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Departments of Internal Medicine (M.S.-M., S.L.C., C.G., E.V., J.J., R.J.U.) and Surgery (D.P.-J., S.E.W., J.I.R.), University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1060
Address all correspondence and requests for reprints to: Randall J. Urban, M.D., Chairman and Professor of Internal Medicine, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0569. E-mail: rurban{at}utmb.edu.
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Abstract |
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Objective and Design: The objective of this study was to determine whether oral administration of a synthetic derivative of testosterone [oxandrolone, Oxandrin (OX)] (7.5 mg orally twice daily for 14 d) to five older women (age, 65 ± 2 yr) would enhance skeletal muscle anabolic biomarkers including mixed muscle fractional synthetic rate (FSR), net phenylalanine balance, androgen receptor, and IGF-I protein expression at d 0, 5, and 14 of treatment. As a positive control, seven older men were examined after 14 d of OX (10 mg orally twice daily).
Setting: The study was performed at the General Clinical Research Center.
Results: Fourteen days of OX significantly increased skeletal muscle FSR in older women (d 0, 0.073 ± 0.006 vs. d 5, 0.092 ± 0.006 vs. d 14, 0.115 ± 0.007%/h) (P < 0.05, d 0 vs. d 14). Conversely, OX stimulated FSR in older men after only 5 d (d 0, 0.061 ± 0.003 vs. d 5, 0.101 ± 0.01 vs. d 14, 0.084 ± 0.01%/h) (P < 0.05, d 0 vs. d 5). Androgen receptor expression was significantly increased in older men by d 14, but had not increased in older women. No change was noted in IGF-I expression in either group. We conclude that the skeletal muscle of older women and men responds to androgen administration, although the time course of anabolism appears to be gender specific.
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Introduction |
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A progressive increase in adiposity and decrease in fat-free mass characterize the aging process in both men and women. This underlying, subtle yet insidious imbalance between protein synthesis and breakdown with aging demands attention as our aging population grows. Interventional therapies such as androgen administration can positively influence protein metabolism and in recent years have been used by clinicians as a method to counter the negative effects of andropause and sarcopenia in aging males. Anabolic hormones such as oxandrolone [Oxandrin (OX)] and testosterone have been shown to increase mixed muscle protein synthesis in young (12) and older men (13) and to improve lean body mass and strength (13).
Little effort has been put forth to find anabolic agents that can be used effectively in older women to stem the effects of age-related muscle loss and strength. Although testosterone effectively stimulates muscle protein synthesis and enhances lean body mass and strength in men, the potential for virilization has limited its use in women. OX, a synthetic analog of testosterone, is an oral anabolic steroid currently used as an adjunctive therapy to promote weight gain in patients after surgery, chronic infections, and severe trauma (14). Additionally, OX is used by clinicians to treat children with growth disorders such as Turner’s syndrome and constitutional delay of growth and puberty (15, 16). Purported to induce fewer androgenic side effects, OX has caught the attention of clinicians for use in patients of both sexes. Numerous clinical studies have demonstrated the positive benefits of OX to various patient populations, including burns (14, 17, 18) and AIDS-wasting myopathy (19, 20). However, although androgens are protein anabolic to skeletal muscle in older men, we do not know whether androgens will induce skeletal muscle anabolism in older women. Therefore, we investigated the physiological, protein metabolic, and molecular effects of 14 d of OX administration on healthy older women, using older men as positive controls.
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Subjects and Methods |
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). All subjects provided written informed consent according to the guidelines established by the Institutional Review Board at UTMB. Eligibility was assessed by performing a medical screening including a physical examination, electrocardiogram, complete blood count, a metabolic panel, a hepatitis panel, an HIV test, and urinalysis. Exclusion criteria were heart or liver disease, hypo- or hypercoagulation disorders, vascular diseases, hypertension, diabetes, cancer, obesity, acute or chronic pulmonary diseases, infectious diseases, or an allergy to iodides. Subjects who had been taking anabolic steroids or corticosteroids in the past 6 months were also excluded, as were subjects engaged in regular aerobic or resistance exercise training. Subjects were asked to maintain their normal activities of daily living throughout the study and were fed a controlled meal by the General Clinical Research Center (GCRC) the evening before each study. Oral OX (BTG Pharmaceuticals, Iselin, NJ) was administered orally at 7.5- and 10-mg doses twice daily for 14 d to the five women (4.43 mg/kg·d) and seven men (3.99 mg/kg·d), respectively. Fourteen days was chosen as the endpoint study day for comparison to d 0 and 5 based upon our previous study showing OX capable of inducing significant changes in muscle protein synthesis in young men after only 5 d of 15 mg/d dosing. Metabolic parameters were measured at baseline (d 0), 5, and 14 d of OX. Because of the brief duration of OX administration (i.e. 14 d), we did not measure hematocrit, blood lipids, or a chemistry panel during or after androgen administration.
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and is similar to a previously published project (12). All subjects were free-living during the 14-d study. Subjects were admitted to the GCRC at the UTMB at 1200 h the day before each metabolic study and were fed a standardized lunch and dinner from the GCRC metabolic kitchen containing 58% carbohydrate, 27% fat, and 15% protein. Each subject was fasted from 2200 h until completion of the 5-h isotope infusion study the following day. On the morning of each study, polyethylene catheters were inserted into the antecubital vein of one arm for purposes of infusion of labeled phenylalanine, in the opposite hand for purposes of arterialized blood sampling, and into the femoral vein and artery of one leg for blood sampling. The femoral artery catheter was also used to infuse indocyanine green (ICG; Akorn, Buffalo Grove, IL) for measurement of leg plasma flow as previously described (21, 22). Leg volume was determined anthropometrically (23).
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50–100 mg) were taken from the lateral portion of the vastus lateralis of the leg, approximately 20 cm above the knee, using a 5-mm Bergström needle as previously described (12, 24). Briefly, biopsies were obtained at 2 and 5 h of tracer infusion for purposes of determining fractional synthetic rate (FSR) based on the incorporation of L-[ring-2H5]phenylalanine into protein over the 3-h span. Amino acid kinetics were determined from arterio-venous blood samples taken at 20-min intervals from 4 to 5 h. The first dose of OX was administered immediately after the completion of the baseline infusion study. At the end of each infusion study, all catheters were removed, and the subjects were fed a meal and discharged to home.
Analytical methods
Blood and muscle processing. Phenylalanine enrichments and concentrations in arterio-venous blood samples were determined using gas-chromatography mass-spectrometry in electron impact mode (Hewlett Packard, Palo Alto, CA) as previously described (12, 25).
Intracellular phenylalanine enrichments and concentrations from vastus lateralis muscle biopsy samples were analyzed using gas-chromatography mass-spectrometry (12, 25).
ICG concentration in infusate and serum samples was measured spectrophotometrically at 
=805 nm.
Hormone assays. The serum concentration of total testosterone was measured with a commercial RIA kit (Diagnostic Products Corp., Los Angeles, CA). The analytical sensitivity and thus reportable range of serum testosterone for this kit was 4–1600 ng/dl. Intraassay coefficients of variation for this assay were less than 5%.
Western blot analysis. Protein was isolated from muscle biopsy samples by slicing frozen muscle into very small pieces using a clean razor blade and thawing the tissue in lysis buffer [150 mM NaCl, 10 mM Tris, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 mM EDTA] containing proteinase inhibitors (1 mM phenylmethylsulfonylfluoride, 1 mM benamidine, 10 µg/ml aprotinin, 50 µg/ml leupeptin, 1 µg/ml pepstatin A) at a concentration of 3 ml of ice-cold lysis buffer per gram of tissue. The tissue was homogenized with a Dounce homogenizer (Kinematica AG, Littau, Switzerland) at 4 C; and centrifuged at 15,000 x g for 20 min, the supernatant was removed and centrifuged again to result in total cell lysate. The androgen receptor (AR) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was incubated with 80 g of cell lysate run on standard SDS-polyacrylamide gel with a working solution concentration range of 1:15–20. The IGF-I antibody (Santa Cruz Biotechnology, Inc.) was incubated with 40 g of cell lysate run on standard SDS polyacrylamide gel with a working solution concentration range of 1:100. The actin "housekeeping" antibody (Sigma, St. Louis, MO) was used with a working solution concentration range of 1:100–200. This antiactin antibody is a broad-based antibody that recognizes an epitope located on the N-terminal region of actin and demonstrates a broad reactivity among multiple actin isoforms in various species. The "housekeeping" antibody was used to correct the results for protein loading of the gel. Western analysis allows the direct measurement of protein expression in the muscle biopsy samples.
Calculations
Two well-established methodologies were used to measure phenylalanine kinetics in human skeletal muscle: phenylalanine net balance (NB) and direct amino acid incorporation via measurement of the mixed muscle FSR (26). A stable isotope of phenylalanine was used for this method because it is an essential amino acid and it is not oxidized in the muscle tissue. NB is therefore calculated as follows: NB = (Ca – Cv)·BF, where Ca and Cv represent the phenylalanine concentrations in the femoral artery and vein, respectively, and BF represents leg blood flow, as determined by the ICG dye dilution method (22). Data are presented per 100 ml of leg volume.
FSR of mixed muscle proteins was determined by measuring the direct incorporation of L-[ring-2H5]phenylalanine into protein, using the precursor-product model: FSR = [(EP2 – EP1)/(Em·t)]·60·100, where EP1 and EP2 are the enrichments of bound L-[ring-2H5]phenylalanine in the first and second muscle biopsies, t is the time interval between biopsies, and Em is the mean L-[ring-2H5]phenylalanine enrichment in the muscle intracellular pool (27). The results are presented as percent per hour. All kinetic parameters presented in this paper were measured with subjects in the postabsorptive state.
Statistical analysis
Statistical analyses were performed with Dunnett’s simultaneous 95% confidence intervals, which yield 97.5% upper and lower limits on each of the primary parameters. Primary parameters include mixed muscle FSR and NB of phenylalanine. Significance levels were adjusted using Bonferroni’s inequalities to account for multiple comparisons made for both primary parameters (yielding simultaneous 95% joint lower confidence limits). Secondary parameters include AR and IGF-I expression. Statistical analysis was carried out using NCSS software (2004) (NCSS, Kaysville, UT). All parameters and descriptive data are presented as means ± SEM.
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Results |
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The mean anthropometric data are displayed in Table 1
. There was no difference from baseline to d 14 of OX administration in both men and women for any of the measurements. However, when differences in height and weight between the men and women are taken into account, women on average were administered more OX (4.43 ± 0.05 vs. 3.99 ± 0.25 mg/kg·d) during the 14 d.
Serum testosterone concentrations
Total serum testosterone concentrations remained unchanged from d 0 (20.8 ± 5.9 ng/dl) to d 5 (23.1 ± 4.3 ng/dl) and d 14 (23.86 ± 4.5 ng/dl) in the women (Fig. 2A). However, the men demonstrated a significant decrease in total serum testosterone concentration from d 0 (315.57 ± 36 ng/dl) to d 5 and 14 (111.5 ± 10.4 and 76.7 ± 12 ng/dl, respectively; P < 0.05) (Fig. 2B).
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Figure 3 displays the NB of phenylalanine measured in the postabsorptive state at d 0, 5, and 14 of OX for both the men and women. Phenylalanine NB improved in both groups from d 0 to d 14. OX significantly increased NB in the positive control group (men) from –30.5 ± 5.3 at d 0 to –14.3 ± 1.5 nmol·min–1·100 ml leg–1 (P < 0.05). Likewise, OX significantly shifted NB in a positive direction in the women from –16 ± 0.88 at d 0 to –6.5 ± 3.2 nmol·min–1·100 ml leg–1 (P < 0.05). Thus, OX shifted both groups from a more protein catabolic state to a more protein anabolic condition.
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FSR of mixed muscle
Figure 4 shows the postabsorptive skeletal muscle FSR data at d 0, 5, and 14 of OX administration. OX administration resulted in a progressive increase in mixed muscle FSR in the women throughout the 14 d (0.073 ± 0.006, 0.092 ± 0.006, and 0.115 ± 0.007%/h), with d 14 FSR significantly different from d 0 (P < 0.05). FSR in the positive control men showed a significant increase by d 5 (0.061 ± 0.003%/h, d 0, and 0.101 ± 0.012%/h, d 5; P < 0.05); however, although still elevated, d 14 FSR in the men was no longer statistically different from basal (0.084 ± 0.01%/h).
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Figure 5 displays the densitometry data for AR measured at d 0, 5, and 14 for both groups. The data are presented as a ratio of the AR band density over the actin band density, respectively. Actin concentration did not change. AR protein expression was significantly increased in the older men by d 14, with no change noted in the women at either d 5 or d 14 (Fig. 5; P < 0.05). IGF-I protein expression remained unchanged after OX administration in both the older women and men.
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Discussion |
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We have previously demonstrated that the skeletal muscle of older (13) and younger (12) men is protein anabolic to long- and short-term androgen administration, respectively. Here, we show that 14 d of a moderate dose of OX is sufficient to stimulate muscle protein synthesis in healthy older women and men, despite being postabsorptive during the pre-post testing conditions. However, the time course of muscle protein anabolism between older women and men appears to be different because FSR was not significantly elevated in the women until d 14. Conversely, FSR in the positive control men was significantly elevated on d 5 but had declined somewhat by d 14. A decline of this proportion in FSR in the men is not unexpected because OX significantly decreased endogenous testosterone production by d 5 in the older men.
Several important factors may account for the anabolic differences between OX and testosterone in women and men and may provide additional clarity to our findings. In particular, testosterone enanthate is administered im in a lipid base that is readily stored in adipose tissue and released slowly, providing a sustained duration of action. Testosterone levels can rise to supraphysiological levels within 24 h, with levels gradually declining to hypogonadal levels (29). In contrast, we showed previously that OX reached peak levels in young men by 10 h but dropped significantly by 18 h (12). Despite the rapidity of the metabolism of OX and the significant fall in serum androgen levels in comparison to that previously seen with testosterone, OX was significantly anabolic to the skeletal muscle of young men (12).
OX stimulated muscle protein anabolism in the older women and men by increasing muscle protein synthesis, as muscle protein breakdown did not change. These findings are consistent with a previous study with young men (12) in that the early and/or short-term response of androgens on skeletal muscle occurs via an increase in muscle protein synthesis. Also similar to findings from our previous 5-d study of OX in young men, outward amino acid transport trended downward from d 0 to d 5 in the older men (data not shown). This finding, combined with a significant increase in muscle protein synthesis at d 5 and no change in intracellular appearance of amino acids (data not shown), provides evidence of increased intracellular reutilization of amino acids in the older men. In the older women, the model-derived muscle protein breakdown data are trending downward by d 14, suggesting that a portion of the early and/or short-term effect of OX in the women may also be affecting proteolysis.
In the present study, we were able to show a significant increase in muscle protein synthesis in women during the fasted state after only 14 d of OX administration. Our previous study using OX in younger men indicated that the potent protein synthetic response of OX was sufficient to ameliorate the net amino acid efflux and protein catabolism associated with an overnight fast (12). We know that during the fasted state muscle protein breakdown is normally much higher than muscle protein synthesis (21, 30). In the present study, our NB data show that muscle protein breakdown is higher than muscle protein synthesis in both older groups in the postabsorptive state. Although OX was able to overcome the catabolic effect of fasting in the women at 14 d, the significant reduction in endogenous testosterone in combination with the fasting condition in the men resulted in a nonstatistically significant elevation in FSR at d 14 in the men.
Unfortunately, the invasive nature of these studies, in combination with the older age of our study population, often limits the number of subjects that meet the rigorous inclusion criteria. Although the small subject numbers in this study are not ideal, we previously showed OX capable of inducing statistically significant increases in the FSR of muscle protein and AR mRNA after 5 d in a group of six young men (12). Moreover, we commonly use and report statistical significance in human studies of protein metabolism with six or seven subjects, regardless of age or intervention (e.g. amino acids) (12, 13, 31, 32, 33, 34). Additionally, although we recognize that the lack of a control group of women is a study limitation and would have permitted a temporally direct comparison with the treatment group, we chose to examine the effects of androgens on skeletal muscle anabolism in older women and compare this response to a positive control group of older men. Furthermore, this was supported by data showing that kinetic parameters such as the FSR of muscle protein remain unchanged in placebo-controlled groups (13, 31). Thus, while the subject numbers in the present study are small and may limit our ability to draw broad conclusions, the purpose of this study was to demonstrate that the skeletal muscle of older women was anabolically responsive to an androgen. Finally, it is important to note the rationale behind the differences in doses administered between the older men and women. As a reminder, the women received 7.5 mg twice a day and the men received 10 mg twice a day (i.e. 20 mg/d). The rationale for this dosing regimen was 2-fold. First, the dosing for the men was selected because it is the maximum approved dose for men, whereas the dosing for the women was chosen to be 7.5 mg twice a day, which when normalized for body weight of the smaller women resulted in a comparable per kilogram of body weight dosing to the men. Because the women were on average 14 kg lighter than the men, once normalized per kilogram body weight, the doses were very similar between the women (4.43 mg/kg) and men (3.99 mg/kg), respectively.
Although the intracellular mechanism stimulating muscle protein anabolism requires further clarification, it is clear that OX improves net protein balance of skeletal muscle. This effect is pronounced in the fasted state because net protein balance becomes less negative in both groups. Thus, OX administration when combined with the anabolic stimulus of a meal may ameliorate the loss of skeletal muscle nitrogen in older women by preventing the loss of intracellular amino acids. This retention may lead to lean muscle mass accretion over time.
The ability to reverse the inevitable losses in lean body mass using an oral anabolic agent has considerable clinical implications. Given that most trauma and burn patients are acutely hypercatabolic and most cancer and AIDS patients are chronically catabolic, efficient reutilization of intracellular amino acids is necessary for continued maintenance of the metabolic state (12, 13, 32). Further research is needed in these groups to better understand the intracellular mechanisms stimulating muscle protein anabolism and the time frame needed for protein accretion to occur.
In summary, the present study demonstrates that the administration of OX in moderate doses will stimulate muscle protein anabolism in women. Moreover, these data suggest that the mechanism in which this is accomplished differs from those previously studied in men. Overall, these data suggest that the skeletal muscle of women is anabolically responsive to an androgen and further suggests that women may achieve similar benefits of longer-term androgen supplementation as demonstrated in their older male counterparts.
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Acknowledgments |
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Footnotes |
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M.S.-M., S.L.C., C.G., E.V., J.J., D.P.-J., S.E.W., J.I.R., and R.J.U. have nothing to disclose.
First Published Online August 8, 2006
Abbreviations: AR, Androgen receptor; FSR, fractional synthetic rate; ICG, indocyanine green; NB, net balance; OX, oxandrolone; SDS, sodium dodecyl sulfate.
Received March 16, 2006.
Accepted July 28, 2006.
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References |
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