Molecular and Cellular Endocrinology, 50 (1987) 149-156

Elsevier Scientific Publishers Ireland, Ltd.

149

MCE 01618

Gamma,-melanotropin promotes mitochondrial cholesterol accumulation in the rat adrenal cortex

Robert C. Pedersen and Alexander C. Brownie Department of Biochemistq Schools of Medicine and Dentistry, State University of New York at Buffalo, Buffalo, NY 14214, U.S.A.

(Received 23 September 1986; accepted 15 November 1986)

Key words; Acyl CoA : cholesterol acyltransferase; Cholesteryl ester hydrolase; Corticosteroidogenesis; Proopiomelanocortin

Summary

Gamma ,-melanotropin ( yX-MSH) facilitates a rapid, dose-dependent, and cycloheximide-insensitive increase in the concentration of mitochondrial free cholesterol in the adrenals of hypophysectomized rats. Physiological concentrations of various synthetic and native preparations of yj-MSH are potent, while y-MSH is not. This cholesterol accumulation coincides with the activation of cholesteryl ester hydrolysis by y3-MSH, while the rates of cholesterol esterification and of mitochondrial cholesterol side-chain cleavage are unaffected. Conversely, ACTH inhibits cholesterol esterification. Therefore, y3-MSH and ACTH together may coordinate a substantial shift in the set-point of cholesteryl ester + cholesterol cycling toward the right. Because ACTH also activates cholesterol side-chain cleavage, this coordinate effect on the flux of cholesterol substrate is manifest as a potentiation of corticosteroidogenesis by y3-MSH. These data extend our previous studies demonstrating that pro-y-MSH polypeptides have an endocrine influence on the rat adrenal cortex.

Introduction

The major prohormone of the pituitary corti- cotroph, proopiomelanocortin (POMC) (Eipper and Mains, 1980), has an amino-terminal region designated N-POMC. Within this sequence is y- melanotropin ( y-MSH) (Nakanishi et al., 1979), so named because of its shared homology with the (Y-

and P-MSH segments of the same prohormone. Although y-MSH itself is not a significant POMC product, larger pro-y-MSH derivatives of N- POMC have been identified in the pituitaries of rat (Browne et al., 1981; Pedersen et al., 1982;

Address for correspondence: Robert C. Pedersen, Depart- ment of Biochemistry, Schools of Medicine and Dentistry,

State University of New York at Buffalo, Buffalo, NY 14214.

U.S.A.

Jackson et al., 1983) cow (Shibasaki et al., 1981) and man (Estivariz et al., 1980; Seidah et al., 1981). These polypeptides are generated from POMC by tryptic-like cleavage at various flanking pairs of basic amino acids. In the neurointer- mediate lobe of the rat (Browne et al., 1981) and bovine (Shibasaki et al., 1981) pituitary, one such product is [Lys’]-y,-MSH. This native glycopoly- peptide consists of y-MSH extended at its amino terminus with a single lysine residue and at its carboxyl end with a sequence of variable, species- dependent length. Circulating polypeptides con- taining the immunoreactive y-MSH sequence have been reported in the rat (Mains and Eipper, 1981; Pedersen et al., 1982) and in humans (Bertagna et al., 1983; Chan et al., 1983; Hale et al., 1984).

Because the y-MSH region of N-POMC is

0303-7207/87/$03.50 0 1987 Elsevier Scientific Publishers Ireland, Ltd

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highly conserved, there is considerable interest in the biological role(s) which pro-y-MSHs may play. We have previously demonstrated (Pedersen and Brownie, 1980) that a brief trypsinization of murine N-POMC generates a product capable of potentiating the steroidogenic effect of ACTH on the adrenal cortex. This action is reproduced by synthetic bovine y3-MSH (Pedersen et al., 1980). Al-Dujaili et al. (1981) observed a similar potenti- ation by purified human N-POMC,_,, on perifused human and rat adrenocortical cells pre- pared from both the inner and the outer zones of the gland.

Although the rat adrenal has specific, high-af- finity receptors for pro-y-MSH (Pedersen and Brownie, 1983), these apparently are not coupled to the adenylate cyclase system (Farese et al., 1983; Pedersen et al., 1983). Moreover, at physio- logical concentrations pro-y-MSHs do not activate cholesterol side-chain cleavage (cholesterol see) or independently stimulate corticosteroidogenesis by normal adrenal tissue (Pedersen and Brownie, 1980; Pedersen et al., 1980). Consequently, pro-y- MSHs are something other than mere ACTH agonists.

In a previous report (Pedersen et al., 1980) we noted that y,-MSH was more effective than an equivalent dose of ACTH at stimulating neutral cholesteryl ester hydrolase (CEH) activity in the rat adrenal. Although the underlying mechanism is still undefined, this suggested substrate augmen- tation as the avenue by which pro-y-MSHs poten- tiate steroidogenesis. The studies reported here were undertaken to test this hypothesis. As indices of cholesterol flux in the adrenal cortex, the fol- lowing were monitored: cholesterol esterification activity catalyzed by acyl CoA : cholesterol acyltransferase (ACAT); CEH activity; mitochon- drial free cholesterol concentration; cholesterol see activity; and plasma corticosterone. The results suggest that pro-y-MSHs may in fact facilitate an increased provision of free cholesterol for the steroidogenic pathway.

Materials and methods

Animal experimentation Female Sprague-Dawley rats (Holtzman)

(160-180 g) were individually caged in rooms kept

at 22°C and illuminated from 06.00 to 18.00 h. Rat chow and water were allowed ad libitum. Transaural hypophysectomies were performed 20-24 h before experiments. Where indicated, pre- treatment with aminoglutethimide phosphate (Ciba; 20 mg/rat) or cycloheximide (Sigma; 10 mg/rat) was performed by i.p. injection 30 min before peptide administration. Except as indicated in Fig. 1, 10 ng of peptide was given i.v. in 0.2 ml saline to animals under light ether anesthesia. Controls received vehicle only. After 10 min the animals were decapitated, trunk blood was col- lected, and the inner zones of the adrenal were rapidly removed by enucleation in situ.

Peptides Synthetic bovine y3-MSH, which corresponds

to N-POMC,,_ 77, has the following sequence: NH ,-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg- Phe-Gly-Arg-Arg-Asn-Gly-Ser-Ser-Ser-Ser-Gly- Val-Gly-Gly-Ala-Ala-Gln-COOH. The residues in boldface connote y-MSH. Authentic y,-MSH from the bovine neurointermediate lobe is glycosylated (Shibasaki et al., 1980) and a lysine residue begins the sequence (i.e., [Lys’]-y3-MSH] (Shibasaki et al., 1981). Rat y,-MSH isolated from the neuroin- termediate pituitary is also glycosylated (Pedersen et al., 1982) and preceded by lysine (Browne et al., 1981), but its carboxyl-terminal sequence differs slightly from the bovine homolog (Drouin and Goodman, 1980; Browne et al., 1981).

Synthetic y-MSH homologs (Ling et al., 1979) were provided by N. Ling and R. Guillemin, Salk Institute, La Jolla. Glycosylated rat [Lys’]-y,-MSH was purified from 50 neurointermediate lobes by a modification of the method of Browne et al. (1981). Briefly, fresh tissue was extracted with 10 ml of an acidic medium (1 N HCl containing 50 ml/l for- mic acid, 10 ml/l trifluoroacetic acid (TFA), and 10 g/l NaCl), defatted with an equal volume of petroleum ether, and concentrated by 3 passes through a pre-equilibrated cartridge of &-silica (SepPak; Millipore). Material of interest was eluted in a cut of 15 --, 25% 1-propanol in aqueous TFA (10 ml/l) and then subjected to HPLC. The sep- aration was carried out on a MicroPak MCH-5 column (Varian) using a linear gradient of acetonitrile (15% + 30%; 0.4%/min) in 0.25 M triethylammonium phosphate, pH 2.1 (Rivier,

151

1978). As monitored by a y-MSH RIA (Pedersen et al., 1982), the major peak of immunoreactive material eluted at 11.5 min and contained no detectable immunoreactive ACTH. Lyophilized aliquots (0.5 1-18) of the preparation were subjected to amino acid and end-group analyses (Jones et al., 1981) to confirm their authenticity.

ACTH,_ 24 (Cortrosyn; Organon) and y-MSH homologs were prepared for injection as previ- ously described (Pedersen and Brownie, 1980).

Cholesterol and enzyme assays Mitochondria, microsomes, and 105 000 X g su-

pernatant from adrenocortical tissue were pre- pared by differential centrifugation (Kramer et.al., 1979).

In experiments designed to monitor the con- centration of mitochondrial cholesterol, pregnen- olone formation was blocked by animal pretreat- ment with aminoglutethimide and by inclusion of the drug (0.75 mM) in the isolation buffer. Mitochondrial cholesterol was extracted (Bligh and Dyer, 1959) and quantitated using a coupled cholesteryl oxidase (Boehringer Mannheim) assay (Gamble et al., 1978) with the fluorogenic indica- tor, 3-( p-hydroxyphenyl)propionic acid (Aldrich; Zaitsu and Ohkura, 1980).

Cholesterol side-chain cleavage activity was de- termined in mitochondrial preparations washed free of residual aminoglutethimide. The assay (Kramer et al., 1979) measures the rate of preg- nenolone formation from endogenous mitochon- drial cholesterol in the presence of a source of reducing equivalents (13 mM isocitrate; Sigma) and an inhibitor of 3/?-hydroxysteroid dehydro- genase/isomerase (10 PM cyanoketone; Upjohn). Pregnenolone was extracted from the incubations with methylene chloride and quantitated by RIA (Bergon et al., 1974).

CEH activity was assayed in the 105000 X g supernatant by a modification (Pedersen and Brownie, 1979) of the double isotope method of Pittman et al. (1975). Microsomal acyl CoA : cho- lesterol acyltransferase (ACAT) activity was mea- sured according to Balasubramaniam et al. (1977), except that 15 min incubations contained 25-50 pg of protein per 0.2 ml of reaction buffer (pH 6.8).

Protein was quantitated by the method of Brad-

ford (1976) using standards of bovine serum al- bumin.

Heat-generated type I absorbance change

The heat-generated type I absorbance change was examined in mitochondrial preparations as previously described (Paul et al., 1976) using an Aminco DW-2 recording spectrophotometer. Cy- tochrome P-450 concentrations were determined by the method of Omura and Sato (1964).

Serum corticosterone

Plasma was prepared from animals not treated with aminoglutethimide. Corticosterone was then assayed as fluorogenic steroid (Silber et al., 1958) with an ethanol-H,SO, reagent against standards of authentic corticosterone (Steraloids) and blanks of charcoal-stripped rat serum.

Miscellaneous

Pregnenolone was a product of Steraloids. Cholesterol, cholesteryl oleate [9,10-3H(N)]oleate (88 mCi/mmol), and cholesteryl [9,10-3H(N)] oleate (10 mCi/mmol) were from Applied Sci- ence. Cholesteryl [l-14C]oleate (55 mCi/mmol), [ l-l4 Cloleoyl-coenzyme A (50 mCi/ mmol), and [7- 3 H(N)]pregnenolone (25 Ci/ mmol) were pur- chased from New England Nuclear. Oleoyl-coen- zyme A and horseradish peroxidase were obtained from Sigma. Other materials were reagent grade or better. All organic solvents except for HPLC-grade acetonitrile (Baker) were redistilled before use.

Results

The administration of bovine y,-MSH (0.1-100 ng, i.v.) to hypophysectomized rats stimulated a concordant, dose-dependent increase in both cyto- solic CEH activity and mitochondrial free cholesterol (Fig. 1). Since y,-MSH does not pro- mote entry of mitochondrial cholesterol into the steroidogenic pathway (Table 1 and Pedersen et al., 1980), cholesterol accumulation was apparent even though aminoglutethimide was not used in this set of experiments.

The effects of y3-MSH and of ACTHi_,, on various components of cholesterol flux in the rat adrenal cortex are compared in Table 1. At 10 min after giving 10 ng bovine y3-MSH to hypophysec-

152

Fig. 1. Dose response of rat adrenocortical CEH activity

(0 . . 0) and mitochondrial free cholesterol concentration

(0 -0) to treatment in vivo with bovine y,MSH. Hypo-

physectomized animals were injected iv. with the indicated

doses of peptide 10 min prior to decapitation. Animals were

not given aminoglutethimide, but the inhibitor was added to

isolation buffer (0.75 mM) and tissue from animals in each

treatment group (n = 4) was pooled for assays. The mean

serum corticosterone concentration for controls was 1.2 pg/dl

and was not significantly altered by any dose of y,-MSH

employed. Values (mean + 2 SD) are from one experiment.

tomized rats (group B), there was a significant

increase in CEH activity ( + 41% vs. controls, group A) but no appreciable effect on cholesterol esteri- fication (ACAT activity). Thus, the overall activity ratio of hydrolysis to esterification (CEH : ACAT)

was shifted from 0.76 in controls to 1.11, a rise that was associated with a 27% increase in the

content of mitochondrial free cholesterol. Gam- ma,-MSH had no effect on the following:

cholesterol association with cytochrome P-450,,, as measured by the heat-generated type I ab- sorbance change (HGI; Paul et al., 1976), cholesterol see activity, or serum corticosterone.

Conversely, an equivalent dose of ACTH,_,, (group C) decreased ACAT activity by 25% vs. controls but did not alter the rate of cholesterol hydrolysis. Therefore, like y,-MSH but for differ- ent reasons, ACTH increased both the CEH: ACAT ratio (to 1.10) and mitochondrial

cholesterol (+20%). However, in contrast to y3- MSH, ACTH also fostered a substantial elevation in the HGI (from 21.6 to 34.6 A3W_420nm/mM P-450) and a 4-fold rise in the rate of cholesterol

TABLE I

ADRENOCORTICAL EFFECTS OF BOVINE ys-MSH AND ACTHi_,, IN HYPOPHYSECTOMIZED RATS

Peptides (10 ng) or saline (controls) were given iv. at 10 min before decapitation to aminoglutethimide-treated (20 mg, i.p.)

hypophysectomized rats. Tissue was pooled from each treatment group before preparation of subcellular fractions. Data were

accumulated from 3-7 experiments, each with 4-5 animals per group, except for HGI (one experiment). In separate experiments,

serum corticosterone was determined in animals not pretreated with aminoglutethimide. The statistical significance between group

means was established by ANOVA.

Treatment group

[Al lB1 [Cl lD1 Controls ys-MSH LVXH,_~, y,-MSH + ACTHi_,

CEH a 1.55 * 0.14 2.18 f 0.21 * 1.67 k 0.17 2.41 kO.18 **

ACAT = 2.03 f 0.27 1.97+0.13 1.52i0.15 n 1.65 +0.15 +I CEH/ACAT ratio 0.76 1.11 1.10 1.46

Mitochondrial b

cholesterol 59.4 +1.8 75.6 k1.0 * 71.0 k2.2 * 92.8 +2.6 + HGI ’ 21.6 17.2 34.6 50.3 Cholesterol a

see 0.39*0.03 0.44 * 0.02 1.70t0.11 3.10*0.10 ** Serum

corticosterone d 2.0 *0.2 2.2 rto.2 35.0 *1.6 56.3 k1.9 **

a nmol product formed/min/mg protein (mean+ SEM).

b nmol cholesterol/mg protein (mean + SEM).

’ A,,_,,, ,/mM P-450.

d pg/dl (mean f SEM).

* P < 0.01 vs. [A]; ** P < 0.01 vs. [Cl; n P < 0.05 vs. [A] and [B]; + P c 0.01 vs. [A], [B] and [Cl.

153

TABLE 2

EFFECT OF CYCLOHEXIMIDE ON THE RAT ADRENOCORTICAL RESPONSE TO ys-MSH AND ACTH,_2,

Rats were hypophysectomized 20-24 h before the experiment. Where indicated, 10 mg cycloheximide (CH) per animal was given i.p.

in 0.2 ml of saline/ethanol (3 : 1, v/v) 30 min before peptide( Gammas-MSH (10 ng) and/or ACTHi_,, (10 ng) were given i.v. in

peptide diluent and animals were killed 10 min later. Controls received diluent only. Aminoglutethimide (0.75 mM) was added to the

tissue isolation buffer. CEH activity and mitochondrial cholesterol were assayed in adrenal tissue pooled from animals in each group

(n = 4). Corticosterone was measured in individual sera.

Treatment

CH ys-MSH ACTH

Serum

corticosterone a

(mean + SEM)

Cholesterol

ester hydrolase

activity b

(mean + 2 SD)

Mitochondrial

cholesterol ’

(mean f 2 SD)

_ _ - 2.2 f 0.6 1.57+0.17 63.3 f0.9

+ _ - l.lkO.3 1.62+0.14 65.3 i 1.6 - + _ 3.75 1.0 2.18k0.21 75.3 * 2.1

+ + _ 4.0 * 1.7 1.97*0.06 76.5i3.5

+ _ + 3.9kO.4 1.46+0.12 78.3 + 2.7

+ + + 2.3 k- 0.4 1.91iO.06 92.9k2.0

= cg/dl. b nmol cholesterol/min/mg cytosolic protein.

’ nmol cholesterol/mg mitochondrial protein.

see, as compared with controls. This was reflected in a serum corticosterone increase from 2.0 to 35.0

p g/dl. When ys-MSH and ACTH were administered

together (group D), their coordinate, reciprocal effects on cholesterol hydrolysis and esterification, respectively, produced a further increase in the

CEH: ACAT activity ratio to 1.46 (192% of con- trols) that was reflected in a 56% elevation in mitochondrial cholesterol. Associated with this mobilization of substrate were even greater incre-

ments, as compared with group B or C, in HGI (to 50.3 A 390_42,,nm/mM P-450), cholesterol see activ- ity (&fold higher than controls), and serum corti-

TABLE 3

ADRENOTROPIC POTENCY OF SELECTED y-MSH HOMOLOGS: (I) STIMULATION OF MITOCHONDRIAL

CHOLESTEROL ACCUMULATION AND (II) POTENTIATION OF ACTH-STIMULATED CORTICOSTEROIDOGENESIS

Data in (I) reflect the independent action of y-MSH homologs (10 ng, i.v.) on mitochondrial cholesterol concentration (’ nmol/mg

mitochondrial protein) in the adrenals of hypophysectomized rats. Data in (II) are the serum corticosterone concentrations (b pg/dl)

achieved in animals receiving the y-MSH homolog alone or together with 10 ng ACTHi_,.,. All of the homologs are synthetic except

for rat [Lys’]-ys-MSH glycopolypeptide. Assays were carried out on samples from individual animals (’ n). The level of significance

between differences in group means was determined in ANOVA.

y-MSH homolog (I) Mitochondrial

cholesterol a

(mean f SEM)

(II) Serum corticosterone b (mean f SEM)

- ACTH + ACTH

[Al PI [Cl PI [El PI [Gl

- y-MSH

[Lys’]-y-MSH

ys-MSH, bovine

ys-MSH, rat

[Lys’]-ys-MSH, rat [Lys’]-ys-MSH, rat glycopolypeptide

58.6 k 1.4 (5) ’ 1.8+0.4 (10) 33.9* 1.9 (9)

57.4+ 2.1 (3) 1.5kO.3 (3) 34.4 + 3.7 (4)

61.1+ 2.2 (4) 1.9*0.5 (3) 37.7 * 2.2 (4)

72.2 + 4.8 (6) * 2.4 f 0.3 (4) 53.9 * 3.3 (6) *

73.6 f 3.4 (4) * 2.0 f 0.2 (3) 58.2 * 2.5 (5) *

76.9* 1.3 (5) * 2.5 + 0.3 (4) 76.2+4.4 (5) **

75.5 * 1.5 (6) * 1.5kO.3 (4) 78.Ok 2.4 (6) **

* P < 0.01 vs. [A], [B], [Cl; ** P < 0.01 vs. [A], [B], [Cl, [D], [El.

154

corticosterone concentration (to 56.3 pg/dl). When cycloheximide was used to inhibit pro-

tein synthesis in vivo prior to peptide administra- tion, the steroidogenic potency of ACTH was ap- propriately blocked (Ferguson, 1963) (Table 2). However, there was no effect of the drug on either CEH activation by ys-MSH or the ‘accumulation of mitochondrial cholesterol in response to either peptide.

In addition to the synthetic bovine yJ-MSH used for the experiments in Fig. 1 and Tables 1 and 2, a variety of other homologs were tested, as they became available, for effect on adrenal cholesterol mobilization and potentiation of ACTH-mediated steroidogenesis (Table 3). These peptides included native rat [Lys’]-y,-MSH glyco- polypeptide (Materials and methods) and several synthetic preparations. When given at a dosage of 10 ng i.v., the following rank order of potency was observed: native rat [Lys’]-y,-MSH z synthetic rat [LysO]-y3-MSH > rat y3-MSH P bovine y,-MSH. Gamma-MSH and [LysO]-y,-MSH, peptides not containing the C-terminal extension of the ys- MSHs, were inactive.

Discussion

In previous studies (Pedersen and Brownie, 1980; Pedersen et al., 1980) we have shown that the potentiating effect of pro-y-MSHs on steroido- genesis by the rat adrenal cortex is associated with an apparent increase in cholesteryl ester hydroly- sis. The present study carries this observation a step further by demonstrating that this increased rate of hydrolysis is coupled with a simultaneous, dose-dependent rise in mitochondrial free choles- terol. There is no effect of yj-MSH on cholesterol esterification. Conversely, ACTH,_ N diminished ACAT activity without altering the rate of cholesterol ester hydrolysis.

Decreased ACAT activity in the rat adrenal cortex (Shima et al., 1972; Civen et al., 1977) and ovary (Schuler et al., 1981) in response to ACTH and LH, respectively, has been described. We believe, however, that the degree to which CEH is under ACTH control is less unambiguous (Peder- sen and Brownie, 1986). It is true that regulation of CEH by ACTH, effected via CAMP-dependent phosphorylation, enjoys considerable support in

the literature (Shima et al., 1972; Beckett and Boyd, 1977; Naghshineh et al., 1978). Also, the pronounced effect of stress on cholesteryl ester hydrolysis (Gidez and Feller, 1969; Trzeciak and Boyd, 1973) is abolished by hypophysectomy (Pedersen and Brownie, 1979). Nevertheless, stimulation of CEH in hypophysectomized rats can be achieved only when ACTH replacement is carried out with supra-physiologic concentrations of the hormone (Pedersen and Brownie, 1979) suggesting that some pituitary factor other than ACTH may be implicated in control of the en- zyme. Our studies support pro-y-MSH as this factor.

An additional explanation for our observations is suggested by evidence that adrenal cytosolic CEH activity may be heterogeneous. A major fraction is apparently unresponsive to CAMP-de- pendent protein kinase (Vahouny et al., 1985). Pro-y-MSHs, which do not stimulate adenylate cyclase (Farese et al., 1983; Pedersen et al., 1983) might somehow communicate preferentially with this distinct esterase activity. The size and state of the intracellular pool of cholesteryl esters may also be of some consequence for the effectiveness of pro-y-MSHs (Pedersen and Brownie, 1987); bovine ys-MSH is reportedly inactive when tested with bovine adrenocortical cells (Cathiard et al., 1985), a tissue notably impoverished in cholesteryl ester stores (Hechter, 1952).

The conversion of cholesterol to pregnenolone is a major control point in adrenal steroid bio- synthesis (Stone and Hechter, 1954) and depends on the ACTH-regulated rate at which cholesterol interacts with cytochrome P-450,, (Simpson et al., 1972; Alfano et al., 1973; Brownie et al., 1973; Bell and Harding, 1974; Paul et al., 1976; Kido and Kimura, 1981). A useful measure of this as- sociation is the HGI absorbance change (Paul et al., 1976), which reflects the cytochrome spin-state transition that accompanies endogenous cholester- ol binding as the mitochondrial preparation is warmed. Although the present study demonstrates that ys-MSH promotes cholesterol accumulation in the mitochondrion, the HGI data indicate this pool does not readily associate with cytochrome P-450,,. Consequently, pro-y-MSHs are not inde- pendently steroidogenic in normal tissue (although for reasons not yet clear, they can promote-steroid

155

production by human aldosteronoma cells in vitro (Lis et al., 1981; Aurrechia et al., 1982)). When ACTH is also present, this barrier is overcome and the pro-y-MSH potentiation of steroid pro- duction, a consequence of increased substrate mobilization, is manifest.

The failure of cycloheximide to blunt the action of y,-MSH (Table 2) is entirely consistent with these observations. In some poorly defined way (Hall, 1985; Pedersen, 1985) inhibitors of protein synthesis interrupt the ACTH-regulated move- ment of mitochondrial cholesterol to the cyto- chrome P-450,, active site (Brownie et al., 1972, 1973; Simpson et al., 1978; Privalle et al., 1983). Cholesteryl ester hydrolysis and translocation to the mitochondrion are proximal to this cyclo- heximide-sensitive point and therefore are unaf- fected by the drug, as previously established by Trzeciak and Boyd (1973) and Mahaffee et al. (1974).

The data in Table 3 suggest that an N-terminal lysine and the C-terminal extended sequence of y,-MSH are required for maximum pro-y-MSH potency. Gamma-MSH itself is not active, and the asparagine-linked oligosaccharide chain associated with the native polypeptide does not enhance ef- fectiveness. It should be noted that the primary circulating pro-y-MSH in rat and man is N-

POMC,_ ,6, which contains the [Lys’]-y,-MSH se- quence and from which [Lys’]-y,-MSH can be cleaved in the neurointermediate lobe (Browne et al., 1981; Shibasaki et al., 1981). N-POMC,_,6 itself was not available to us for study, but others (Al-Dujaili et al., 1981) have previously demon- strated its inherent ACTH-potentiating activity. The dosage of pro-y-MSHs used to test for cholesterol mobilization (10 ng/rat, i.v.; Table 3) is at the high end (- lo-” M) of the physiologi- cal range (Pedersen et al., 1982) but significant effects of bovine y,-MSH were detectable at con- centrations as low as 0.1 ng (Fig. 1).

In summary, the apparent reciprocal modula- tion of CEH and ACAT activities by pro-y-MSH and ACTH, respectively, may define an intriguing mode of dual regulatory control over adrenal steroidogenesis. It has been suggested (Jamal et al., 1985, Suckling, 1985) that the adrenal cortex harbors a ‘futile’ cycle of cholesterol esterification and hydrolysis similar to that reported in the

macrophage (Brown et al., 1980). An active bi-cycle of this description, sensitive to intracellular regu- latory elements under coordinated hormonal con- trol, would provide an especially responsive mech- anism for generating steroidogenic substrate on demand. This hypothesis deserves further investi- gation.

Acknowledgements

We thank Drs. R. Guillemin and N. Ling, The Salk Institute, La Jolla, for their generous gifts of synthetic y,-MSH-related peptides. The expertise of Mr. L. Joseph with the animal experimentation is gratefully acknowledged. Competent technical assistance was also provided by Ms. M.P. Russo, Ms. S. Siembida, Mr. R. Linsmair, and Mrs. E. Lawson. These studies were supported by U.S. P.H.S. Research Grant AM 18141 and HL 06975 to A.C.B. and Research Career Development Award HD 00613 to R.C.P.

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