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Biochimica et Biophysics Acta, 360 (1974) 193-204 @I Elsevier Scientific Publishing Company. Amsterdam - Printed in The Netherlands

BBA 56464

HORMONAL REGULATION OF CUTANEOUS LIPOGENESIS: EFFECTS OF HYPOPHYSECTOMY, POSTERIOR-HYPOPHYSECTOMY AND a-MELANOCYTE-STIMULATING HORMONE TREATMENT

MARY F. COOPER, ANTHONY J. THODY and SAMSHUSTER

Department of Dermatology, Wellcome Laboratories for Research into Skin Disease, University of Newcastle upon Tyne, Newcastle upon Tyne (U.K.)

(Received February 15th, 1974)

Summary

The effects of hypophysectomy, posterior-hypophysectomy and ol-melano- cyte-stimulating hormone on skin lipogenesis were studied in rat ear biopsies incubated in vitro with [U- 1 4 C] glucose. In skin from intact rats, 90% of the labelled lipid was present in the dermis and localised mainly in pilosebaceous units. The most heavily labelled lipids in dermis were the wax monoester- sterol ester fraction and triglyceride; in epidermis relatively more isotope was incorporated into polar lipids and free sterols. Hypophysectomy reduced the labelling of all dermal lipids, the greatest changes occurring in the wax ester- sterol ester fractions and squalene. In contrast, epidermal labelling was slightly increased. Posterior-hypophysectomy reduced dermal labelling of sterols, free fatty acids, wax esters-sterol esters and squalene, but had little effect on polar lipids and appeared to increase the labelling of glycerides. In posterior-hypo- physectomised rats a dose of 30 pg cr-melanocyte-stimulating hormone/100 g body weight per day restored dermal wax ester biosynthesis to normal within 5 days.

Introduction

The sebaceous gland is one of the most active sites of cutaneous lipogene- sis [ 1,2] . Sebaceous activity in the rat is under hormonal control, and is in- fluenced by a variety of steroid and peptide hormones including androgens [ 31, anterior pituitary hormones acting via their target glands [4], and cw-melano- cyte-stimulating hormone (a-MSH) from the neurointermediate lobe of the pituitary [ 5,6] .

Abbreviation: a-MSH, or-melanocyte-stimulating hormone.

194

The hormonal regulation of lipid biosynthesis in the skin has so far re- ceived little study. In 1963 Wilson [ 71 showed that sterol ester biosynthesis in rat skin was enhanced by testosterone or progesterone and decreased by oestra- diol. Burgess and Wilson [S] also reported an increase in squalene biosynthesis following testosterone treatment. More recently Prottey et al, [9] have de- scribed enhanced lipogenesis in dermis of rat skin after topical methyl testoster- one treatment. Other more detailed studies of the androgenic regulation of sebaceous metabolism have been confined to modified sebaceous structures such as mouse preputial gland [lO,ll] or the costovertebral gland of the hamster [ 12,131.

The effects of peptide hormones on sebaceous lipogenesis have not been investigated. Of the pituitary hormones which influence sebum secretion, cu-MSH is strongly sebotrophic [6] and, unlike the gonadotrophins and thyroid- stimulating hormone, probably acts directly on the sebaceous glands. However, the metabolic events in sebaceous tissue following a-MSH treatment are not known.

To investigate the hormonal re~lation of skin lipid metabolism in greater detail, we have studied lipogenesis in vitro from [U-l 4 C] glucose in rat ear skin. In this paper we report the effects of hypophysectomy, posterior-hypophy- sectomy and cy-MSH replacement on dermal and epidermal lipogenesis.

Mater&Is and Methods

Materials Solvents were Analar grade and were redistilled before use. Chloroform

was re-stabilised by the addition of 2% (v/v) methanol. Oleic acid, cholesterol, cholesteryl oleate and n-hexadecane were from

B.D.H. Chemicals Ltd; monoolein, diolein Grade II, triolein Grade I and stearic acid stearyl ester Grade I (an example of a wax monoester) were from Sigma, and squalene from Koch-Light Laboratories Ltd. Egg lecithin, egg lysolecithin, egg phosphatidylethanolam~e, bovine phosphatidylse~ne, bovine sphingomy- elin, wheat germ phosphatidylinositol, ox heart cardiolipin, and phosphatidic acid from egg lecithin were all Grade I from Lipid Products, South Nutfield, Surrey.

Wax diesters type I and II were isolated from rat sebum by preparative thin-layer chromatography on silicic acid, using as the solvent benzene-hexane (1: 1, v/v) [ 141. [U-l 4 C] glucose (255 Cifmole), [’ 4 C] cholesterol (58 Cilmole) and glyceryl tri-[’ 4 C] oleate (38 Ci/mole) were from the Radiochemical Centre (Amersham, Bucks); I1 4 C] choline-labelled lecithin and lysolecithin (117 Ci/ mole) from I.C.N. Tracerlab.

All other reagents were Analar or the best available grade.

Male Wistar rats were maintained on standard Oxoid breeding diet. Hypo- physectomy or posterior-hypophysectomy was carried out by the parapharyn- geal method. Those rats which were posterior-hypophysectomised failed to lose weight, and testicular size remained normal. The extent of hypophysecto- my was confirmed at autopsy by measurement of adrenal and testicular weight and by macroscopic and microscopic examination of the sella turcica.

195

Ear biopsies were taken at least three weeks after operation, when the rats were 15-20 weeks of age. Hormone injections were subcutaneous, in a vehicle containing NaCl (0.9%), Tween 80 (0.4%), sodium carboxymethylcellulose (0.5%) and benzyl alcohol (0.9%). Control groups received injections of vehicle alone.

Synthetic a-MSH was a generous gift of CIBA-Geigy Ltd.

Tissue incubations 3-mm punch biopsies were taken through the outer portion of the ear,

from surface to surface, while the animals were under light ether anaesthesia. The intact punch biopsies, weighing 4-6 mg, were incubated individually in 11 mm X 25 mm polystyrene specimen vials, with the biopsies completely immersed in reaction medium. The reaction mixture contained 2 mM [U-’ 4 C] glucose (5 Ci/mole) in a final volume of 0.5 ml Krebs-Ringer-bicar- bonate containing benzyl penicillin (100 pg/ml) plus streptomycin sulphate (100 E.cg/ml). The reaction mixtures were gassed continuously via syringe needles with O2 --CO2 (95: 5, v/v). Incubation was at 37°C without shaking, and usually for 3 h. Controls consisting either of unincubated mixtures or boiled tissue were included with each experiment, and in all cases negligible amounts of radioactivity were recovered in lipid extracts from control tubes.

Separation of dermis and epidermis After incubation the ear punches were rinsed in saline and then soaked in

0.5% acetic acid for 2 h at room temperature to loosen the epidermis. The discs of epidermis from each side of the ear punch were then peeled off by gentle blunt dissection using small forceps and a No. 11 scalpel blade. Histological examination of the separated tissues showed that this procedure removed all the epidermis together with the top portions of the pilosebaceous follicles, but that virtually all sebaceous glands remained in situ in the dermis.

Lipid extractions The tissues were transferred to glass stopped tubes containing 1.9 ml of

chloroform-methanol-water (1:2:0.8, by vol.) and homogenised using a Poly- tron model PTlO. The homogenates were allowed to stand overnight at 4°C and the lipids. were then extracted by the method of Bligh and Dyer [ 151. Total lipid extracts in chloroform were sampled for radioactivity measurement, and the remainder of the extract was stored under nitrogen at -15” C.

Thin-layer chromatography To determine the percentage distribution of isotope among the different

lipid classes, aliquots of total lipid extract were analysed by repeat two-dimen- sional thin-layer chromatography on 20 cm X 20 cm Eastman Chromagram Sheets (silicic acid + organic binder, on a poly(ethylene terephthalate) backing sheet). Before use, the plates were. washed overnight in re-distilled ether and were activated at 100°C for 30 min, either before or after the ether wash. It was noted, however, that satisfactory separations could also be obtained using unactivated plates.

The load consisted of the lipid extract plus a mixture of carrier lipids

196

(detailed under Materials) in quantities sufficient to be made visible by iodine (lo-50 pg). Plates were developed twice in Solvent 1, twice in Solvent 2 and once in Solvent 3 (see Fig. 1). A 3.5-cm strip of plate, containing the origin, was then cut off and developed once in Solvent 4 (Skipski et al. [16] ) to separate the polar lipids.

The lipid spots were located with iodine vapour, which was allowed to evaporate off before cutting the plates for scintillation counting. Fig. 1 shows the results of a typical separation using carrier lipids alone.

Radioactivity measurements Portions of plate containing the lipids spots were cut out and placed in

polyethylene counting vials, to which were added 10 ml of a toluene-ethanol- based scintillator [ 171. Radiactivity was measured in a Packard Tri-Carb liquid scintillation counter, with quench correction by the external standard channels ratio method. Preliminary experiments showed that with this procedure, the recovery of radioactivity from [’ 4 C] triolein, [’ 4 C] cholesterol, [’ 4 C] lecithin or [’ 4 C] lysolecithin spotted on to plates ranged from 94 to 100%.

Aliquots of total lipid extract in chloroform were pipetted into glass vials. The solvent was removed by gentle heating, and scintillator was added for radioactivity measurement as above.

Expression of results All isotope incorporation data have been expressed as dpm incorporated

per 3-mm biopsy (equivalent to dpm per unit skin surface area) rather than as dpm per mg tissue. This was to allow comparison with sebum secretion data (which are surface area related) and also to avoid any problems caused by changes in skin thickness and weight (such as could result from alterations in connective tissue content) under different hormonal conditions. In practice,

solvent 3 (once)

solvent

CotTie)

1 solvent

CtwTce)

i f

solvent 1 (twice) Fig. 1. Thin-layer chromatography of neutral and polar lipids on silicic acid using solvent systems as follows: Solvent 1, light petroleum (40-60°C)-diethyl ether-acetic acid (50:50:1, by vol.); Solvent 2. light petroleum (40-60°C)-benzene (70: 30, v/v): Solvent 3, light petroleum (40-60’0. After develop- ment in Solvent 3 the plate is cut along the dotted line, and the strip containing the origin developed in Solvent 4 (chloroform-methanol-acetic acid-water [25:15:4:2, by vol.) [161).

197

however, no significant differences have been found in ear punch biopsy weights as a result of hypophysectomy or posterior-hypophysectomy.

Autoradiography For autoradiography experiments, ear punches were incubated for 3 h

with [U-l 4 C] glucose (5 Ci/mole). The tissue was then cryostat sectioned, washed thoroughly and mounted with Kodak AR10 stripping film. Exposure was for two weeks.

Results

Sites 0 f cutaneous lipogenesis Autoradiographs of tissue incubated with [’ 4 C] glucose showed that most

of the incorporated radioactivity was localised in the pilosebaceous units and in epidermis (Fig. 2).

The results of separating the skin into dermis and epidermis are shown in Table I. About 90% of the isotope incorporated into lipids was recovered from the dermis containing the sebaceous glands.

Time course and pattern of lipogenesis Fig. 3 shows the time course of ’ 4 C incorporation into dermal and epider-

Fig. 2. Autoradiograph of ear skin incubated with [U-14C] glucose. e, epidermis; d, demis; s. sebaceous gland (X 406U).

198

TABLE I

LIPOGENESIS FROM [U-14C] GLUCOSE IN RAT EAR SKIN

Ear punch biopsies from intact rats were incubated *with 2 mM [U-14C1 glucose as described in Materials

and Methods. Results are expressed as mean + S.E. (n = 4, Expt 1; n = 5, Expt 2).

Experi-

ment

1 2

14C incorporated into total lipids Dermis as

(dpm/3-mm biopsy per 3 h) percentage ._ -.-_____ of total

Dermis Epidermis _._-._ -

45480 i 2080 4100 i: 1130 91.7 38020 t 2820 3610 z!z 480 91.3

mal lipids of skin from an intact rat. Incorporation in both dermis and epider- mis was linear for at least 4 h.

Time courses for the most heavily labelled lipids in dermis are shown in Fig. 4. The pattern of ’ 4 C incorporation was essentially the same at each time interval; the fraction containing sterol esters and wax monoesters (fatty acyl esters of long-chain fatty alcohols [ 181) was the most highly labelled, and there was also active incorporation into the “triglyceride” fraction, into free fatty acid and into the wax diester classes, type I (diesters of a hydroxy fatty acid with one unsubstituted fatty acid and one long-chain alcohol 1141) and type II (fatty acyl diesters of long-chain diols [ 141).

Time (hl

1 2 3 4 5

3 4 5

Fig. 3. Time course of 14C incorporation into total lipids of dermis (0) and epidermis (0) of ear skin from an intact rat. Results are expressed as dpm per biopsy, and linear regressions are shown for dermis and epidermis.

Fig. 4. Time course of 14C incorporation into individual lipid classes in dermis of ear skin from an intact rat. Results are expressed as dpm per biopsy and are the mean of duplicate incubations.

199

TABLE II

DISTRIBUTION OF 14C IN LIPID CLASSES IN DERMIS AND EPIDERMIS

Ear punch biopsies from intact rats were incubated with [II- 14C1 glucose as described in Materiels and Methods. Results are expressed as percentage of total dpm incorporated into lipids (mean k SE.. n = 5. dermis; n = 4, epidermis).

Lipid class Dermis (+ sebaceous glands) (%)

Epidermis (“0)

Lysolecithin Sphingomyelin Choline glycerophosphatides Serine + inositol f ethanolamine glycerophosphatides Phosphatidic acid + cardiolipin Total polar lipids

Free steral Monoglyceride 1,2-Diglyceride 1,3-Diglyceride “Triglyceride”* Free fatty acid Wax diester type I Wax diester type II Wax monoester * sterol ester Squalene Unidentified* *

0.35 C 0.08 0.87 + 0.24 0.50 k 0.09 1.69 rt0.55 8.58 * 0.36 11.56 ?r 1.94 3.10 + 0.47 8.89 + 0.50 0.33 + 0.09 .V.ll Y!Z 1.76

12.86 t 0.34 32.11 4 4.39

2.08 + 0.31 10.58 rt 3.42 1.17 + 0.15 2.37 + 0.31 4.52 + 0.82 4.43 t 0.75 1.52 + 0.22 2.76 It 1.05

20.95 k 2.46 9.04 k 3.16 9.58 It 0.73 11.03 rt 1.98 8.59 2 1.18 5.42 + 1.36 3.67 + 0.45 2.59 + 0.56

32.86 + 2.01 14.35 c 5.75 1.49 -e 0.20 1.09 rt 0.34 0.97 zk 0.22 4.25 + 0.54

* The fraction designated LLtriglyceride” could also contain such compounds as glyceryl ether diesters, which may be present in rat sebum [IS].

** Unidentified lipid. two spots running either side of monoglyceride in Solvent I.

Table II shows the distribution of ’ 4 C between individual lipid classes in dermis and epidermis after 3 h incubation. The pattern of epidermal labelling showed marked differences from that of dermis, with relatively more labelling in polar lipids and free sterols and less labelling in the triglyceride and the wax ester-sterol ester fractions.

Effects of hypophysectomy Hypophyseetomy significantly reduced 1 4 C incorporation into total lipids

of dermis (Table III). By contrast, there was a small increase in epidermal labelling (significant in one experiment). This epidermal increase appeared to be a general one, not confined to any one lipid class, but the low levels of radioactivity in these extracts did not permit an accurate comparison for in- dividual lipids.

The effects of hypophysectomy on dermal lipids are shown in Table IV. Labelling of all neutral and polar lipid fractions was reduced; the greatest changes occurred in the wax diester classes, the combined wax monoester and sterol ester fraction, and squalene,

Effects of posterior-hypophyseetomy Lipogenesis was also measured in a small group of posterior-hypophy-

sectomised rats (three animals only). Removal of the neurointermediate lobe of

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TABLE III

EFFECTS OF HYPOPHYSECTOMY AND POSTERIOR-HYPOPHYSECTOMY ON 14C INCORPORA- TION INTO TOTAL LIPIDS OF DERMIS AND EPIDERMIS

Ear punch biopsies were incubated with [U- 14C] glucose as described in Materials and Methods. Results are expressed as mean f S.E. and P values are given relative to the control (intact or sham-hypophysectomised)

group. N.S., not significant.

Experi- Treatment Dermis Epidermis

ment (dpmi3-mm fdpm/3-mm biopsy per 3 h) biopsy per 3 h)

___-

1 Intact (n = 4) 45480 f 2080 4100 k 1130 Hypophysectomised (n = 5) 19540f1190 5560 + 350

P < 0.001 N.S.

2 Sham-hypophysectomised tn = 5) 31550 ? 1280 4510 k 430

Hypophysectomised (n = ‘7) 15870 + 1080 6640 4 400 P <O.OOl P < 0.01

Posterior-hypophysectomised (n =3) 27 510 f 2680 4330 t 1370 N.S. N.S.

-

TABLE IV

EFFECTS OF HYPOPHYS~CTOMY AND POSTERIOR-HYPOPHYSECTOMY ON DERMAL LIPO- GENESIS

Ear punch biopsies from control (sham-hypophysectomised), hypophysectomised and posterior-hypo- physectomised rats were incubated with [U-14C]glucose as described in Materials and Methods. Results are expressed as mean rf S.E. (n = 5, control; n = 7. hypophysectomised; n = 3, posterior-hypophysecto- mised). N.S.. not significant.

Lipid class (dpm/3-mm biopsy per 3 h) Percentage change from -. -.. control Control HYPO- Post-hypo- ~

physecto- physecto- HYPO- Post-hypo- mised mised physecto- physecto-

mised mised

Lysolecithin 29Ort 50 13ort 10 150 * 20 - 55* - 48 N.S. Sphingomyelin 440 z!z 90 2302 30 18OIt 30 - 48N.S. - 59* Choline gfycerophosphatides 3020 z!z 140 2300 -t 240 2920 + 190 - 24* - 3 N.S. Serine + inositol ~yeerophosphatides 400f 60 25Oi: 30 300* + - 38’ - 18 Ethanolamine glycerophosphatides 770 r 10 560 + 40 760 _+ 60 - 27* - 1 N.S. Phosphatidic acid + cardiolipin 350 + 30 250 ?r 20 290 4 40 - 29’ - 17 N.S. Total polar lipids 5310 4 180 3590 rt: 330 4360** - 32’ - 18

Free sterol 640+ 80 430f 40 300 + 60 - 33* - 53* Monoglyceride 490 rt 80 31oi: 10 420 t 30 - 37 N.S. - 14 N.S. 1.2-DigIyceride 1370 + 70 990 z?z 70 1740 2140 - 28* + 27* 1,3-Diglyceride 330 t 40 170 ?r: 20 190 i: 30 - 48* - 42*

“Triglyceride” 6740 rt- 500 4050 k 460 9190 + 1300 - 40* + 36 N.S. Free fatty acid 3090 f 120 1530 f: 140 2380 + 300 - 50* - 26*

Wax diester type I 2590 k 210 920+110 213Ort260 -64* - 18 N.S. Wax diester type II 96Ozf: 70 36Ort 60 740 t 80 - 63* - 23 N.S. Wax monoester + sterot ester 81705750 2730+330 48704350 -67* - 40* Squalene 370 + 60 802 20 130 + 10 - 78* - 65” Unidentified 420 It 40 19Ok 30 190 i: 20 - 55* - 55*

_..

* Significant at P = 0.05 or below. ** Mean of two values.

201

the pituitary did not affect epidermal lipogenesis, but there was a small reduc- tion in dermal labelling (Table III). Although this change in total dermal lipo- genesis was not statistically significant, a number of significant changes were seen in the labelling of individual lipid classes (Table IV). Free sterols, free fatty acids, the combined wax monoester and sterol ester fraction and squalene all showed significant reductions in labelling. Wax diester labelling was also reduced, although this change was not significant. In the major polar lipid fractions very little change occurred. The 1,2-diglyceride fraction showed a small but significant increase in labelling, and “triglyceride” labelling was also increased (although the latter change was not significant).

Effects of a-MSH replacement The effects of ol-MSH treatment on the biosynthesis of wax esters and

sterol esters in dermis were studied in the posterior-hypophysectomised and completely hypophysectomised rats. Both groups received a dose of 30 pg CX-MSH/lOO g body weight per day, and ear biopsies were taken at intervals up to 12 days after starting injections. A sham-operated control group received injections of vehicle alone. In this experiment considerable day-to-day variation was found in the mean values for the control group (see legend to Fig. 5) even though the standard errors for any one day were small. For simplicity, there- fore, the results for each experimental group are expressed as a percentage of the control mean at each time interval.

In both experimental groups, ’ 4 C incorporation into wax and sterol esters was significantly (P < 0.025) below the control mean before starting injections, and remained below control level (P > 0.05) up to day 3. After 5 days of treatment, however, labelling was restored to normal in the posterior-hypophy- sectomised rats (Fig. 5). In the completely hypophysectomised group ’ 4 C incorporation did not return to normal and was still significantly (P < 0.001) below the control mean after 12 days of treatment.

01 ’ ’ , I I I I I -1 0 2 4 6 6 10 12

Time of treatment (days) Fig. 5. Effects of a-MSH on the biosynthesis of wax and stem1 esters in dermis. Posterior-hypophysecto- mised (0) and completely hypophysectomised (0) rats were injected with CX-MSH (30 Pg/lOO g body weight per day) starting day 0. The mean value for the sham-operated control group showed day-to-day

variability (range 4000 dpm-17 000 dpm per biopsy) and the results are therefore expressed as a percen-

tage of the control mean for each day + S.E. (n = 3. posterior-hypophysectomised: n = 7. hypophysecto- mised).

202

Discussion

The measurement of lipogenesis in rat ear biopsies provides a simple and reproducible method for studying hormone-induced changes in skin lipid metabolism. Glucose was chosen as the radioactive substrate for these studied because it is probably the major physiological precursor for de novo lipid biosynthesis in skin [19,20]. In addition it was hoped by the use of glucose in substrate quantities to avoid any problems caused by changes in pool size when a precursor such as acetate is used.

In intact rats the mean rate of incorporation of glucose carbon into total skin lipids was approximately 60 ng atoms/cm2 per h, or 1600 ng atoms/g tissue per h. This is similar to rates reported by other workers [20,21], but is an order of magnitude below the sebum secretion rate for rat skin, assuming an average rate of 40 mg/day per 300 g rat [22] and a body surface area of about 200 cm2 [18]. This discrepancy may at least partly reflect the non-physiological nature of in vitro systems, but it is also probable that some sebum is formed from precursors other than glucose, such as amino acids [20] and dietary lipids [ 231. The in vitro method nevertheless gives a useful measure of physiolo- gical activity, as the changes in total lipogenesis reported here (Table III, Fig. 5) parallel those observed in sebum secretion rate [6, 241.

Different patterns of lipid labelling were found in the dermis and epider- mis. The relatively higher incorporation of ’ 4 C into free sterols and polar lipids in epidermis (Table II) supports the findings of other workers for rodent [2,9,25] and human [1,26] skin, and presumably represents active membrane synthesis during cell proliferation and keratinisation. In dermis, the observed pattern of incorporation resembles the composition of rat sebum [ 181, except for a somewhat greater percentage of isotope in the “triglyceride” fraction (which could also contain such compounds as glyceryl ether diesters [ 181).

Hypophysectomy reduced the labelling of all dermal lipid classes, but relatively greater decreases were found in the wax ester-sterol ester fraction compared with glycerides and polar lipids (Table IV). This could perhaps indi- cate that wax ester biosynthesis in the sebocyte is affected to a greater degree than is the turnover of other lipids as the sebaceous cells divide, differentiate and undergo lysosomal autolysis [ 271. Some glyceride and polar lipid biosyn- thesis could, however, occur in non-sebaceous dermal components (although histological examination of the ear tissue showed few adipose cells), and with results from whole dermis it is not possible to say definitely whether the pattern of sebaceous lipogenesis is altered by hypophysectomy. Changes have been reported in the relative proportions of the wax and sterol ester classes of rat skin surface lipid following hypophysectomy [ 281; as no obvious change was seen in the relative labelling of these classes in dermis (Table IV) it is possible that an increased epidermal contribution accounts for the surface lipid changes. The suggestion that type I diester waxes originate principally from the epidermis [28] is, however, not supported by our findings (Table II) and the significance of the observed alteration in surface lipids therefore remains unclear.

The finding of increased epidermal lipogenesis after hypophysectomy is of some interest. Large increases in skin lipogenesis are found in essential fatty

acid deficiency, and are reversibly by topical prost~landin treatment [29 1. ~ypophyse~tomy can reduce prost~l~din biosynthesis f30,31] and it is thus possible that that change in epidermal labelling seen here is also a result of impaired protaglandin metabolism.

Removal of the neurointermediate lobe of the pituitary had a less marked effect on dermal lipogenesis than did total hypophysectomy, but in general the pattern of changes was similar (Table IV). The increase observed in 1,2-digly- ceride and “triglyceride” labelling is difficult to interpret.cr-MSH and related peptides have little action in the rat on lipolysis 132,331 or adipose tissue lipogenesis [ 341, and it therefore seems unlikely that changes in dermal adipose cells due to lack of (w-MS&l could account. for these results.

In the posterior-hypophysectomised rats a dose of 30 Erg ~-MS~/lOO g body weight per day quickly restored dermal wax ester and sterol ester biosyn- thesis to normal (Fig. 5). The same dose given. to completely hypophysecto- mised rats had a noticeably smaller effect, and this suggests that the presence of the anterior pituitary may be required for a-MSH to exert its full effect on sebaceous lipogenesis. The influence of the anterior pituitary on sebum secre- tion is mediated mainly through the thyroid, adrenals and gonads [ 351, and in the male rat gonadotrophins are of particular importance for maintaining sebaceous activity, via the stimulation of testicular androgen secretion [4]. Recent experiments suggest that LY-MSH and testosterone may act synergisti- cally to increaase sebum secretion (Thody, A.J. and Shuster, S., unpublished). Androgens are known to stimulate sebaceous lipogenesis [9,10], and the demonstration that a-MSH can also influence skin lipid metabolism will permit a more detailed study of the relationship between these hormones in control- ling sebaceous function.

Acknowledgements

The authors wish to thank Mrs Linda Bush for excellent technical assis- tance. We also thank Mr D. Wilkins and Mrs L. Carrick for assistance with autoradiography and animal operations.

This work has been supported by Grants from the Wellcome Trust and the Medical Research Council.

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