The Direct Effect of ACTH on the Rat Preputial Gland*

The Direct Effect of ACTH on the Rat Preputial Gland*


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The role of the pituitary in maintaining the and that sebaceous structures were directly integrity of sebaeeous structures has been ap- stimulated by the hormone. The paired prepupreciated for some time. As early as 1941, Noble tial gland of the rat served as a model seba-

and Collip (1) implicated various pituitary ceous structure in these experiments. extracts in preputial gland growth. Subsequent


studies by Lasher et al (2) showed that the pituitary was necessary for growth of sebaeeous

1. Effect of ACTH in vivo Albino, male rats (Sprague-Dawley derivates by the Charles River Breeding Laboraprinciples have been examined. Stimulation of supplied tory) weighing 150 gms (7 weeks of age) were sebaceous structures has been demonstrated castrated and adrenalectomized; completeness of with growth hormone (3), prolactin (4) and a surgical procedures was verified in each instance at

glands. Since the earliest observations, the effects of a number of different pituitary

fraction isolated from tbc pituitary residue post-mortem. The endocrine ablations served to a) remove all sources of androgenic hormones thereby

after acetic acid extraction (5) by Lorincz and Lancaster. The latter principle has only been

providing relatively unstimulated preputial glands and b) provide animals in whom effects not medi-

characterized in terms of its "sebotrophic" ated by adrenal cortical hormones could be

effects and growth hormone and prolactin are examined. All animals were maintained on Purina rat chow and 0.85% saline ad libilum. In each exrecognized as general somatotrophic hormones. periment daily subcutaneous injections of 10 units

Thus, the report by Jacot and Selyc (6) in of acthar gel (Armour's ACTH 40 units/ml) were 1951, of an extra-adrenal effect of ACTH on begun 12 days post-surgery in one group of anithe rat preputial gland is of particular interest mals, while a group of weight-matched controls

since unlike the others, this hormone has were treated with saline injections. Injections were given for either 4 or 6 days. specific trophic effects on a specific target The animals were sacrificed by a blow on the organ: the adrenal.

head and the paired preputial glands were immedi-

Since these latter studies were performed ately excised and cleaned of surrounding fat and before highly purified preparations of the hormone were available, absence of contaminants could not be assured. Moreover, in recent years ACTH has been shown to exert a multiplicity of actions on carbohydrate and lipid metabo-

lism which are not mediated by the adrenal (7). The possibility that the response of the preputial gland was secondary to these more generalized metabolic alterations rather than directly to ACTH could not be excluded. To answer these questions, the present studies

fascia. Preformed sebum was gently expressed from

the main duct. The glands were bisected longitu-

dinally along the main duct and weighed. The

glands were incubated either in pairs or individually in 1 ml of Krebs-Ringer bicarbonate (KRB) supplemented with 2 mg. of glucose-U-C'° with a specific activity of 0.5 pv/mg. The incubation was carried out at 37° C for 90 minutes in a Dubnoff metabolic shaker, in an atmosphere of 95% 02 and 5% CO2. The incubation vials were sealed with rubber serum caps which permitted the introduction of gas and other substances.

At the termination of the experiment, 0.2 ml

hyamine was introduced with a *21 needle from were undertaken. Highly purified preparations a 1 ml syringe into the sealed vessel into a plastic of ACTH were employed in vivo and in vitro to cup suspended beneath the rubber cap and 1 ml document that contaminants were not involved 0.1 N HC1 was similarly introduced into the me-

dium in the main compartment of the vessel.

* From the Department of Dermatology of Har- Trapping of involved C°°02 in the hyamine was vard Medical School at the Massachusetts General completed by mild mechanical agitation for 90 Hospital, Boston, Massachusetts. minutes at room temperature. The hyamine-con-

This paper was supported by Grant *' GM- taming cups were then transferred to counting

06271 of the National Institutes of Health, vials for assay of the radioactivity. An extract of Bethesda, Maryland.

Presented in part at the Twenty-fifth Annual total tissue lipids was then prepared after homogMeeting of The Society for Investigative Derma- enization of glands in chloroform-methanol as pretology, Inc., San Francisco, California, June 21, viously described (8). An aliquot was assayed for radioactivity. Error introduced by occlusion of





TABLE I Effect of ACTH administered in vivo on the rat preputial gland Adrenalectomized castrate a" rats injected sub-

cutaneously with 10 u acthar gel or saline daily. One group treated for 4 consecutive days; another

group for 6 days. Excised glands incubated in KRB plus glucose U-C'4 (1.0 X 10' counts/yessels)in vitro. Control



4 days: Mean values SEM No. animals 7 224 Rat weight (g) Gland weight (mg) 36 Nitrogen: ,gm/ 13.0 mg gland

C"02 : Counts/


10 2

7 216 51

10 6

0.2 11.0


<.01 ns

300 3800






min/100 zgm N

Total lipid C'4: 480 Counts/min/100 pgm N



C"02 : Counts/ min/100 gm N Total lipid C'4:



1.0 13.4

7 ns 5 <.01 1.0 ns


300 2600



100 940



200 67

renal androgens do not seem to contribute significantly to maintenance of preputial gland under these experimental conditions.

One gland of each pair was incubated in KRB-

glucose U-C" (vide supro); this served as the

control gland. The other gland was incubated in identical medium to which 1 unit of ACTH was added. Ineubations and analysis of C"O, and lipid C" and tissue nitrogen were carried out as above.

Three highly purified preparations of ACTH

were employed: Oxycellulose adsorbed ACTH with

a specific activity of 103 units/mg (Wilson Co. lot # 101900) and 130 units/mg. (Wilson Co. lot 104529) respectively and chemically homogenous ACTH with specific activity of 107 units/mg, prepared and kindly supplied by Dr. Aaron Lcrner of Yale University. The purity of this latter preparation has been documented elsewhere (10). RESULTS

The effect of the administration of ACTH in vieo on various parameters was evaluated by

statistical analysis of the difference between the means of treated groups and control groups after 4 and 6 days of treatment respectively. In experiments where ACTH was added in vitro results were expressed for each experimental gland in relation to the contralateral control. Je, the latter

6 days: Mean values SEM 6 No. animals 195 Rat weight (g) Gland weight (mg) 43 Nitrogen: ,gm/ 13.6 mg gland

glands results from superimposing adrenalectomy on castration; thus in short-term experiments ad-


experiments confidence levels were based on "Stu-

dent" t values, derived from the common logarithms of the ration of experimental/control glands (11).

1. Effect of ACTH in vivo

Counts/min/lOO pgm N

Ten units of ACTH gel administered daily for either 4 or 6 days to castrate adrenalectomixed rats did not result in significant differ-

* ns = not significant.

respective control groups. However, a sta-


ences in total body weight in comparison to the

tistically significant increase of 41 and 55% in labelled water soluble compounds is negligible. weight of preputial glands occurred after 4 and Tissue nitrogen was determined in the remaining

fat free tissue residue by a modified Ncsslcr 6 days (Table I). Tissue nitrogen content paralleled the gain in weight and thus the eonmethod (9). All radioactive assays were performed in the centratiou of nitrogen (figmN/mg tissue) was Tri-carb liquid scintillation counter using a system

containing 4 gm PPO (2:5 diphenyloxazole) and

not significantly altered.

Metabolism of glucose U-C1' by excised

50 mg vovop (1:4 his 2 phenyl oxazole benzcne) in glands was stimulated by pretreatment with 1 liter toluene-ethanol (88:12).

2. The Effect of ACTH in vitro Paired preputial glands were obtained from male albino rats weighing 150 to 200 gm. The animals were castrated 6 to 12 days before the experiment in order to provide glands minimally stimulated by androgens. Adrenals were left intact in order to minimize prior exposure to endogenous ACTH. Experiments in the authors' laboratory have shown

ACTH in vivo. Evolution of C"O, appeared to increase proportionately with changes in gland

weight. Thus the counts in CO, per 100 gm tissue nitrogen were not significantly altered. Incorporation of radioactivity into tissue lipids was markedly augmented, however, and on the basis of tissue nitrogen exceeded control values

by 206 and 170% respectively after 4 and 6 that little if any further atrophy of preputial days of ACTH treatment. No significant dif-



TABLE II Effect of ACTH on the rat preputial gland in vitro Paired glands from castrated c? rats incubated in KRB plus glucose-U-C" (1.0 X 10 counts/min/ vessel) with and without 1 unit of various preparations of ACTH. Mean results for control glands expressed as counts/mm/mg tissue. Results for ACTH treated glands expressed as ratio of eontralateral control. Mean ratios analyzed statistically for SEM and p values calculated from the common log. Control Glands counts/mm/mg ACTH

ACTH Glands

ACT11/rontrol X lot


No. animals

Lipid C"

Wilson, 103 u/mg , 101900 10 pgm/ml Wilson, 130 u/mg $ 104529 7.7 ,igm/ml










2 6

686 309


118 121





Homogeneous 107 u/mg








Total Lipid C"





125 122

7 8





(Lerner) 10 ,,gm/ml

ferences between effects after 4 days and 6 AUTH to excised glands in the presence of days of ACTH were noted for any parameter.

isotopically labelled glucose effected enhanced incorporation of radioactivity into lipids with-

Effect of AUTH in vitro

out producing a consistent increase in U"O, Thus while the effect of AUTH on The addition of AUTH to the incubating evolution. lipid metabolism may be elicited in vivo as well medium also stimulated glandular metabolism. Results presented in Table II show the means as in vitro, effects on the oxidation of glucose of the percentage change induced by AUTH to UO, are less consistent and appear to parallel changes in tissue mass more than augmenfor each pair of glands. tation of. lipogenesis. The present data do not One unit of AUTH in 4 experiments utilizing

permit discrimination between the possibilities that this disparity exists because conversion of duced similar results. In each experiment there was a variable and statistically insignificant glucose to lipids is a more sensitive and thus more easily demonstrable index of AUTH acincrease in C"O, evolution. All three preparation than its effect in oxidative metabolism; or tions, however, resulted in significant augmen- because there is a real difference between imtation of lipid radioactivity. The effect ob- mediate effects of AUTH on lipid and oxidaserved with homogeneous ACTH was at least tive metabolism. In the latter instance the as great if not greater than that obtained with direct effects of AUTH might be exerted on the less pure Wilson preparation. lipid metabolism and changes in oxidative

3 different preparations of the hormone pro-


metabolism would reflect secondary alterations linked to tissue hyperplasia.

The present studies have confirmed earlier In any event, the results obtained in vitro observations that the administration of AUTH substantiate a direct action of AUTH on to castrate, adrenaleetomized rats causes an glandular metabolism. The documentation of increase in the weight of the preputial gland. similar effects with equivalent dosages of differThis is accompanied by a proportional in- ing potency and homogeneity makes it unlikely crease in tissue nitrogen. Excised glands from that they arc due to trace contaminants. The pretreated animals exhibit an enhanced oxida- findings constitute the first demonstration of an tion of glucose TI-U1' to U"O, which is propor- in vitro or direct action of pituitary hormones tional to the increase in tissue mass. Lipogene- on sebaceous tissue. The metabolic response is sis is augmented disproportionately and is a sensitive index of changes which have been significantly greater per unit tissue nitrogen in previously documented by slower and less direct pretreated animals. The direct addition of effects on tissue growth. As such, it potentially



provides a sensitive assay of responses of seba- androgenie stimulation. In other words, the ceous structures to other hormones of pituitary elaboration of permissive quantities of various pituitary factors in the course of normal endoorigin. To what extent the effect of ACTH is rele- crine interactions could provide the appropriate vant to the physiological or pathological regu- metabolic setting for the expression of specific lation of sebaeeous glands remains to be eluci- androgenie stimulation of sebaceous glands. dated. Certain inferences are warranted, however: 5TJMMARY AND CONCLUSIONS

1. Various pituitary factors have now been implicated in sebaceous gland growth. These

The earlier report that the administration of ACTH to castrate adrenaleetomized rats causes

include such generalized growth factors as growth hormone and prolaetin and crude growth of the preputial gland has been confractions derived from pituitary gonadotrophie hormones (12). To date, however, ACTH is the only pituitary hormone which has been shown to stimulate sebaeeous structures directly and independently of effects on its specific target gland. In addition, preliminary evidence in our laboratory suggests that MSH has effects similar to ACTH in dosages of 10 micrograms/mI in vitro (13). Except for thyrotrophin, which has been reported in one study to have no effect in pituiteetomized, gonadeetomized rats (5), other pituitary hormones with trophie effects for specific endocrine structures have not been examined. Thus on the basis of available evidence, sebaeeous glands, like adipose tissue,

firmed. Concomitant augmentation of lipogenesis and carbohydrate oxidation in vitro in glands excised from pretreated animals has been docu-

mented. That the effect is directly upon the sebaceous structure has been documented by exhibition of ACTH in vitro. The enhanced lipogenesis so induced affords a sensitive index of hormonal action. Contributions by traces of

contaminants was ruled out by the use of several different highly purified preparations of ACTH. It is suggested, on the basis of available evidence, that a variety of pituitary hormones act to stimulate sebaeeous structures and that this action plays a permissive role in sebaeeous gland

seem to respond to a variety of pituitary growth by providing an appropriate meta-

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