Inhibition of 5-alpha reductase blocks prostate effects of testosterone without

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1 Articles in PresS. Am J Physiol Endocrinol Metab (September 14, 2004). doi: /ajpendo Borst et al. E R1-1 Inhibition of 5-alpha reductase blocks prostate effects of testosterone without blocking anabolic effects Stephen E. Borst 1,2, Jun Hak Lee 1 and Christine F. Conover 2 1 Department of Applied Physiology & Kinesiology, University of Florida, Gainesville 2 Geriatric Research, Education and Clinical Center, Malcom Randall VA Medical Center, Gainesville, Florida Corresponding author: Stephen Borst, Ph.D. VA Medical Center, GRECC SW Archer Rd. Gainesville FL phone: FAX: seborst@ufl.edu Running head: Anabolic effects of testosterone and 5- reductase inhibitor Copyright 2004 by the American Physiological Society.

2 Borst et al. E R1-2 Abstract We studied the effect of the 5- reductase inhibitor MK-434 on responses to testosterone (T) in orchiectomized (ORX) male Brown Norway (BN) rats aged 13 months. At 4 weeks after ORX or SHAM surgery, a second surgery was performed to implant pellets delivering 1 mg T/day or placebo pellets. During the second 4 weeks of the study, rats received injections of MK-434 (0.75 mg/day) or vehicle injections. Treatment with T elevated serum T to 75% above that for SHAM animals (p = 0.002) and did not affect serum dihydrotestosterone (DHT) or serum estradiol. T-treatment also caused an elevation of prostate T and a marked elevation of prostate DHT. During the second half of the study, ORX rats lost an average of ± 4.62 g of body weight. T completely prevented weight loss, and the effect was not inhibited by MK-434 (p < 0.001). ORX produced a non significant trend toward a small (5%) decrease in the mass of the gastrocnemius muscle (p = ). This trend was also reversed by T and the effect of T was not blocked by MK-434. T caused a significant 16% decrease in subcutaneous fat which was not blocked by MK-434 (p < 0.05). Finally, T caused a 65% decrease in urine excretion of deoxypyridinoline, a marker of bone resorption, and again the effect was not blocked by MK-434 (p < ). In contrast, T caused a greater than 5-fold increase in prostate mass and the effect was almost completely blocked by MK-434 (p < ). This study demonstrates that 5- reductase inhibitors may block the undesirable effects of T on the prostate, without blocking the desirable anabolic effects of T on muscle, bone, and fat. Key words testosterone, dihydrotestosterone, 5- reductase, anabolic, prostate, body composition, bone resorption

3 Borst et al. E R1-3 Introduction We have recently reviewed the many trials evaluating testosterone (T) administration as a strategy for combating loss of muscle mass and strength in elderly men (5). Most studies report no more than a modest increase in lean body mass and only a few report any increase in strength. These findings do not necessarily indicate that older men are unresponsive to T. Most studies have been conducted with replacement doses of T, and higher doses have not been considered for fear of accelerating underlying prostate cancer (6, 19, 37). Recently, Magliano et al. reported that a very high dose of 600 mg T/ week produced substantial increases in muscle mass, and that increases were equal in younger vs. older men (22). T is the principal androgen acting in tissues lacking 5-reductase. In tissues expressing 5reductase, T is converted to dihydrotestosterone (DHT) and in those tissues, DHT is the principal androgen. 5-reductase is highly expressed in the prostate, but not in muscle or bone (17). This observation led us to the hypothesis that inhibitors of 5-reductase might block the undesirable effects of T on the prostate, without blocking the desirable effects of T on muscle, fat and bone. We examined the prostate and anabolic effects of a high dose of 1 mg T/day, alone and in combination with a 5- reductase inhibitor. For the latter, we chose MK-434 because its reported potency in rats (32) is higher than that of finasteride (12, 30, 33). Our study was conducted with Brown Norway (BN) male rat, an established model of age-induced hypogonadism (1, 23). Orchiectomy (ORX) of BN males causes a marked reduction in prostate mass and high-turnover osteopenia, which characterized by an elevation in urine deoxypyridinoline (Dpd), a marker on bone resorption (13, 43). Phillip et al. (27) and Prakasam et al. (28) found that T prevents castration-induced losses in the width of the tibial epiphyseal growth plate and in femoral bone mineral density in Sprague-Dawley and Wistar rats,

4 Borst et al. E R1-4 respectively. Carson et al. (10) and Wimalawansa et al. (46) reported that T administration prevents loss of muscle mass and bone mineral density in hindlimb-suspended BNxF344 and Sprague-Dawley rats, respectively. Materials and Methods Animals and experimental design. Barrier-raised and viral pathogen-free Brown Norway (BN) male rats aged 13 months were obtained from Zivic-Miller Laboratories (Zellenople, PA). Experimental procedures conformed to the ILAR Guide to the Care and Use of Experimental Animals and were approved by the Institutional Animal Care and Use Committee at the Gainesville VA Medical Center. Closed orchiectomy (ORX) was performed on 32 rats and involved removal of the testes, epididymis and epididymal fat pads. Sham surgery was performed on 8 rats. After surgery, rats received a nutritional supplement (Jello-O plus protein and fat) daily for 2 days. After 25 days, rats were housed overnight in metabolic cages and urine was collected for analysis of deoxypyridinoline (Dpd). At 28 days, a second surgery was performed for subcutaneous implantation of pellets designed to deliver 1 mg testosterone/day for 30 days (Innovative Research of America, Sarasota FL). At the time of the second surgery, we also began 28 days of treatment with MK-434 (0.75 mg/day in 0.15 ml of 90% DMSO, 10% ethanol, s.c.). MK-434 was a gift from Merck Inc., Rahway NJ. There were 5 experimental groups: SHAM, ORX, ORX+T, ORX+MK, and ORX+T+MK. Rats not receiving testosterone (T) pellets received placebo pellets. Rats not receiving MK-434 received vehicle injections. After 25 days of drug treatment, rats were again housed in metabolic cages for urine collection. After 28 days of drug treatment, rats were euthanized and tissues collected. Steroid hormone assays were performed using kits obtained from Diagnostic Systems Laboratories, Webster, TX. Serum androgens and estradiol were extracted according the

5 Borst et al. E R1-5 manufacturer s instructions. Serum T was measured using an enzyme immunoassay (EIA) kit with a sensitivity of 3.8 pg/ml and an inter-assay coefficient of variation (CV) of 11%. Serum DHT was measured using a radioimmunoassay (RIA) kit with a sensitivity of 4 pg/ml and an inter-assay CV of 4.5%. Prostate androgens were extracted according to the protocol of Theobald et al. (40). Serum estradiol was measured using an RIA kit with a sensitivity of 2.2 pg/ml and an inter-assay CV of 7%. Urine deoxypyridinoline (Dpd) is a degradation product of type I collagen and a specific marker for bone resorption. Dpd was measured in urine samples using a Pyrilinks-D EIA kit with a sensitivity of 1.1 nmol/l and an inter-assay CV of 4% (Quidel Corp, Santa Clara, CA). Because the concentration of urine solutes may be altered by water excretion, Dpd was normalized to urine creatine and reported as nm Dpd/mM creatinine. Creatinine was measured using a colorimetric assay kit with a high sensitivity (as 10-fold dilution of samples is required) and an inter-assay CV of 2% (Sigma Chemical Co., St. Louis, MO). Free fatty acid (NEFA) concentration was measured using a colorimetric assay kit that relies on fatty acids as substrate for enzymatic acylation of CoA (Wako Chemicals, Neuss, Germany). Statistics: Two-way and repeated measures ANOVA were performed using PRISM software (GraphPad Software Inc., San Diego, CA). Results During ORX surgery, an average of ± 0.26 g of tissue was removed (testes, epididymis and most of epididymal fat pad). In the first 4 weeks following surgery, ORX rats gained 5.20 ± 2.71 g over their post-surgical weight, while SHAM rats gained ± 4.49 g (figure 1, left panel). During the second 4 weeks of the study, following surgical pellet implantation, SHAM+vehicle rats maintained body weight (average loss of 0.56 ± 4.08 g), while ORX+vehicle

6 Borst et al. E R1-6 rats lost an average of ± 4.62 g. ORX-induced loss of body weight was prevented by T (p < ) and the effect of T was not blocked by MK-434 (p = 0.910, figure 1, right panel). No differences in serum NEFA were observed among the experimental groups (0.212 ± meq/l for SHAM, ± meq/l for ORX+veh, ± meq/l for ORX+T, ± meq/l for ORX+MK, and ± meq/l for ORX+MK+T). T-induced prostate enlargement was blocked by MK-434 (figure 2, left panel). ORX caused a 51% decrease in prostate mass (p < 0.01). In ORX rats, T caused a 5.4 fold increase in prostate mass, which was almost completely blocked by MK-434 (p < ). T caused non-significant trend toward a 5% increase in mass of the gastrocnemius muscle (figure 2, center panel, p = , ANOVA) and the effect was not blocked by MK-434 (p = 0.925). T caused a significant 16% decrease in the inguinal depot of subcutaneous fat (IWAT, p = ) and the response was not blocked MK-434 (figure 2, right panel, p = for MK, p = for interaction). Urine deoxypyridinoline (Dpd) was measured at 25 and 55 days (figure 3). Dpd was normalized urine creatinine and expressed as nm Dpd/mM creatinine. At 25 days, ORX caused a 75% increase in Dpd/creatinine (p < 0.001). At 55 days, T caused a 65% decrease in urine Dpd/creatinine (p < ) and the effect was not blocked by MK-434 (p = 0.670). The effects of treatment on serum androgens are shown in figure 4. ORX reduced serum T by 76% (p < 0.002). In ORX rats, T-administration elevated serum T to a level that was somewhat higher that what was observed for SHAM rats (p ). Serum DHT was not significantly affected by treatment. Serum E2 concentrations were very low and were not significantly affected by treatment (1.67 ± pg/ml for SHAM, ± 7.22 pg/ml for ORX+veh, ± pg/ml for ORX+T, 5.09 ± 1.56 pg/ml for ORX+MK, and ± pg/ml for ORX+MK+T). The effects of treatment on prostate androgens are shown in

7 Borst et al. E R1-7 figure 5. ORX caused a 40% decrease in prostate T (p < ) and a 90% decrease in prostate DHT (p < ). MK-434 alone had no effect on prostate androgens. T administration to ORX animals elevated prostate T by 3- to 4-fold, producing higher concentrations than were observed in SHAM animals (p < ). T administration to ORX animals increased prostate DHT 26-fold (p = ) an effect that was almost completely blocked by MK-434 (p = ). Discussion The most important findings of this study were as follows. 1). ORX resulted in a loss of body weight, a very small loss of muscle mass, a significant increase in bone resorption and a decrease in prostate mass. 2). In ORX rats, T administration elevated serum T and prostate T and DHT to above that observed in SHAM rats. T administration prevented the ORX-associated losses of body weight and muscle mass and caused a small reduction in subcutaneous fat. T also suppressed bone resorption to a level below that of SHAM animals and caused marked prostate enlargement. 3). MK-434 almost entirely blocked T-induced prostate enlargement, but did not block any of the anabolic effects of T on body weight, muscle mass, fat mass or bone resorption. Inhibition of T-induced prostate enlargement by MK-434 was probably due to inhibition of 5- reductase, as MK-434 caused small changes serum and prostate T, but a marked decrease in prostate DHT. Taken together, these findings indicate that co-administration of T and MK-434 produces desirable anabolic effects on muscle bone and fat, without undesirable prostate enlargement. Although T and DHT mediate separate effects in different tissues, there is strong evidence that they act through the same receptors. Conversion to DHT amplifies the effects of T in several ways. First, DHT has a higher affinity for the androgen receptor than does T (16).

8 Borst et al. E R1-8 Second, binding of DHT stabilizes the androgen receptor, slowing the rate of receptor degradation (47). Third, conversion of T to DHT prevents its conversion to androsteindione, a much weaker androgen (15). Virilization of external genitalia during development depends on DHT (2). However, in adult men the effects of DHT appear to be generally undesirable. The latter include increased body hair, acne, male-pattern baldness and prostate enlargement. Suppression of DHT with the 5-reductase inhibitor finasteride does not appear to have major undesirable effects. Finasteride does not have catabolic effects on muscle or bone (23). In contrast, the effects of T which are not mediated by DHT are generally considered to be desirable. These include increased muscle mass and bone mineral density, deepening of the voice, increased libido, spermatogenesis, and increased hematocrit (desirable in the absence of polycythemia). Thus obtaining only the effects of T which are not mediated by DHT is an attractive therapeutic target. Recent evidence suggests that some of the anabolic effects of administered T may depend on aromatase activity and tissue conversion of T to estradiol. The essential role of estradiol in regulating bone mass in males was confirmed with the identification young adult men who cannot produce estradiol (E2) due to a lack of P-450 aromatase activity (25), or who cannot respond to E2 due to defective E receptor (36). These men have significantly reduced bone mineral density (BMD) despite normal or above-normal androgen concentrations. E2 administration improves BMD in these men, whereas T is ineffective (9). In men, it is estimated that eighty-five percent of E2 is produced by peripheral conversion of circulating androgens and that adipose tissue is the main site of conversion (35). The aromatase inhibitor anastrazole increases bone resorption in normal young men, as indicated by elevations in urinary Dpd and N- telopeptide (21). Thus the effects of administered T on adipose tissue and bone may be

9 Borst et al. E R1-9 mediated, in part by E2. We did not observe any elevation of serum E2 following T administration. However, we can not rule out the possibility that bone E2 was elevated and thus the possibility that T-induced suppression of bone resorption is mediated by local aromatase activity. In rats, ORX is known to reduce prostate mass (13, 43) and to increase bone resorption (13, 43). Whether ORX reduces muscle mass depends on the strain of rat. Vandenput et al. showed that ORX of 12 month old Wistar rats reduced lean body mass, as assessed by DEXA (41). Brown et al. found that ORX of 7 month old Sprague-Dawley rats caused no change in the weight of several muscles (8). Administration of T increases prostate mass in both ORX and intact rats (10). Several groups have shown that T prevents ORX-induced loss of bone (27, 28, 42). In male rats, the short term effects of ORX on muscle include loss of glycogen stores (29) and reduced protein synthesis (21) and both of these effects are prevented by T administration. Rogozkin reported that several anabolic steroids stimulate muscle protein synthesis in intact rats (31). Van Zyl has reported that T treatment significantly increased treadmill exercise performance in intact rats (44). Our choice of MK-434 as a 5- reductase inhibitor was based on the fact that it has greater potency in the rats than does finasteride. While finasteride is effective in the range of 5 to 40 mg/day (12, 30, 33), Seo et al. (32) have shown that 1 mg MK-434/day suppresses DHT. This is the first report showing that a 5- reductase inhibitor blocks the effect of T on the prostate, without blocking the anabolic effects of T. In the present study, rats lost weight after ORX surgery and recovered their initial body weight by 4 weeks. In contrast, after the second surgery for implantation of pellets, rats not treated with T had a progressive loss of body weight, while those treated with T maintained body weight. It is not clear why weight loss was greater after the second surgery, which was probably

10 Borst et al. E R1-10 less traumatic than the first. We did not measure food intake and weight loss may have had a central component. Weight loss may be attributable to the use of DMSO as a vehicle for injections, although all 5 experimental groups received DMSO. It is also not clear which body compartment accounted for the weight loss. ORX rats that were not treated with T had only slightly lower muscle mass, while visceral fat was unchanged and subcutaneous fat was increased. It is possible that the prevention of weight loss by T was due to an increase in skin, internal organs, muscles that we did not measure, or fluid retention. Although T produces substantial anabolic effects in young and middle aged hypogonadal men (3, 7, 38, 45), the anabolic effects of T replacement therapy in older men have been harder to demonstrate. Most studies of T replacement in older men have reported small increases in lean mass (4, 6, 19, 37, 39). Brill et al. (6), Kenny et al. (19), Clague et al. (11), and Snyder et al. (37) have all reported that replacement doses of T fail to increase strength in elderly hypogonadal men. In contrast, two groups have reported increased strength in elderly men following T replacement. Ferrando et al. (14) administered replacement doses of T enanthate for 6 months to men aged greater than 60 with low circulating T. Treatment was designed to raise nadir T levels into the range of 17 to 28 nm. At 6 months there were significant increases in muscle strength and no increase in prostate mass. Sih et al. (34), who found that T replacement caused a 5 kg increase in grip strength, amounting roughly to a 10% improvement. Although it is still not clear whether T replacement increases strength in older men, a recent abstract by Magliano et al. (22) reported that elderly men have a strong anabolic response to high doses of T. In this randomized controlled trial, both younger (aged years) and older men (aged years) were treated for 20 weeks with T at doses of 125, 300 and 600 mg/week. In the two age groups, significant and equal increases in muscle mass were observed. Strength and safety

11 Borst et al. E R1-11 were not assessed in the study and, in general, higher doses of testosterone have not been considered in older men because of the risk of accelerating underlying prostate cancer. Initial fears that T replacement would promote prostate cancer were lessened by the findings of Hajjar et al. (18), who treated 45 elderly men with a replacement dose of T and found no increase in prostate cancer during a 2-year follow-up. However, concerns for the effect of T on the prostate have been rekindled by the recent release of 40-year data from the Baltimore Longitudinal Study on Aging showing a positive correlation between blood testosterone levels and the risk of prostate cancer (26). Concerns are increased by the high prevalence of earlystage prostate cancer in elderly men. Approximately 10% of men will develop clinically manifest prostate cancer in their lifetime and ~3% will die of the disease (24). However, autopsy data show that 42% of men over the age of 60 have early-stage prostate cancer (24). In addition to prostate effects, other risks are associated with T replacement in older men, including fluid retention, gynecomastia, worsening of sleep apnea, polycythemia (39). It is not clear if these other effects are mediated by DHT. We conclude that 5- reductase inhibitors block at least some of the undesirable effects of T, while allowing desirable anabolic effects to occur. Our results suggest that administration of higher doses of T in combination with a 5- reductase inhibitor might be a safe and effective strategy for combating sarcopenia in elderly men. Acknowledgments This research was supported by a Department Veterans Affairs Merit Award to S.E.B. We thank Drs. Richard E. Peterson and H. Michael Theobald for consultation regarding measurement of prostate androgens.

12 Borst et al. E R1-12 References 1. Banerjee PP, Banerjee S, and Brown TR. Increased androgen receptor expression correlates with development of age-dependent, lobe-specific spontaneous hyperplasia of the brown Norway rat prostate. Endocrinology 142(9): , Bartsch G, Rittmaster RS, and Klocker H. Dihydrotestosterone and the concept of 5reductase inhibition in human benign prostatic hyperplasia. Eur Urol 37: , Bhasin S, Storer TW, Berman N, Yarasheski KE, Clevenger B, Phillips J, Lee WP, Bunnell TJ, and Casaburi R. Testosterone replacement increases fat-free mass and muscle size in hypogonadal men. J Clin Endocrinol Metab 82(2):407-13, Blackman MR, Sorkin JD, Munzer T, Bellantoni MF, Busby-Whitehead J, Stevens TE, Jayme J, O'Connor KG, Christmas C, Tobin JD, Stewart KJ, Cottrell E, St Clair C, Pabst KM, and Harman SM. Growth hormone and sex steroid administration in healthy aged women and men: a randomized controlled trial. JAMA 288(18): , Borst SE. Interventions for Sarcopenia and Muscle Weakness in Older People. Age & Ageing (in press) Brill KT, Weltman AL, Gentili A, Patrie JT, Fryburg DA, Hanks JB, Urban RJ, and Veldhuis JD. Single and combined effects of growth hormone and testosterone administration on measures of body composition, physical performance, mood, sexual function, bone turnover, and muscle gene expression in healthy older men. J Clin Endocrinol Metab 87(12): , 2002.

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14 Borst et al. E R George FW. Androgen metabolism in the prostate of the finasteride-treated, adult rat: a possible explanation for the differential action of testosterone and 5 alphadihydrotestosterone during development of the male urogential tract. Endrocrinology 138(3): , George FW and Noble JF. Androgen receptors are similar in fetal and adult rabbits. Endocrinology 115(4): , Gormley GJ. Finasteride: a clinical review. Biomed & Pharmacother 49: , Hajjar RR, Kaiser FE, and John EM. Outcomes of long-term testosterone replacement in older hypogonadal males: a retrospective analysis. J Clin Endocrinol Metab 82(11): , Kenny AM, Prestwood KM, Gruman CA, Marcello KM, and Raisz LG. Effects of transdermal testosterone on bone and muscle in older men with low bioavailable testosterone levels. J Gerontol Med Sci. 56A:M266-M272, Kochakian CD. Definitions of androgens and protein anabolic steroids. Pharmacol Therap 1: , Leder BZ, LeBlanc KM, Schoenfeld DA, Eastell R and Finkelstein JS. Differential effects of androgens and estrogens on bone turnover in normal men. J Clin Endocrinol Metab 88(1):204-10, Magliano L, Woodhouse LJ, Bhasin S, and Storer TW. Testosterone dose-dependently increase skeletal muscle mass (SMM) in healthy men. Med & Sci Sports Exer 36(5):S238, Abstract #1661, Matsumoto AM, Tenover L, McClung M, Mobley D, Geller J, Sullivan M, Grayhack J, Wessells H, Kadmon D, Flanagan M, Zhang GK, Schmidt J, Taylor AM, Lee M,

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16 Borst et al. E R1-16 rats given the 5 alpha-reductase inhibitor finasteride. Endrocrinology 136(2): , Rogozkin V. Metabolic effects of anabolic steroid on skeletal muscle. Med Sci Sports Exercise 11(2): , Seo SI, Kim SW, and Paick JS. The effects of androgen on penile reflex, erectile response to electrical stimulation and penile NOS activity in the rat. Asian J Androl 4:169-74, Shao TC, Kong A, Marafelia P and Cunningham GR. Effects of finasteride on the rat ventral prostate. J Androl 14(2):79-86, Sih R, Morely JE, Kaiser FE, Perry HM, Patrick P, and Ross C. Testosterone replacement in older hypogonadal men: a 12 month randomized, controlled trial. J Clin Endocrinol Metab 82: , Simpson ER. Aromatase: biologic relevance of tissue-specific expression. Semin Reprod Med 22(1):11-23, Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, Williams TC, Lubahn DB and Korach KS. Estrogen resistance caused by a mutation in the estrogenreceptor gene in a man. N Engl J Med 331: , Snyder PJ, Peachey H, Hannoush P, Berlin JA, Loh L, Holmes JH, Dlewati A, Staley J, Santanna, Kapoor SC, Attie MF, Haddad JG, and Strom BL. Effect of testosterone treatment on body composition and muscle strength in men over 65 years of age. J Clin Endrocrinol & Metab 84: , Storer TW, Magliano L, Woodhouse L, Lee ML, Dzekov C, Dzekov J, Casaburi R and Bhasin S. Testosterone dose-dependently increases maximal voluntary strength and

17 Borst et al. E R1-17 leg power, but does not affect fatigability or specific tension. J Clin Endocrinol Metab 88(4): , Tenover JL. Testosterone replacement therapy in older adult men. Int J Androl 22: , Theobald HM, Roman BL, Lin TM, Ohtani S, Chen SW and Peterson RE. 2,3,7,8- tetrachlorodibenzo-p-dioxin inhibits luminal cell differentiation and androgen responsiveness of the ventral prostate without inhibiting prostatic 5alphadihydrotestosterone formation or testicular androgen production in rat offspring. Toxicol Sci 58(2):324-38, Vandenput L, Boonen S, Van Herck E, Swinnen JV, Bouillon R, and Vanderschueren D. Evidence from the aged orchidectomized male rat model that 17 beta-estradiol is a more effective bone-sparing and anabolic agent than 5-alpha-dihydrotestosterone. J Bone Miner Res 17(11): , Vandenput L, Swinnen JV, Van Herck E, Verstuyf A, Boonen S, Bouillon R, and Vanderschueren D. The estrogen receptor ligand ICI 182,780 does not impair the bonesparing effects of testosterone in the young orchidectomized rat model. Calcif Tissue Int 70(3): , Vanderschueren D, Van Herck E, Suiker AM, Visser WJ, Schot LP, and Bouillon R. Bone and mineral metabolism in aged male rats: short and long term effects of androgen deficiency. Endocrinology 130: , Van Zyl CG, Noakes TD, and Lambert MI. Anabolic-androgenic steroid increases running endurance in rats. Med Sci Sports Exerc 27(10): , 1995.

18 Borst et al. E R Wang C, Swerdloff RS, Iranmanesh A, Dobs A, Snyder PJ, Cunningham G, Matsumoto AM, Weber T, and Berman N. Transdermal testosterone gel improves sexual function, mood, muscle strength, and body composition parameters in hypogonadal men. Testosterone Gel Study Group. J Clin Endocrinol Metab 85(8): , Wimalawansa SM, Chapa MT, Wei JN, Westlund KN, Quast MJ, and Wimalawansa SJ. Reversal of weightlessness-induced musculoskeletal losses with androgens: quantification by MRI. J Appl Physiol 86(6): , Zhou ZX, Lane MV, Kemppainen JA, French FS, and Wilson EM. Specificity of ligand-dependent androgen receptor stabilization: receptor domain interactions influence ligand dissociation and receptor stability. Mol Endocrinol 9(20): , 1995.

19 Borst et al. E R body weight (g) sham ORX+veh ORX+T ORX+MK body weight (% of 4 weeks ORX+T+MK 92 * SHAM+veh ORX+veh ORX+T ORX+MK ORX+T+MK * * * time (weeks) time after pellet implantation (weeks) Figure 1. ORX-induced loss of body weight is prevented by T and the effect is not blocked by MK-434. BN male rats aged 13 months received ORX or SHAM surgery at week 0. After 4 weeks, we implanted T or placebo pellets and began daily injection of MK-434 or vehicle. ORX rats were assigned to 4 groups (vehicle, T, MK and T+MK) in a manner which produced similar mean body weights in the 4 groups (see left panel). However, there was a considerable range in body weight within each group. For this reason, body weight following drug treatment is expressed as a percentage of pretreatment weight (see right panel). Compared to SHAM+vehicle, ORX+vehicle and ORX+MK animals lost body weight (*indicates p < , repeated measures ANOVA). SHAM+vehicle, ORX+T and ORX+T+MK animals maintained body weight. Results are presented as means ± SEM, N = 5 for SHAM and 8 for other groups.

20 Borst et al. E R1-20 grams * # SHAM+veh ORX+veh ORX+T ORX+MK ORX+MK+T * # grams ( * ) ( * ) mass (g) * * 0.0 prostate mass 0 1 gastroc muscle mass Figure 2. MK-434 blocks effects of T on prostate, but not muscle and fat. Left panel: ORX reduced prostate mass by 51% indicates p < 0.01 vs. SHAM). 28-day treatment of ORX animals with T caused a 5.4 fold increase in prostate mass (gray bars), which was significantly inhibited by MK-434 (gray, striped bar) (*indicates p < for T, #indicates p < for MK, p < for interaction, 2-way ANOVA). Center panel: T caused a trend toward a 5% increase in mass of the gastrocnemius muscle (gray bars). ( * ) Indicates p = for T. Right panel: T caused a small (16%), but significant decrease in the inguinal depot of subcutaneous fat (IWAT; gray bars, p = 0.031). The effect of T was not blocked by MK434 (gray striped bar). No treatment effects were observed in visceral fat (VFAT). Results are presented as means ± SEM, N = 5 for SHAM and 8 for other groups. 0 IWAT VFAT

21 Borst et al. E R1-21 Alternate version of figure 2 grams SHAM+veh * ORX+veh ORX+T ORX+MK * # # ORX+MK+T prostate mass gastroc muscle mass 1.5 grams 1 SHAM+veh ORX+veh ORX+T (* ) ( * ) ORX+MK ORX+T+MK grams SHAM+veh ORX+veh IWAT VFAT * * ORX+T ORX+MK adipose tissue Figure 2. MK-434 blocks effects of T on prostate, but not muscle and fat. Left panel: ORX ORX+MK+T reduced prostate mass by 51% indicates p < 0.01 vs. SHAM). 28-day treatment of ORX animals with T caused a 5.4 fold increase in prostate mass, which was significantly inhibited by MK-434 (*indicates p < for T, #indicates p < for MK, p < for interaction, 2-way ANOVA). Center panel: T caused a trend toward a 5% increase in mass of the gastrocnemius muscle. ( * ) Indicates p = for T. Right panel: T caused a small (16%), but significant decrease in the inguinal depot of subcutaneous fat (IWAT, p = 0.031). The effect of T was not blocked by MK434 (gray striped bar). No treatment effects were observed in visceral fat (VFAT). Results are presented as means ± SEM, N = 5 for SHAM and 8 for other groups.

22 Borst et al. E R1-22 SHAM Dpd/creatinine (nm/mm # # # # * * ORX+veh ORX+T ORX+M K ORX+M K+T 0 4th week 8th week Figure 3. T inhibits bone resorption in ORX rats, and the effect is not blocked by MK-434. During the first four weeks of the experiment, ORX caused a 75% increase in urine excretion of Dpd (#indicates p < vs. SHAM). During the second four weeks, T administration caused a 65% decrease in Dpd (*indicates p < vs. placebo) and the effect was not blocked by MK-434. Results are presented as means ± SEM, N = 5 for SHAM and 8 for other groups.

23 Borst et al. E R1-23 Alternate version of figure th week 8th week Dpd/creatinine (nm/mm) # # # # * * 0 SHAM ORX+veh ORX+T ORX+MK ORX+MK+T Figure 3. T inhibits bone resorption in ORX rats, and the effect is not blocked by MK-434. During the first four weeks of the experiment, ORX caused a 75% increase in urine excretion of Dpd (#indicates p < vs. SHAM). During the second four weeks, T administration caused a 65% decrease in Dpd (*indicates p < vs. placebo) and the effect was not blocked by MK-434. Results are presented as means ± SEM, N = 5 for SHAM and 8 for other groups.

24 Borst et al. E R * serum androgen (pg/ml) # # T DHT * 0 S H AM ORX+ v e h OR X+ T ORX+ M K ORX+ M K+T Figure 4. Effects of ORX, T, and MK-434 on serum androgens. ORX caused a 76% decrease in serum T (#indicates p = vs. SHAM) with no change in serum DHT. T treatment caused a significant increase in serum T (*indicates p = vs. placebo). Results are presented as means ± SEM, N = 5 for SHAM and 8 for other groups.

25 Borst et al. E R1-25 adrogen content (pg/tissue) T DHT # * & * $ 0 ^ SHAM ORX+veh ORX+T ORX+MK ORX+T+MK Figure 5. Effects of ORX, T, and MK-434 on prostate androgens. ORX caused a 40% decrease in prostate T (#indicates p < vs. SHAM) and a 90 Results are presented as means ± SEM, N = 5 for SHAM and 8 for other groups.