer two hypotheses to explain this effect, one based upon competition between endogenous cholesterol

and exogenous 25-hydroxycholesterol for side-chain cleavage and another based upon production of inhibiting substances, probably from 25hydroxycholesterol.

In bovine adrenal mitochondrial preparations, 25-hydroxycholesterol inhibits

the side-chain cleavage of labeled cholesterol (Raggatt and Whitehouse, 1966; Simp- son and Boyd, 1967). In rat adrenal mitochondria, competition between cholesterol and 25-hydroxycholesterol was shown by Jefcoate et al. (1974). Our results indi- cate that the inhibition of side-chain cleavage by AGI is much greater for 25-hy- droxycholesterol than for endogenous cholesterol. The dilution of the fraction of mitochondrial cholesterol available for side-chain cleavage, by 25-hydroxycholes- terol, might explain the observed effect of 25-hydroxycholesterol in the presence of ACTH and AGI. The following evidence is available concerning the existence of sterols with an adverse action on cellular metabolism.

1. Patients with CLAH die within a few months after birth with only very few exceptions (Prader and Siebemnan, 1957; O’Doherty, 1964; Tsutsui et al., 1970; Kirkland et al., 1973). This is surprising as patients with the more common forms of adrenal insufficiency and even patients with congenital adrenal absence can be treated with relative case (Sperling et al., 1973; Pakravan ct al., 1974; DiGeorge, 1975). Therefore one is tempted to conclude that in CLAH the defect adrenal pro- duces one or more substances that are responsible for the severity of this disease. Such a production of toxic metabolites is not uncommon with inborn error-s of metabolism (Bondy and Rosenberg, 1974). In CLAH these supposedly toxic mctabolites (“adrenotoxins”) could be produced from cholesterol which accumu- lates in the adrenal (Tsutsui et al., 1970). Similar processes might be induced by AGI. It should be noted, however, that no detailed studies on the sterol composi- tion in CLAH adrenals are available at present.

2. 5-Cholene-3fl,24-diol (a C-24 sterol) inhibits the effect of ACTH on corti- costerone production in isolated rat adrenal cells (fig. 3).

H.E. Fake et al. 113

3. 3&2Oa-Dihydroxy-5-cholenic acid (also a C-24 sterol) has strong anti-aldo- sterone properties (British patent, 1962).

4. Several sterols, including 2~~h~rdroxycholestero1, inhibit growth of cultured cells (Chen et al., 1974).

Production of C-24 sterols from cholesterol is a common process in mammalian liver, an example is the production of bile acids (Danielsson and Sjovall, 1975). Small amounts of sterols with hydroxylated side-chains, which might be intermedi- ates in the production of C-24 sterols, are normally present in human cord blood (Eberlein, f965), aorta and brain (Van Lier and Smith, 1967, 1969). ~thou~h less probable, one should consider the possibility that even in short-term experinlents

certain sterols might inhibit one or more processes involved in corticosterone bio-

synthesis. Acute toxic effects of 25hydroxycholesterol itself on our cells are im- probable, as this sterol is a good substrate for steroid production in the absence of AGI.

ACKNOWLEDGEMENTS

Mrs. Mary Ames;r,-Heinrich, Mr. A.A.M. Kempers and Dr. D.A. Price are grate- fully ackno~fledged for their help with the preparation of the manuscript, and Mr. J.G.M. Huijmans for his assistance with part of the exper~~~lenta~ work.

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