Testicular effects on food intake, body weight, and body composition in male hamsters

Physiology & Behavior, Vol. 27, pp. 637-640. Pergamon Press and Brain Research Publ., 1981. Printed in the U.S.A.

Testicular Effects on Food Intake, Body Weight, and Body Composition in Male Hamsters WENDELIN

N . S L U S S E R 1 A N D G E O R G E N. W A D E 2

D e p a r t m e n t o f Psychology, University o f Massachusetts, A m h e r s t , M A 01003 R e c e i v e d 17 A p r i l 198I SLUSSER, W. N. AND G. N. WADE. Testicular effects on food intake, body weight, and body composition in male hamsters. PHYSIOL. BEHAV. 27(4)637-640, 1981.--Castration significantly increased body weight gain in male hamsters. Treatment with testosterone propionate (TP), 5a-dihydrotestosterone propionate (DHTP), or estradiol benzoate (EB) prevented the castration-induced weight gains. All of these body weight changes were seen in the absence of significant changes in food intake, suggesting an important role for metabolic actions of testicular steriods. Castration increased carcass lipid content and decreased protein content. Both of these effects were prevented by TP treatment. DHTP treatment prevented only the decrease in carcass protein, and EB treatment prevented only the increase in carcass lipid. The adiposity-reducing action of testosterone may be mediated by aromatized (estrogenic) metabolites, and 5t~-reduced metabolites could have some anabolic effects. There was a highly-significant negative correlation between carcass lipid content and epididymal fat pad lipoprotein lipase activity, raising the possibility that in male hamsters testicular effects on adiposity may not be mediated via changes in lipoprotein lipase activity. Some other possible metabolic effects of testicular hormones are considered. Hamsters Testosterone Carcass composition

Estradiol

Dihydrotestosterone

G O N A D A L steroids have been shown to influence food intake, body weight, and carcass composition in rodents [9,24]. However, the effects of these hormones on body weight and food intake can vary depending on the species studied. F o r example, male rats and golden hamsters differ in their responses to castration and androgen replacement therapy. Castration decreases food intake and body weight gain in male rats [2, 9, 26], but castrated male hamsters are significantly heavier than gonadally-intact animals [10,23]. In both species these effects of castration are reversed by treatment with testosterone propionate (TP) [2,10]. In rats TP treatment increases lean body mass at least in part by stimulating protein synthesis in tissues such as skeletal muscle, liver, and kidney [1,9]. It is likely that these anabolic actions are mediated by unmetabolized testosterone, because testosterone is much more effective at increasing body weight than its principal metabolites such as 5c~-dihydrotestosterone or estradiol [2]. Also, there is relatively little in vivo conversion of testosterone to these compounds in skeletal muscle [1]. In addition to its protein anabolic actions, testosterone can reduce carcass lipid content in male rats [9,20]. It has been suggested that this adiposity-reducing effect of TP is mediated by aromatized (estrogenic) metabolites formed either in the hypothalamus [16] or in peripheral tissues such as adipose tissues [3]. Several lines o f evidence are consis-

Body weight

Food intake

tent with this hypothesis: (1) Treatment with estradiol benzoate (EB) mimics the effects of TP and reduces adiposity in female rats [4]. (2) The nonaromatizable androgen, 5adihydrotestosterone propionate (DHTP) does not affect carcass lipid content of castrated male rats [20]. (3) Adipose tissues and hypothalamus contain the aromatase enzymes necessary to form estrogens from testosterone [12,13]. It is possible that the adiposity-reducing effect of testosterone (via estrogens) is due in part to decreased adipose tissue lipoprotein lipase (LPL) activity. L P L hydrolizes circulating triglycerides and is an important determinant of their rate of storage by adipose tissues [17]. EB or TP treatments which reduce adiposity also reduce adipose tissue L P L activity in rats ]3, 4, 25]. These effects of TP can be blocked by concurrent administration of an aromatase inhibitor [3]. Little is known about the effects of castration and hormone replacement on food intake, carcass composition, or adipose tissue L P L activity in male hamsters. In view of the multiple weight-regulatory differences between rats and hamsters (e.g., [21]), we felt that it might be of some value to gather these kinds of data. In the following experiment we examined the effects of castration and replacement with an aromatizable androgen (TP), a nonaromatizable androgen (DHTP), or an estrogen (EB) on eating and body weight in male hamsters.

ZNow at Columbia University, New York, NY. 2Send reprint requests to George N. Wade.

C o p y r i g h t © 1981 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/81/100637-04502.00/0

638

SLUSSER AND WADE METHOD

Sixty male golden hamsters were purchased from Charles River Breeding Laboratories (Wilmington, MA) and housed singly in wire-bottom cages under a light cycle of 14 hr light and 10 hr dark per day (lights on at 0500 hr). The animals had ad libitum access to tap water and Purina rodent chow pellets and were given two weeks to adapt to these housing conditions. The animals were two months old at the start of the experiment. After adaptation, body weight (to the nearest 1 g) and food intake (to the nearest 0.1 g) were measured twice a week. Chow pellets were placed directly on the wire cage bottoms. Spilled food was collected, dried, and weighed. The hamsters did not pouch the food pellets. After collection of one week of baseline data, fifty-three of the hamsters were castrated via a single scrotal incision under methoxyflurane (Metofane; Pitman-Moore) anesthesia, and seven hamsters were sham-operated. The castrated animals were divided into six groups matched for baseline food intake and body weight. (Mean body weight: 80_+1 g for all seven groups.) The castrated groups received the following hormone treatments: 100/~g TP/day, 1000/~g TP/day, I00/xg DHTP/day, 1000/zg DHTP/day, 5/zg EB/day, or 0.1 ml sesame oil vehicle/day. After 52 days of injections the animals were killed with an overdose of sodium pentobarbital (Nembutal). One epididymal fat pad per animal was removed and homogenized for LPL assay. The carcasses were shaved, eviscerated, and analyzed for water, lipid, and protein content by a modification [4] of the method of Leshner et al. [ 11 ]. Adipose tissue LPL activity was assayed by a modification [3, 4, 6] of the method of Schotz et al. [19]. All measures were analyzed by one-way analyses of variance followed by Newman-Keuls post-hoc tests where appropriate. RESULTS

Body Weight Gain Castration significantly increased body weight gain over the 52 days of the experiment, and treatment with TP, DHTP, or EB prevented the castration-induced body weight gains, F(6,53)=6.74, p<0.01 (Fig. 1). None of the steroidtreated groups differed significantly from the sham-operated controls. Food Intake Neither castration nor treatment with any of the three steroids had any significant effect on food intake over the 52 days of the experiment, F(6,53)=0.53, NS. Carcass Composition Carcass components are presented as percentages of the wet carcass (Fig. 2). None of the experimental manipulations had any significant effect on carcass water content, F(6,53)=0.93, NS. Castration produced a small, but significant decrease in carcass protein content (Fig. 2), and this decrease was partially prevented only by the high (1000 p~g/day) doses of TP and DHTP, F(6,53)=3.08, p<0.05. These two androgen-treated groups were not significantly different from either the sham-operated males or the oiltreated castrates. Neither the low doses of TP and DHTP nor the EB had any effect on carcass protein content.

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Castration significantly increased carcass lipid content (Fig. 2), and this effect of castration was prevented only by the high dose of TP or by EB, F(6,53)=4.56,p<0.01. If these data are presented as protein/lipid ratios, it is clear that only the high dose of TP completely prevented the castrationinduced changes in carcass composition. TP treatment prevented both the increase in lipid content and the decrease in protein content induced by castration. Treatment with EB was only partially effective, because estrogen prevented the changes in lipid, but not protein, content. Lipoprotein Lipase Activity Epididymal fat pad LPL activity was reduced significantly by castration (Fig. 3). Treatment with either the high dose of TP or with EB prevented this decrease in LPL activity. The high dose of DHTP reduced LPL activity in the castrates, F(6,53)= 10.51, p<0.01. The same results are seen if the data are presented as L P L activity per mg wet tissue. If carcass lipid content and epididymal fat pad LPL activity are compared across all 60 hamsters there is a highlysignificant negative correlation between the two measures (r= - .49, p <0.0005). DISCUSSION

Food Intake and Body Wei~,,ht We were able to confirm the findings of earlier investigators who showed that castration increased body weights of male hamsters [10,23] and that this weight increase could be prevented by treatment with TP [10]. We also found that

A N D R O G E N S A N D H A M S T E R BODY W E I G H T

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FIG. 2. Effects of steroids on carcass composition in male hamsters. See legend to Fig. 1 for key to hormone treatments, a: p<0.05 vs sham-operated males; B: p<0.05 vs oil-treated castrates, Newman-Keuls tests,

FIG. 3. Effects of steroids on lipoprotein lipase activity in epididymal fat pads of male hamsters. Data are presented as/zmoles free fatty acid released per hour per mg tissue protein. See legend to Fig. 1 for key to hormone treatments. Asterisk indicates p<0.05 vs oil-treated castrates, Newman-Keuls tests.

either EB or DHTP could prevent castration-induced weight gains. The weight-reducing actions of EB treatment contrast with the lack of effectiveness of estrone reported by Kowalewski 110]. This difference could be due to: (1) the use of different estrogens (estradiol vs estrone), (2) differences in dosage (55 p.g EB/day vs 20 p~g estrone per kg four times a week), or (3) our use of esterified estradiol (EB) compared with unesterified estrone. The fact that DHTP seems to be as effective as TP in preventing weight gain in hamsters contrasts with the situation in rats where TP is substantially more effective in preventing the body weight effects of castration than is DHTP [2]. (Both androgens increase body weight in rats.) This finding raises the possibility that reduction of testosterone to 5a-dihydrotestosterone could play a role in testicular effects on body weight in hamsters, but probably not in rats (cf. [2,20]). All of these effects of hormone manipulation on body weight were seen in the absence of significant changes in food intake. This result is similar to the findings that manipulation of ovarian hormones in female rats can alter body weight without affecting food intake [5, 13, 18]. These data are consistent with the suggestion that metabolic effects of gonadal steroids play a significant role in regulation of body weight and composition [25].

terone mediate its actions on carcass lipids in hamsters, just as we have suggested in male rats [2, 3, 16, 20]. Two findings from this experiment are consistent with this possibility: (1) A substantially lower dose of EB (5/xg/day) than of TP (1000 txg/day) was required to reduce adiposity. (2) Even a very high dose of the nonaromatizable DHTP had no effect on carcass lipid content. Additional research will be necessary to test this aromatization hypothesis. Castration also caused a small reduction in carcass protein content, probably a consequence of the withdrawal of anabolic androgen(s). High doses of TP or DHTP, but not EB, partially prevented the decrease in carcass protein. Again the effectiveness of DHTP suggests that in hamsters, but not rats [2,20], 5c~-reduced metabolites of testosterone could play some role in its anabolic effects. Although TP was the only hormone we tested which prevented the effects of castration on both carcass lipid and protein content, both effects could be mediated by metabolites of the hormone. There is substantial evidence that the actions on carcass lipids are due to estrogenic metabolites, and there is some evidence that a 5a-reduced metabolite may have anabolic actions. It should be noted that a higher dose of TP is necessary to alter carcass composition in hamsters (1000 /zg/day) than in rats (<200 ~g/day) [2,9]. Similarly, female hamsters are less responsive to the body weight effects of EB than are female rats [12]. Castration, which increased carcass lipid content, reduced epididymal fat pad L P L activity. EB and the high dose of TP reduced carcass adiposity but increased epididymal adipose tissue L P L activity. In fact, there was a highlysignificant negative correlation between epididymal fat pad LPL activity and carcass lipid content. This is just the opposite of what is found in rats where EB and TP reduce both carcass adiposity and gonadal fat pad L P L activity [3, 4, 22].

Carcass Composition and LPL Activity The increased body weight of castrated male hamsters is due in part to true growth, that is, to increased body length [8]. Our data indicate that another portion of this weight gain is due to increased carcass lipid content. Either the high dose of TP or EB was able to prevent this increase in adiposity. It is possible that aromatized (estrogenic) metabolites of testos-

640

SLUSSER AND WADE

It is not p o s s i b l e to d e t e r m i n e f r o m the p r e s e n t d a t a w h e t h e r t h e s e s t e r o i d - i n d u c e d c h a n g e s in e p i d i d y m a l fat p a d L P L activity are a c c o m p a n i e d by similar c h a n g e s in o t h e r fat pads. If they are, it w o u l d suggest t h a t in h a m s t e r s t e s t i c u l a r effects on b o d y fat levels are not m e d i a t e d by c h a n g e s in a d i p o s e tissue L P L activity. W e h a v e s h o w n p r e v i o u s l y t h a t in rats p r o g e s t e r o n e c a n i n c r e a s e c a r c a s s lipid c o n t e n t without affecting a d i p o s e tissue L P L activity (e.g,, [4]). Other metabolic actions of testicular hormones may be of p r i m a r y i m p o r t a n c e for b o d y w e i g h t a n d c o m p o s i t i o n c h a n g e s in h a m s t e r s . T h e s e c o u l d include c h a n g e s in lipogenesis or lipolysis o r c h a n g e s in the m e t a b o l i c activity o f b r o w n fat. B r o w n fat is e x t r e m e l y well d e v e l o p e d in h a m -

sters [7] a n d has b e e n h y p o t h e s i z e d to play a n i m p o r t a n t role in caloric e x p e n d i t u r e , especially o f lipids [8]. It w o u l d seem to be w o r t h w h i l e to e x a m i n e the effects o f g o n a d a l steroids o n b r o w n a d i p o s e tissue in h a m s t e r s . ACKNOWLEDGEMENTS Supported by Research Grant NS 10873 and Research Career Development Award NS 00090 from the NINCDS, Research Grant AM 20785 from the NIAMDD, and Research Scientist Development Award MH 00321 from the NIMH. We are grateful to Joy Littlejohn and Christina Decoteau for expert assistance and to Janet Gray, Antonio Nunez, Susan Schwartz and Linda Siegel for advice and discussions.

REFERENCES

1. Bardin, C. W. and J. F. Catterall. Testosterone: A major determinant of extragenital sexual dimorphism. Science 211: 1285-1294, 1981. 2. Gentry, R. T. and G. N. Wade. Androgenic control of food intake and body weight in male rats. J. comp. physiol. Psychol. 90: 18--25, 1976. 3. Gray, J. M., A. A. Nunez, L. I. Siegel and G. N. Wade. Effects of testosterone on body weight and adipose tissue: Role of aromatization. Physiol. Behav. 23: 465-469, 1979. 4. Gray, J. M. and G. N. Wade. Food intake, body weight, and carcass adiposity in female rats: Actions and interactions of progestins and anti-estrogens. Am. J. Physiol. 240: E474--E481, 1981. 5. Hervey, E. and G. R. Hervey. Energy storage in female rats treated with progesterone in the absence of increased food intake. J. Physiol. 200:118P-119P, 1968. 6. Hietanen, E. and M. R. C. Greenwood. A comparison of lipoprotein lipase activity and adipocyte differentiation in growing male rats. J. Lipid Res. 18: 480-490, 1977. 7. Himms-Hagen, J. Cellular thermogenesis. A. Rev. Physiol. 38: 315-351, 1976. 8. Himms-Hagen, J. Obesity may be due to a malfunctioning of brown fat. Can. med. Ass. J. 121: 1361-1364, 1979. 9. Kochakian, C. D. Definition of androgens and protein anabolic steriods. Pharmac. Ther. B. 1: 14%177, 1975. 10. Kowalewski, K. Effect of pre-pubertal gonadectomy and treatment with sex hormones on body growth, weight of organs and skin collagen of hamsters. Acta endrocr. 61: 48--56, 1969. 11. Leshner, A. I., V. A. Litwin and R. L. Squibb. A simple method for carcass analysis. Physiol. Behav. 9: 281-282, 1972. 12. Morin, L. P. and A. S. Fleming. Variation of food intake and body weight with estrous cycles, ovariectomy, and estradiol benzoate treatment in hamsters. J. comp physiol. Psychol. 92: 1-6, 1978. 13. Mueller, K. and S. Hsiao. Estrus- and ovariectomy-induced body weight changes: Evidence for two estrogenic mechanisms. J. comp. physiol. P.Lvchol. 94:1126-1134, 1980. 14. Naftolin, F., K. J. Ryan and Z. Petro. Aromatization of androstenedione by the anterior hypothalamus of adult male and female rats. Endocrinology 90: 295-298, 1972.

15. Nimrod, A. and K. J. Ryan. Aromatization of androgens by human abdominal and breast fat tissue. J. olin. Endrocr. Metah. 40: 367-372, 1975. 16. Nunez, A. A. , L. I. Siegel and G. N. Wade. Central effects of testosterone on food intake in male rats. Physh~l. Behav. 24: 46%472, 1980. 17. Robinson, D. S. The function of the plasma triglycerides in fatty acid transport. In: Comprehensive Bh~chemistIT, Vol. 18, Lipid Metabolism, edited by M. Florkin and E. H. Stotz. Amsterdam: Elsevier, 1970, pp. 51-116. 18. Roy, E. J. and G. N. Wade. Role of food intake in estradiolinduced body weight changes in female rats. Hormone,~ Behav. 8: 265-274, 1977. 19. Schotz, M., A. Garfinkel and R. J. Huebotter. A rapid assay for lipoprotein lipase. J. Lipid Re.~. I 1: 68-69, 1970. 20. Siegel, L. I. , A. A. Nunez and G. N. Wade. Effects of androgens on dietary self-selection and carcass composition in male rats. J. comp. physiol. Psy,chol. 95: in press, 1981. 21. Silverman, H. J. and I. Zucker. Absence of post-fast food compensation in the golden hamster (Meso~'ricetus altrattts). Physiol. Behav. 17: 271-285, 1976. 22. Steingrimsdottir, L., J. Brasel and M. R. C. Greenwood. Hormonal modulation of adipose tissue lipoprotein lipase may alter food intake in rats. Am. J. Physiol. 239: E162-E167, 1980. 23. Swanson, H. H. Effects of pre- and post-pubertal gonadectomy on sex differences in growth, adrenal and pituitary weights of hamsters. J. Endocr. 39: 555-564, 1967. 24, Wade, G. N. Sex hormones, regulatory behaviors, and body weight. In: Advances in the Study qfBehavior, Vol. 6. edited by J. S. Rosenblatt, R. B. Hinde, E. Shaw and C. G. Beer. New York: Academic Press, 1976, pp. 201-279. 25. Wade, G. N. and J. M. Gray. Gonadal effects on food intake and adiposity: A metabolic hypothesis. Physiol. Behav. 22: 583-593, 1979. 26. Wang, G. H. , C. P. Richter and A. F. Guttmacher. Activity studies on male castrated rats with ovarian transplants and correlation of the activity with the histology of the graphs. Am. ,I. Physiol. 73: 581-599, 1925.