the effect of glucocorticoids on adipocyte corticotropin receptors and adipocyte responses

268

Biochimica et Biophysiea Acta, 672 (1981) 268--279 © Elsevier/North-Holland Biomedical Press

BBA 29495

THE EFFECT OF GLUCOCORTICOIDS ON ADIPOCYTE CORTICOTROPIN RECEPTORS AND ADIPOCYTE RESPONSES

CATHERINE M. BEHRENS and J. RAMACHANDRAN *

Hormone Research Laboratory, 1088 HSW, University of California, San Francisco, CA 94143 (U.S.A.)

(Received October 1st, 1980)

Key words: Hormone binding; Corticotropin receptor; Glucocorticoid regulation; (Adipocyte)

Summary

A specific and sensitive assay for determining the binding of adrenocorticotropin (ACTH) to isolated rat adipocytes has been developed and utilized to study the effect of glucocorticoids on ACTH receptor. Measurement of the binding of tritiated ACTH (spec. act. 90 Ci/mmol) to adipocytes isolated from normal, adrenalectomized, and adrenalectomized dexamethasone-treated rats indicated that there are no differences among these three populations in either the magnitude or the affinity of the binding reaction. The binding interaction was found to be of high affinity (Kd = 5.23 + 1.92" 10 -9 M) and paralleled closely the stimulation of lipolysis (Kin = 2.09 + 0.35 • 10 -9 M). About 16 300 receptors were calculated to be present per adipocyte. Hormone-induced cyclic 3',5'-adenosine monophosphate production remained intact after adrenalectomy, thereby confirming that receptors are not lost during steroid deprivation. The lipolytic response did, however, become less sensitive to both ACTH and epinephrine following adrenalectomy. Pre-treatment of adrenalectomized rats with dexamethasone resulted in an increase in basal and hormone-stimulated levels of cyclic AMP and glycerol production to super-normal values. In adipocyte ghost preparations, ACTH and epinephrine sensitive adenylate cyclase activity was not decreased by adrenalectomy and dexamethasone administration did not result in a selective enhancement of ACTH sensitive adenylate cyclase activity. Our results indicate that glucocorticoids do not cause their permissive effects by specific regulation of the ACTH receptor on the adipocyte.

* To w h o m correspondence should be addressed. Abbreviation: ACTH, adrenocorticotropin.

269

Introduction

The role which steroids exert in allowing the full expression of a hormone- induced cellular response has been well documented for a number of tissues, among which are liver [2,3], heart [4], skeletal muscle [5] and adipose tissue [3--9]; the molecular basis of this 'permissive' effect, however, remains to be elucidated. The lipolytic response of the rat adipocyte is a good case in point since although its steroid dependence has been demonstrated repeatedly [3,5--9], the site(s) of this regulation is yet to be defined precisely. In 1970, Braun and Hechter [1] reported that adrenalectomy resulted in a specific loss of ACTH-stimulated adenylate cyclase activity in the rat adipocyte ghost, while the epinephrine- and glucagon-induced activities were unaffected in the steroid- deprived state. Furthermore, administration of dexamethasone to adrenalectomized rats restored the sensitivity of the ghost cyclase to ACTH, whereas the response to epinephrine remained unchanged. However, other studies suggest that glucocorticoids may no t have the specific effect on the ACTH response of adipocytes implied by the results of Braun and Hechter. Allen and Beck [9] found that the lipolytic response of the isolated adipocyte to epinephrine is reduced after adrenalectomy and this loss could be prevented by administration of hydrocortisone. However, they observed no reduction in epinephrine- induced adenylate cyclase activity in adipose tissue homogenates from adrenalectomized rats. Allen and Beck [9] also demonstrated a decrease in both epinephrine and ACTH-stimulated intracellular cyclic AMP levels following adrenalectomy, although the response to ACTH was depressed to a greater extent than the response to epinephrine. In addition, the effect of adrenalectomy on the activity of both hormones was enhanced when the experiments were conducted in the absence of a phosphodiesterase inhibitor. Fain [12] has reported that epinephrine-induced adenylate cyclase activity could be increased in adipocyte ghosts by incubation of adipocytes with dexamethasone and growth hormone in vitro, whereas the response to ACTH under these conditions was more erratic, and the observed enhancement was not statistically significant. Finally, Exton et al. [3] observed a decrease in the magnitude of the lipolytic response to both ACTH and epinephrine following adrenalectomy in spite of an accompanying increase in cyclic AMP accumulation.

This present study was undertaken to determine whether glucocorticoids exert their permissive effect through the regulation of the ACTH receptor population of the adipocyte. A binding assay was developed which could reli- ably measure the direct interaction of [3H]ACTH with its adipose cell receptors. The magnitude and affinity of the ACTH binding to adipocytes isolated from normal, adrenalectomized, and adrenalectomized dexamethasone-treated rats have been determined. The results of the binding experiments and their correlation to adipose cell response are described in this article.

Materials and Methods

Tritiation o f ACTH. Synthetic 3,5-diiodoTyr2'23ah-ACTH was prepared in this laboratory [13] and generously provided by Professor C.H. Li. [3,5-3H]- Tyr2'23ah-ACTH ([3H]ACTH) was prepared by catalytic dehalogenation of 3,5-

270

diiodoTyr2'23~h-ACTH with pure tritium in the presence of palladium, as previously described [14]. [3H]ACTH was purified by gel filtration on Sepha- dex G-25 and ion-exchange chromatography on carboxymethyl cellulose. The major product which emerged in the position of synthetic human ACTH was obtained in the overall yield of 30%. [3H]ACTH was found to be homogeneous by paper electrophoresis at pH 3.7 and 6.7 and comigrated with synthetic human ACTH. Electrophoresis of a tryptic digest of [3H]ACTH at pH 6.7 showed that the radioactivity was equally distributed in the tryptic peptides corresponding to ~-8-ACTH and a~2-39-ACTH. The ultraviolet absorption spec- trum of [3H]ACTH was identical to that of synthetic human ACTH, indicating that the iodine atoms on the tyrosine residues were quantitatively removed. The amino acid composition of an acid hydrolysate of [3H]ACTH agreed well with that of human ACTH.

The biological activity of [3H]ACTH was assessed in two isolated target cell systems. The ability of [3H]ACTH to stimulate glycerol release in isolated rat adipocytes or corticosterone synthesis in isolated rat adrenocortical cells was indistinguishable from that of synthetic human ACTH. The potency of [3H]- ACTH was similarly found to be identical to that of synthetic human ACTH in both assays. The specific radioactivity of [3H]ACTH was found to be 90 Ci/mmol (200 dpm/fmol) or approx. 80% of the theoretically attainable radioactivity.

Steroidogenic activity was determined by incubating the hormone with isolated rat adrenocortical cells as previously described [ 15,16]. Corticosterone was measured by a specific radioimmunoassay using antiserum characterized in this laboratory [17]. Lipolytic activity was measured using adipocytes isolated from rat epididymal fat pads by digestion with collagenase, as described previously [18,19]. Glycerol release was quantitated by the chromatropic acid analysis according to Korn [20].

Binding studies with intact cells. Adipocytes were isolated as for lipolysis from the epididymal fat pads of Sprague-Dawley rats (160--180 g) and were suspended in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 4% bovine serum albumin purified as described [21] and 0.01% lima bean trypsin inhibitor (Worthington). Aliquots of the adipocytes (50--70 mg dry weight per ml) were incubated with [3H]ACTH alone or in the presence of excess unlabeled hormone. To insure minimal adsorptior~ of the peptide to test tubes and pipets [14] all hormone solutions were prepared in the incubation buffer containing 4% bovine serum albumin in sfliconized glass test tubes or polypropylene tubes. Incubations were carried out at room temperature for 90 min, and separation of the bound from free hormone was accomplished by centrifuging the adipocyte suspension at ~ 70 × g for 30 s and carefully removing the infranate with a pasteur pipet. The cells were resuspended in an equivalent volume of ice-cold incubation buffer, and a 0.5 ml aliquot of the resuspended ceils was filtered through Unipore polycarbonate membranes (25 mm diameter, 1 # poresize; Bio-Rad, Richmond, CA) presoaked in ice-cold incubation buffer. The filt- ter was then washed with 1 ml of ice-cold incubation buffer. The entire procedure could be completed within 1 min. All determinations were made in triplicate and filtration of triplicates was performed simultaneously on a filtration manifold (Hoefer Instruments, San Francisco, CA). The amount of radioactiv-

271

ity bound to the cells was determined by placing the polycarbonate filter in 5 ml of PCS scintillant (Amersham-Searle), incubating at 60°C for 1 h and counting in a Packard liquid scintillation spectrometer, Model 3320. All binding experiments were performed at least twice.

To obtain the amount of specifically bound [3H]ACTH, radioactivity bound to the cells in the presence of excess unlabeled ACTH (500 pg/ml) was sub- tracted from the total radioactivity bound in the absence of unlabeled hormone. Consistently, non-displaceable radioactivity amounted to 0.03--0.06% of the total radioactivity added.

For the competitive displacement experiment, bovine pancreatic insulin (25 U/mg) was obtained from Calbiochem, glucagon from Schwarz-Mann and synthetic human ~-end0rphin was a gift from Professor C.H. Li.

Adrenalectomy and glucocorticoid replacement protocol. Male Sprague-Daw- ley rats (140--180 g) were used in all experiments and when adrenalectomized were maintained on drinking water containing 0.9% NaC1. To ensure that endo- genous steroid stores had been cleared, adrenalectomized rats were maintained at least 5 days post-operatively before any treatment was initiated. Administra- tion of dexamethasone (Sigma) was carried out essentially according to Braun and Hechter's protocol [1], described briefly as follows and with the qualifica- tions noted. A total dose of 0.5 mg dexamethasone/100 g body weight was injected intra-peritoneaUy in 5 injections (0.2 ml each) administered over a 48 h period. Because of difficulties encountered in dissolving the dexamethasone in an aqueous solution, the following vehicle was adopted: dexamethasone was dissolved in 95% ethanol to a final concentration of 5 mg/ml and 1 ml of this mixture was added to 4 ml of a solution of 5% bovine serum albumin in 0.9% saline to yield dexamethasone at a concentration of I mg/ml. Placebo groups received the same series of injections containing 20% ethanol in 5% bovine serum/0.9% saline. Animals were killed 5 h after the final injection in all experiments.

Biological assays. Lipolysis was measured as described above. Cyclic AMP production in intact adipocytes was assayed by incubating ~ells (20--40 mg dry weight per tube) in Krebs-Ringer buffer 4% bovine serum albumin containing 0.1% lima bean trypsin inhibitor, 1 mM theophylline (Calbiochem) and the appropriate hormone. The reaction was terminated at 15 min by the addition of 0.1 M sodium acetate buffer, pH 4.0. The cyclic AMP levels produced were quantitated by the Gilman procedure [22] with modifications suggested by Johnson et al. [23]. Adenylate cyclase activity in adipocyte ghosts was assayed as described by Birbaumer et al. [24]. All incubations were performed in the presence of 10 mM theophyUine and with an ATP-regeneration system consist- ing of phosphocreatine (Calbiochem) and creatine kinase (Calbiochem); cyclic AMP was measured by the Gilman method [22]. L-Epinephrine was obtained from Calbiochem. Bovine serum albumin was purified as described previously [21]. All other reagents were of the highest quality available and were used without further purification. Protein determinations were performed according to the procedure of Lowry et al. [25]. The DNA content of the adipocytes was measured according .to Cerriotti [26]; cells were prepared for analysis by extraction of lipid in chloroform/methan01 (2 : 1) and calf-thymus DNA in 0.1 N NaOH was used as the standard.

272

Results

One of the primary concerns of this study was to develop an assay which could be used with confidence to monitor the biologically relevant binding of ACTH to its adipocyte receptors. In the course of developing such an assay, it soon became apparent that the extraordinary propensity of ACTH to bind to the surfaces of containers and filters would present a major problem. Table I illustrates that [3H]ACTH binds significantly to several types of membranes which are commonly employed in binding studies to separate bound hormone from the free. Polycarbonate filters exhibited the lowest affinity for ACTH and were therefore used in all studies of binding of the tritiated hormone to adipose cells.

Binding of [3H]ACTH to isolated rat adipocytes was found to be rapid (Fig. I). The binding was complete within 10 min and remained stable for at least 90 rain. In addition, the magnitude of the binding of [3H]ACTH was proportional to the number of cells present, as shown in Table If.

The specificity of the binding of [3H]ACTH to the adipocytes was also examined. Other basic peptides such as ~.endorphin, insulin and glucagon did not displace bound [3H]ACTH, whereas unlabeled ACTH caused a 62% displacement at an equivalent concentration.

The interaction of [3H]ACTH with isolated rat adipocytes as a function of radioactive hormone concentration is shown in Fig. 2. The stimulation of lipolysis by ACTH in the same batch of isolated adipocytes is also presented in the figure for comparison; it should be noted that the binding and lipolytic activity of ACTH were determined under identical experimental conditions, with the exception that the binding was measured at room temperature rather than at 37°C. From Fig. I it is apparent that the lipolytic response parallels closely the binding of [3H]ACTH.

Non-linear least-squares analysis of the data in Fig. 2 gives a value of 5.23 + 1.92 nM for the K d for the interaction of [3H]ACTH with rat adipocytes, compared to a value of 2.09 + 0.35 nM for half-maximal stimulation of lipolysis.

T A B L E I

A D S O R P T I O N O F A C T H T O V A R I O U S M A T E R I A L S

1 m l 3 .3 n M [ 3 H ] A C T H in i n c u b a t i o n b u f f e r w a s either filtered through the various membranes pre - s o a k e d in ice-cold incubation buffer or allowed to stand in tubes for 9 0 r a i n and then aspirated. The fil- t e r s and the t u b e s w e r e w a s h e d w i t h 2 X 1 -ml ice-cold buffer, mixed with seintillant and counted.

Mate r i a l P e r c e n t [ 3 H ] ACTH bound

A. Filters Cel lu lose a c e t a t e (Mi l l /pore) 0 .4 D E A E ( W h a t m a n ) 0 .2 Glass f i b e r ( W h a t m a n ) 0 .7 P o l y v i n y l c h l o r l d e (Mi l l lpore) 3 .3 Polycarbonatc ( B l o - R a d ) 0 . 0 6

B. Tubes Polypropylene 0 . 0 5 Polystyrene 0 .3

2 7 3

80

-~ 6O

~n

4 0 z

:z: I - o 20

o

o

- o - -

I I I I 20 40 60 80

TIME ( minutes )

Fig. 1. Kinetics of binding of [3H]ACTH to adipocytes. Isolated adipocytes were incubated with [3H]- ACTH (3.3 nM) at room temperature for the t imes indicated. Separat ion of bound from free hormone was accomplished by aspirat ion of incubat ion infranate fol lowed by f i l t rat ion through polycarbonate membranes, as de ta i l ed in the text . Non-specific b inding was corrected by determining binding in the presence of unlabeled ACTH (500 #g/ml) and subtract ing this value from the to ta l radioact ivi ty bound. Approx. 60 mg/ml dry weight of adipocytes were present per tube.

Using the estimate of 7800 adipocytes per mg dry weight of cells [27], it can be calculated that there are approx. 16 300 binding sites for ACTH per cell.

The binding of [~H]ACTH to adipocytes isolated from normal, adrenalectomized, and adrenalectomized dexamethasone-treated rats was compared; these results are illustrated in Fig. 3. It is apparent that there is no significant differ- ence in either the affinity of ACTH for its receptor or in the total number of receptors present under these conditions. The fat content of the adipocytes was seen to decrease significantly following adrenalectomy; a similar observation has been noted in the literature [28]. Since the dry weight of the cells could therefore no longer be used as a reliable index of cel] number, binding was nor-

TABLE II

BINDING OF [3H]ACTH AS A FUNCTION OF CELL NUMBER

Isolated rat adipocytes were incubated wi th [3H]ACTH (3.3 nM) at room temperature for 90 rain and the amoun t of [3H]ACTH bound specifically was determined by f i l t ra t ion through polycazbonate filters. All values represent the mean of tr ipl icate determinat ions .

Cells [3H]ACTH bound (mS dry weight) (fmol)

10 1 .25 20 3 .11 30 3 .83

2 7 4

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I 2 3 4 5 6 7 8 9 I0 II 12 HORMONE CONCENTRATION (nM)

Fig. 2. B i n d i n g o f [ 3 H ] A C T H a n d s t i m u l a t i o n o f ] ipo lys i s as a f u n c t i o n o f [ 3 H ] A C T H c o n c e n t r a t i o n . Iso- l a t e d a d i p o c y t e s we re i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s o f [ 3 H ] A C T H a t r o o m t e m p e r a t u r e f o r 9 0 m i n f o r b i n d i n g d e t e r m h u t t i o n s a n d f o r 1 2 0 r a in a t 3 7 ° C f o r t h e l i p o l y t i c a s say ; b o t h d e t e r m i n a t i o n s we re p e r f o r m e d o n t h e s a m e b a t c h o f ce l ls a n d in t h e s a m e i n c u b a t i o n b u f f e r s . A p p r o x . 5 0 m g / m l d r y w e i g h t o f a d i p o c y t e s w e r e p r e s e n t p e r t u b e . All p o i n t s p r e s e n t a re m e a n va lues o f t r i p l i ca t e i n c u b a t i o n s ; S.E. is a lso i n d i c a t e d . T h e l ines r e p r e s e n t t h e b e s t f i t d e r i v e d f r o m w e i g h t e d n o n - l i n e a r l e a s t - squa re s ana lys i s . [ S H ] A C T H b o u n d , o o; g l y c e r o l re lease , l . . . . . -@.

malized to cell DNA content. Approx. 1.1 /~g DNA was found to be present per 100 000 adipocytes.

The data obtained from the binding experiments correlates well with the results of the biological assays. Table III demonstrates that adrenalectomy does not result in a loss of sensitivity to either the ACTH or epinephrine-induced

1 2 5

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276

T A B L E IV

H O R M O N E - I N D U C E D A D E N Y L A T E C Y C L A S E A C T I V I T Y I N A D I P O C Y T E G H O S T P R E P A R A -

T I O N S F R O M N O R M A L . A D R E N A L E C T O M I Z E D A N D A D R E N A L E C T O M I Z E D D E X A M E T H A S O N E - T R E A T E D R A T S

All p e r c e n t a g e s were ca lcu la ted f r o m the m e a n of triplicate determinations. Basal levels w e r e : 2 .08 ± 0 .11 ,

1.6 ± 0 .03 , 1.7 + 0 .18 n m o l cyc l ic A M P / r a g p r o t e i n 15 / r a in f o r t he n o r m a l , a d r e n a l e c t o r a i z e d a n d adrenal - e c t o m i z e d dexamethasone- treated populat ions, respectively. Cycl ic A M P p r o d u c t i o n w a s m e a s u r e d in the pre sence o f 10 m M t h e o p h y l l i n e .

H o r m one C o n c e n t r a t i o n (M)

Cycl ic AMP P r o d u c t i o n p e r c e n t a g e inc rease ove r basal

N o r m a l A d r e n a l e c t o r n i z e d A d r e n a l e c t o m i z e d

d e x a m e t h a s o n e - t r e a t e d

A C T H

E p i n e p h r i n e

5 • 10 -7 0 0 50 ± 6

10 -6 7 5 ± 6 109-+ 6 2 5 2 ± 13 5 • 10 -6 154 ± 12 211 + 25 350 ± 16

10 - s 2 2 0 ± 24 283 + 12 370 ± 21 5 • 10 - s 2 5 0 + 28 3 3 0 + 22 4 1 5 + 25 7 • 10 -7 0 0 60 + S

2 - 1 0 - 6 3 8 ± 4 6 8 ± 3 3 8 0 ± 16

7 • 10 -6 1 2 0 ± 12 154 + 12 3 8 0 ± 24 2 • 10 - s 225 ± 10 194 ± 18 375 ± 10

cyclic AMP accumulation. The translation of the cyclic AMP production into the lipolytic response was however diminished following adrenalectomy, par- ticularly for ACTH stimulation. Pre-treatment of adrenalectomized rats with dexamethasone resulted in an enhancement of both ACTH and epinephrine- induced cyclic AMP production and lipolytic responses to super-normal levels. Of particular interest, however, is the dramatic increase in basal levels of both cyclic AMP and glycerol production for this group (Table ~III).

In light of the fact that glucocorticoid manipulation did not alter the ACTH receptor population of the adipocyte nor enhance selectively the ACTH- induced cyclic AMP production, the specificity of dexamethasone pre-treatment on adipocyte ghost adenylate cyclase was re-investigated in this study. The experiments of Braun and Hechter [1] were repeated. Glucocorticoid administration to adrenalectomized rats did not result in a selective increase in ACTH-induced adenylate cylcase activity, since the epinephrine response was also enhanced by dexamethasone pre-treatment. Table IV compares the hormone-sensitive adenylate cyclase activity of ghost preparations from normal, adrenalectomized, and adrenalectomized dexamethasone-treated rats; it can be seen that adrenalectomy did not decrease either ACTH or epinephrine-induced activities when the assay was conducted in the presence of theophylline and that pre-treatment with dexamethasone resulted in an enhancement of the normal response.

Discussion

The ability to use glucocorticoid manipulation to effect the modulation and regulation of a specific receptor population, as proposed by Braun and Hechter

277

[1], would provide an attractive experimental system for the study and char- acterization of the ACTH receptor. In order to test this hypothesis, we developed a binding assay which could be used to monitor directly the biologically relevant ACTH receptor population of the adipocyte. Since iodination of tyrosine residues of ACTH results in considerable loss of biological activity [13, 29,30], we resorted to catalytic dehalogenation of 3,5-diiodoTyr2'23~h-ACTH in the presence of tritium for the preparation of radioactive ACTH. This procedure yielded [3H]ACTH of the highest specific radioactivity (90 Ci/mmol) prepared to date, without any loss of activity. However, binding studies with radioactive hormone indistinguishable from the unlabeled hormone in all physicochemical and biological properties still presented severe problems; the strong tendency of ACTH to adhere to non-receptor materials cannot be over- emphasized. This interaction of ACTH with non-specific surfaces could, however, be minimized by the use of polycarbonate filters for the separation of bound and free hormone. The small amount of radioactivity adsorbed to polycarbonate Unipore filters (0.03--0.06%) could not be displaced by unlabeled ACTH even at concentrations as high as 10 -4 M, and probably represented the extent of damaged hormone in the [3H]ACTH preparation. The larger amounts of [3H]ACTH bound to other non-biological surfaces such as polystyrene could be readily displaced by excess unlabeled hormone and this observation is reminiscent of the behavior of radioactive insulin [31,32].

The binding of [3H]ACTH to isolated rat adipocytes was found to be proportional to the number of cells. Moreover, the interaction was rapid, reaching equilibrium within 10 min at room temperature. Saturation of binding was observed at the same relatively low concentrations of the radioligand which are needed for stimulating lipolysis. Additionally, the binding interaction of [3H]- ACTH paralleled closely the stimulation of lipolysis (Fig. 2) and the K a for the association of [3H]ACTH with adipocyte receptors is comparable to the Km for lipolysis. The observed binding of [3H]ACTH to rat adipocytes was also quite specific since two peptide hormones, glucagon and insulin, which have been shown to stimulate physiological responses in adipocytes and to bind to puta- tive receptors on these cells [33,34], failed to displace [3H]ACTH. Another highly basic peptide hormone, ~-endorphin, also had no effect on the interaction of [3H] ACTH with adipocytes.

The results of this study have indicated that the binding of [3H]ACTH to adipocytes in the concentration range over which lipolysis is elicited can be accounted for by a single class of binding sites; in addition, saturation of binding and maximal stimulation of lipolysis are obtained at comparable concentrations of the hormone (Fig. 1). These results suggest that for the lipolytic response there are no spare receptors for ACTH on rat adipocytes. Lang et al. [27] also found that binding of Phe2,4,5-dehydro[4,5-3H]norvaline4~Z-24-ACTH (spec. act. 7.42 Ci/mmol) closely paralleled the stimulation of lipolysis by this analog. It may be inferred from the present investigation that stimulation of lipolysis in isolated rat adipocytes by ACTH is mediated by a class of receptors with a K d of 5.23 + 1.92 nM and that there are about 16 300 of these receptors per cell. This is very much lower than the estimate of 2.12 • l0 s sites per cell reported by Lang et al. [27], but comparable to the number of binding sites found for other hormones [ 35].

278

The binding procedure was used to monitor the ACTH receptor population of the rat adipocyte following adrenalectomy and dexamethasone administration. The binding experiments indicate clearly that adrenalectomy with sub- sequent glucocorticoid deficiency does not result in a decreased number of ACTH receptors on the adipocyte. Consequently, the dramatic reduction of ACTH-induced lipolysis following adrenalectomy which has been observed by many investigators and confirmed in this study as well, cannot be attributed to glucocorticoid regulation of the receptor population. Moreover, pre-treatment of adrenalectomized rats with dexamethasone does not increase either the affinity or the magnitude of the binding interaction; therefore, receptor modulation cannot explain the enhanced biological response observed following dexamethasone administration.

The effect of glucocorticoids on cyclic AMP accumulation and adenylate cyclase activity remains a controversial point. The experiments with intact adipocytes indicate that when assayed in the presence of theophylline there is no loss of sensitivity to hormone-induced cyclic AMP production following adrenalectomy (Table IV); the concentration of ACTH or epinephrine required to attain half-maximal stimulation of the cyclic AMP response remains unchanged. These experiments confirm that there is no decrease in the receptor population of the adipocyte and thereby provide a physiologic correlate to the binding results discussed above.

Our results with adipocyte ghost preparations from adrenalectomized rats indicate that there is no selective glucocorticoid effect on the ACTH-sensitive adenylate cyclase. Our observation of an enhanced epinephrine-sensitive adenylate cyclase (as well as an enhanced ACTH response) in dexamethasone- treated adrenalectomized rats is not in agreement with the data of Braun and Hechter [1]. Table IV also indicates that when compared to ghosts isolated from intact rats, the membrane preparations from the adrenalectomized group responds to both ACTH and epinephrine stimulation with normal cyclic AMP production. It is apparent that the inclusion of a phosphodiesterase inhibitor in the adenylate cyclase assay has minimized the effects of glucocorticoid deprivation and that pre-treatment with dexamethasone has effected a general enhancement of cyclic AMP production in the ghost preparation. These results confirm the data obtained from the experiments with intact adipocytes.

Although the data discussed above have indicated clearly that glucocorticoids do not play a role in the modulation and regulation of the ACTH receptor on the adipocyte, it is equally important to note that the receptor population does not seem to be sensitive to autoregulation, a phenomenon which has been observed for a number of hormone-receptor systems [36--38]. With the release of the negative feedback control which would occur following adrenalectomy, ACTH levels increased above normal circulating levels. However, the number of ACTH receptors on the adipocyte is not altered following adrenalectomy treated states in which circulating ACTH levels would be much lower. In light of this information, determination of whether ACTH regulates its own receptor on the adrenal cells could perhaps offer some insight into the signifi- cance and/or evolution of the ACTH receptor of the adipocyte.

279

Acknowledgments

We wish to thank Professor C.H. Li for his interest and encouragment and J.D. Nelson for skilled animal surgery. This work was supported by grants from the National Institutes of Health (CA 16417), the Northern California Chapter of the ARCS Foundation and the Patent Fund of the University of California, San Francisco.

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the effect of glucocorticoids on adipocyte corticotropin receptors and adipocyte responses

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