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0021-972X/00/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2000 by The Endocrine Society Vol. 85, No. 1 Printed in U.S.A. Androstenedione Does Not Stimulate Muscle Protein Anabolism in Young Healthy Men* BLAKE B. RASMUSSEN, ELENA VOLPI, DENNIS C. GORE, ROBERT R. WOLFE AND Departments of Surgery (B.B.R., D.C.G., R.R.W.) and Internal Medicine (E.V.), University of Texas Medical Branch, and the Metabolism Department, Shriners Hospital (B.B.R., E.V., R.R.W.), Galveston, Texas 77550 2 H5]phenylalanine, blood sampling from femoral artery and vein, and muscle biopsies. Plasma testosterone, androstenedione, LH, and estradiol concentrations were determined by RIA. After ingestion of oral androstenedione, plasma testosterone and LH concentrations did not change from basal, whereas plasma androstenedione and estradiol concentrations were significantly increased (P , 0.05). Compared to a control group, androstenedione did not affect muscle protein synthesis and breakdown, or phenylalanine net balance across the leg. We conclude that oral androstenedione does not increase plasma testosterone concentrations and has no anabolic effect on muscle protein metabolism in young eugonadal men. (J Clin Endocrinol Metab 85: 55–59, 2000) ABSTRACT Androstenedione is the immediate precursor of testosterone. Androstenedione intake has been speculated to increase plasma testosterone levels and muscle anabolism. Thus, androstenedione supplements have become widely popular in the sport community to improve performance. This study was designed to determine whether 5 days of oral androstenedione (100 mg/day) supplementation increases skeletal muscle anabolism. Six healthy young men were studied before the treatment period and after 5 days of oral androstenedione supplementation. Muscle protein turnover parameters were compared to those of a control group studied twice as well and receiving no treatment. We measured muscle protein kinetics using a three-compartment model involving infusion of L-[ring- A that even if androstenedione does not stimulate testosterone release, it may directly stimulate muscle protein synthesis. The purpose of this study was to assess 1) whether 5 days of oral androstenedione supplementation increases plasma testosterone concentration, and 2) whether oral androstenedione has an anabolic effect on skeletal muscle. NDROSTENEDIONE is the immediate precursor to testosterone during steroid biosynthesis (1). Recently, it has been speculated that exogenous androstenedione is converted to testosterone and thus may have an anabolic effect on skeletal muscle. Numerous studies have shown that testosterone increases muscle size (2– 6) by increasing muscle protein synthesis (4, 7–9) in healthy and hypogonadic men, and that it increases maximum voluntary muscle strength in eugonadal men (3). Because of these speculations, but without any published reports on the effects of exogenous androstenedione on muscle protein synthesis, androstenedione has, on the one hand, been banned from use by the Olympic Committee and many professional sports organizations and, on the other hand, become a widely popular, commercially available, oral supplement. However, a 1966 study from Horton and Tait (10) evaluated the pharmacokinetics of androstenedione and showed that if androstenedione was given through the gastrointestinal route, only about 2% was converted to testosterone. On the other hand, androstenedione is a steroid possessing an androgenic activity of 10 –20% relative to the activity of testosterone (11). Thus, it is possible Materials and Methods Study subjects Six healthy male volunteers (age, 32 6 4 yr; height, 179 6 2 cm; weight, 77 6 6 kg; body mass index, 23.9 6 1.6 kg/m2) were studied in the postabsorptive state before and after androstenedione administration. The leg volume, estimated using an anthropometric approach (12), was 12.3 6 1.6 L. Six healthy volunteers (three women and three men) were also studied twice in the postabsorptive state and used as a control group for determinations of muscle protein kinetics (age, 31 6 3 yr; height, 168 6 3 cm; weight, 67 6 5 kg; body mass index, 23.7 6 1.2 kg/m2; leg volume, 9.5 6 0.5 L). All subjects gave informed, written consent before participating in the study, which was approved by the institutional review board of the University of Texas Medical Branch (Galveston, TX). Each subject was screened for determination of health status at the General Clinical Research Center at the University of Texas Medical Branch. Subjects were recreationally active, although none was involved in consistent resistance exercise training. None of the subjects was taking any form of medication, creatine, amino acid supplements, or anabolic steroids or was on an excessive protein diet. Received June 4, 1999. Revision received July 28, 1999. Rerevision received October 7, 1999. Accepted October 14, 1999. Address all correspondence and requests for reprints to: Robert R. Wolfe, Ph.D., Shriners Hospital, Metabolism Department, 815 Market Street, Galveston, Texas 77550. E-mail: [email protected]. * This work was supported by NIH Grant DK-33952 and Shriners Grant 8490. It was conducted at the General Clinical Research Center at the University of Texas Medical Branch (Galveston, TX), funded by Grant M01-RR-00073 from the National Center for Research Resources, NIH, USPHS. This study was presented in part at the Experimental Biology Meeting, April 1999, Washington, D.C. Protocol Each subject was studied twice. In the subjects given androstenedione, the first study was performed to acquire baseline data, and the second study was performed after 5 days (5–7 days after the initial study) of androstenedione administration (Fig. 1a). The six subjects included in the control group were also studied twice. The control group was used to assess the variability of muscle protein kinetics within the same subject on two different occasions. The subjects did not engage in phys- 55 56 JCE & M • 2000 Vol 85 • No 1 RASMUSSEN ET AL. because there is no known psychological component to the rate of muscle protein turnover. The second study for the treatment group was initiated on day 5 of oral androstenedione administration. It was identical to the first study, outlined above. After basal blood samples were taken, the subjects were administered the morning dose of androstenedione, and the tracer infusion was started. Blood samples for model kinetics and hormone concentrations and muscle biopsies were taken as described for the initial study. A time course for androstenedione and testosterone concentrations after the administration of oral androstenedione was measured in one representative subject. The second study for the control group was also identical to the first infusion protocol, described above. Analysis FIG. 1. A, The study design for the treatment group included two studies separated by 5 days of oral androstenedione administration. The control group did not receive androstenedione. B, The infusion protocol used before and after androstenedione treatment and for the control group. ical exercise the day before being studied. They were asked to maintain their normal dietary patterns, and they kept a notebook detailing their food intake and physical activity. The night before each study the subjects were admitted at the General Clinical Research Center of the University of Texas Medical Branch. After 2200 h, the subjects were allowed only water ad libitum. After an overnight fast, a Teflon catheter was placed in a forearm vein for isotope infusion, another catheter was placed in a wrist vein of the opposite arm for arterialized blood sampling, and femoral arterial and venous catheters were placed. A blood sample was drawn for background phenylalanine enrichment and androstenedione, testosterone, estradiol, and LH determinations (Fig. 1b). A primed (2 mmol/kg), continuous infusion (0.05 mmol/kgzmin) of l-[ring-2H5]phenylalanine (98% enriched; Cambridge Isotope Laboratories, Woburn, MA) was then started and maintained throughout the study. After 2 h of infusion, an initial muscle biopsy was taken from the vastus lateralis, approximately 20 cm above the knee, using a 5-mm Bergström biopsy needle (Stille, Stockholm, Sweden). To measure leg blood flow, an indocyanine green (ICG) infusion was started (0.5 mg/ min) in the femoral artery 4 h after the start of the isotope infusion and continued for 30 min. Blood samples were collected at 10-min intervals from the femoral vein and the heated wrist vein. Subsequently, the ICG infusion was stopped, and four arterial and venous blood samples were collected every 10 min for analysis of phenylalanine enrichment. Additional blood was collected at the beginning and end of the fifth hour of the study to measure hormone concentrations. At the end of the study a muscle biopsy needle was inserted into the leg in the opposite direction from the previous biopsy, and a second biopsy was taken. After the first study, each subject in the treatment group was given androstenedione (Ultimate Nutrition, Farmington, CT; 50 mg/capsule; minimum purity, 99.5%) for 5 days at a dosage of 100 mg/day, which is double that suggested by the manufacturer. Androstenedione was taken orally each morning with breakfast. We confirmed that the capsules given to the subjects contained 50 mg pure androstenedione by gas chromatography/mass spectrometry (GC/MS) analysis. The control group did not take oral androstenedione. A placebo was not given, Blood phenylalanine enrichments and concentrations were determined by GC/MS (model 5973, Hewlett-Packard Co., Palo Alto, CA) after purification of the amino acids (13) and derivatization to tertbutyldimethylsilyl derivative. Isotopic enrichments are expressed as the tracer to tracee ratio (13). Free muscle intracellular phenylalanine enrichments were measured by GC/MS after extraction and purification as previously described (13). The enrichments of protein-bound phenylalanine were measured after hydrolysis of the extracted muscle proteins (13) using the external standard curve approach (14). Leg blood flow was determined from blood samples collected during the continuous infusion of ICG (15, 16). Sera from blood samples were analyzed in a spectrophotometer with absorbance set at l 5 805 nm. The coefficient of variation of each ICG measurement (intrasubject) was less than 5%. Plasma androstenedione, testosterone, LH, and estradiol concentrations were determined by RIA (Diagnostic Products, Los Angeles, CA). According to the manufacturer, the cross-reactivity of androstenedione on the estradiol assay is not detectable. Calculations The three-compartment model of leg muscle amino acid kinetics used in this study has been described previously (17). Use of this model allowed us to determine the rate of utilization of phenylalanine for muscle protein synthesis and its intracellular appearance from muscle protein breakdown. The model assumptions are addressed in Refs. 17 and 18. The parameters of the three-compartment model of leg amino acid kinetics used to determine the phenylalanine kinetics are defined as follows (Fig. 2): phenylalanine entry into leg: Fin 5 CA 3 BF (1); phenylalanine exit from leg: Fout 5 CV 3 BF (2); net balance across the leg: NB 5 (CA 2 Cv) 3 BF (3); muscle inward transport: FM,A 5 {[(EM 2 EV)/(EA 2 EM)] 3 CV 1 CA} 3 BF (4); muscle outward transport: FV,M 5 {[(EM 2 EV)/(EA 2 EM)] 3 CV 1 CV} 3 BF (5); A-V shunting: FV,A 5 Fin 2 FM,A (6); protein breakdown: FM,O 5 FM,A 3 [(EA/EM) 2 1] (7); and protein synthesis: FO,M 5 FM,O 1 NB (8). Components of the kinetic parameters are defined as follows: CA and CV, concentrations of phenylalanine in the artery and vein, respectively; BF, leg blood flow; and EA, EV, and EM, enrichments (tracer/tracee) of phenylalanine in the femoral artery and vein, and intracellular muscle, respectively. The muscle protein fractional synthetic rate (FSR) was calculated using the precursor-product model previously described (19, 20). Statistical analysis Data are expressed as the mean 6 sem. Pre-post differences in hormone concentrations in the androstenedione group were assessed using the two-tailed paired t test. Differences in muscle protein kinetics between control and androstenedione were analyzed using ANOVA for repeated measures. Pairwise multiple comparisons were carried out using the t test with Bonferroni’s inequalities. Differences were considered significant at P , 0.05. Results Ingestion of androstenedione in the treatment group caused the plasma concentration of androstenedione to increase approximately 3-fold when measured 4 h after inges- ANDROSTENEDIONE DOES NOT PROMOTE ANABOLISM 57 FIG. 3. Time course of androstenedione and testosterone concentrations after ingestion of 100 mg androstenedione in one representative subject. FIG. 2. Three-compartment model of leg amino acid kinetics. Free amino acid pools in femoral artery (A), femoral vein (V), and muscle (M) are connected by arrows, indicating unidirectional amino acid flow between each compartment. Abbreviations are identified in the text. TABLE 1. Plasma androstenedione, testosterone, estradiol, and LH concentrations in normal men before and after 5 days of treatment with oral androstenedione Hormone Androstenedione (nmol/L) Basal Last hour of infusion study Testosterone (nmol/L) Basal Last hour of infusion study LH (IU/L) Basal Last hour of infusion study Estradiol (nmol/L) Basal Last hour of infusion study Pretreatment Oral androstenedione 7.5 6 0.7 7.0 6 0.8 12.8 6 0.8 37.2 6 10.7a 21.0 6 2.6 24.5 6 4.7 19.9 6 1.1 24.5 6 3.3 1.95 6 0.30 1.51 6 0.38 1.85 6 0.22 1.73 6 0.22 0.10 6 0.01 0.10 6 0.01 0.12 6 0.02 0.17 6 0.02a Data are expressed as the mean 6 a P , 0.05 vs. pretreatment. SEM. androstenedione (P 5 0.070, pre vs. post within the androstenedione group; P 5 0.521, pre vs. post within the control group). Muscle protein synthesis tended to be higher with time in both groups (P 5 0.051). Although the synthesis was slightly elevated in the androstenedione group, the change was not different from that observed in the control group, nor was the change as large in magnitude as the increase in breakdown. Net muscle balance of phenylalanine across the leg, a measure of net protein deposition, did not change over time in either group. However, net balance tended to be more negative in the androstenedione group (time by treatment interaction; P 5 0.093), indicating an increase in net protein catabolism rather than an improvement in anabolism as predicted (Table 3). Muscle protein synthesis was measured by the precursorproduct method (FSR) in all of the subjects in the androstenedione group and in three subjects in the control group (Fig. 4). Consistent with the data from the three-compartment model, no differences were found between groups, and no time effect was seen in either the treatment group (0.074 6 0.015%/h vs. 0.077 6 0.023%/h) or the control group (0.056 6 0.017%/h vs. 0.057 6 0.008%/h). Discussion tion (P , 0.05). Nonetheless, plasma testosterone and LH concentrations did not change (P 5 NS). On the other hand, androstenedione ingestion significantly increased the estradiol concentration (P , 0.05; Table 1). The time courses for androstenedione and testosterone concentrations after androstenedione ingestion for one representative subject are shown in Fig. 3. Blood flow, phenylalanine enrichments, and concentrations were not significantly different between the control and treatment groups and were unaffected by time (Table 2). The muscle amino acid kinetic data are reported in Table 3. Phenylalanine transport into and out of the leg did not change with time in both groups, with no differences between the groups. There was a trend for an overall increase in muscle protein breakdown with time (time effect, P 5 0.086), which was entirely attributable to the group receiving To our knowledge this is the first report of the response of muscle protein metabolism to androstenedione supplementation in human subjects. The data show that 5 days of oral androstenedione supplementation do not stimulate muscle protein anabolism or increase plasma testosterone concentrations. Rather, oral androstenedione is aromatized to estradiol and, probably reduced and conjugated for excretion by the liver (10, 11). As the plasma androstenedione concentration increased more than 3-fold, our data also indicate that androstenedione has no direct anabolic effect on skeletal muscle. Our failure to demonstrate an effect of androstenedione on either testosterone concentrations or muscle protein synthesis is probably not due to the study design. For example, we have shown that 5 days after an im injection of 200 mg testosterone enanthate in young men whose activity level and physical characteristics were similar to those of the vol- 58 JCE & M • 2000 Vol 85 • No 1 RASMUSSEN ET AL. TABLE 2. Leg blood flow, phenylalanine concentrations, and enrichments in normal men before and after 5 days of treatment with oral androstenedione Control Blood flow (mL/min z 100 mL leg vol) Concentrations (nmol/mL) Femoral artery Femoral vein Enrichments (%) Femoral artery Femoral vein Muscle Data are expressed as the mean 6 Androstenedione P value (by ANOVA) Pre Post Pre Post Effect of time Effect of treatment Interaction 3.9 6 0.4 3.1 6 0.3 3.1 6 0.4 3.5 6 0.1 0.390 0.660 0.066 55 6 3 62 6 4 55 6 3 61 6 3 48 6 3 52 6 3 49 6 3 55 6 3 0.716 0.661 0.097 0.072 0.641 0.328 7.05 6 0.53 5.51 6 0.33 4.12 6 0.43 7.45 6 0.28 5.52 6 0.24 4.09 6 0.56 7.38 6 0.28 5.81 6 0.21 4.43 6 0.21 7.20 6 0.21 5.19 6 0.16 4.46 6 0.32 0.747 0.202 0.991 0.912 0.962 0.514 0.380 0.188 0.911 SEM. Control subjects did not receive androstenedione. TABLE 3. Leg muscle phenylalanine kinetics in normal men before and after 5 days of treatment with oral androstenedione Control Phenylalanine inflow (Fin) Phenylalanine outflow (Fout) Inward transport (FM,A) Outward transport (FV,M) A-V shunting (FV,A) Protein breakdown (FM,O) Protein synthesis (FO,M) Net balance (NB) Androstenedione P values (by ANOVA) Pre Post Pre Post Effect of time Effect of treatment Interaction 212 6 24 238 6 26 112 6 29 138 6 29 101 6 23 69 6 10 44 6 9 225 6 7 165 6 9 183 6 10 97 6 14 115 6 16 68 6 12 78 6 11 59 6 12 218 6 2 151 6 22 164 6 23 75 6 12 88 6 13 76 6 16 48 6 7 35 6 5 213 6 2 170 6 9 189 6 9 130 6 19 150 6 20 39 6 22 74 6 6a 55 6 6 219 6 2 0.334 0.353 0.230 0.197 0.041 0.086 0.051 0.964 0.187 0.141 0.946 0.768 0.251 0.180 0.496 0.142 0.045 0.026 0.045 0.015 0.896 0.356 0.800 0.093 Data are expressed as nanomoles per min/100 mL leg vol (mean 6 a P 5 0.070 for time effect within the androstenedione group. FIG. 4. Muscle FSRs of control and androstenedione treatment groups. Subjects were studied twice in the postabsorptive, resting state. pre, Initial study; post, second study (5 days after ingestion of 100 mg/day androstenedione for the androstenedione treatment group). No differences were detected between the control group and the treatment group or between pre and post values. unteers of the present study, plasma testosterone was significantly elevated, and muscle protein synthesis increased 2-fold (7). We have also detected an anabolic effect on muscle protein synthesis when a synthetic anabolic hormone (oxandrolone) was given orally for 5 days, even though testosterone plasma concentrations significantly declined, and oxandrolone concentrations increased only to approximately 2 ng/dL. (21). To further substantiate the validity of our study SEM). design, we have included a control group, showing that muscle protein synthesis is not affected by prior determination of the same value. Although there was a trend for muscle protein synthesis to increase, no significant differences were found between androstenedione and control groups using two different methods (three-pool model and precursor-product approach). On the contrary, there was a trend for muscle protein breakdown to be elevated, which was entirely attributable to androstenedione intake. The trend for an increase in protein breakdown after androstenedione treatment may have been the consequence of the increase in estradiol. In fact, it has been reported that long term exposure to estrogens decreases muscle fiber size in rats (22). Overall, the trend for an increase in both protein breakdown and synthesis indicates that androstenedione tended to increase muscle protein turnover. However, the increased turnover did not lead to muscle protein anabolism, as the increase in protein breakdown exceeded the increase in protein synthesis. A similar situation takes place in catabolic states such as sepsis or burn injury, in which muscle protein breakdown is significantly elevated (23, 24), and muscle protein synthesis increases as well, although not enough to counteract the catabolic effect of increase in breakdown. The stimulation of muscle protein synthesis in this circumstance is probably due to the increased availability of intracellular amino acids secondary to accelerated rate of breakdown. The trend for an increase in muscle protein turnover may also indicate tissue remodeling and, hence, improvement in muscle function, but a recent study found no improvement in muscle function after an- ANDROSTENEDIONE DOES NOT PROMOTE ANABOLISM drostenedione supplementation (25). Regardless of the mechanisms accountable for the response to androstenedione ingestion in the present study, the increase in protein turnover observed did not lead to muscle protein anabolism. The failure of androstenedione intake to raise testosterone concentrations is somewhat surprising. A study showed that approximately 5% of circulating androstenedione is converted to testosterone (26). Considering that the daily production of testosterone is about 5 mg/day (26) and that we administered 100 mg/day androstenedione, if 5% of the oral androstenedione was converted to testosterone, we should have observed a doubling of testosterone concentrations. As this was not the case, our results could have been due to a suppression of endogenous testosterone secretion by androstenedione. However, the plasma LH concentration was not suppressed by androstenedione administration, suggesting that androstenedione per se or the derived estrogens did not inhibit the hypothalamicpituitary-Leydig cell axis. Our results are consistent with recent data from another laboratory (25) showing that androstenedione does not increase testosterone concentrations during short term administration (100 mg/day) or when given during resistance training (300 mg/day). In addition, the same study reported no differences between the androstenedione supplement group and a placebo group after 8 weeks of resistance training for the following measures: volitional muscle strength, mean cross-sectional area of type 2 muscle fibers, and lean body mass (25). Thus, it is likely that a significant proportion of ingested androstenedione is reduced and conjugated by the liver before reaching peripheral testosterone-converting tissues. In fact, a kinetic study has shown that 5.9% of the androstenedione administered iv was converted to testosterone, whereas the corresponding value was only 1.8% when androstenedione was given via the gastrointestinal route (10). This difference was due to the fact that 89% of the orally administered androstenedione was converted to testosterone glucuronide, which was excreted through the urinary tract (10). In conclusion, 5 days of oral androstenedione administration at double the dosage suggested by the manufacturer does not increase plasma testosterone concentration, nor does it have an anabolic effect on skeletal muscle. Our inability to demonstrate the putative beneficial effects of androstenedione on skeletal muscle protein contradicts the popular idea that androstenedione is an ergogenic aid for athletes. To the contrary, the increase in estrogens, the possible interaction or competition with androgen receptors, and the possible carcinogenic effect of prolonged androgen intake make androstenedione consumption inadvisable in healthy eugonadal men. 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