Testosterone (TST) is a steroid hormone that is not freely soluble in blood. Consequently, the majority (~98%) of this hormone is bound to either sex hormone-binding globulin [~40% (SHBG)] or albumin (11); therefore, only the unbound or free TST is considered to be biologically active where it can readily diffuse across the sarcolemma, allowing it to interact with intracellular androgen receptors (AR) and activate downstream androgen-signaling mechanisms. Previous studies have shown that elevated serum TST increases AR expression in proliferating myoblasts (23) and may be associated with elevated myofibrillar protein synthesis.
Resistance exercise is known to induce acute increases in serum TST levels that can vary depending on exercise-related variables such as intensity (27), volume (13), an acute bout (1), sequential bouts (22,28), and type of muscular contractions (4,11). Only one of these studies investigated AR mRNA expression after a single bout of resistance exercise (4); however, apparently no studies have examined the effects on AR mRNA and protein expression after sequential bouts of resistance exercise. The increased TST observed following resistance exercise is typically not associated with significant changes in SHBG levels (13). This is noteworthy because both the unbound/active fraction of TST and the total TST level is increased with resistance exercise. Consistent elevations in serum TST resulting from resistance exercise may enhance androgen-AR interactions within skeletal muscle and, along with increased number of AR, may up-regulate the expression of various muscle-specific genes (15). This is due to the fact that the binding of TST to its ligand-binding domain transforms the AR to an active transcription factor that regulates gene expression by interacting in a ligand-specific manner with a specific regulatory DNA sequence known as the androgen response element (ARE) located within the promoter of muscle-specific genes (19). Therefore, the central element of androgen signaling in skeletal muscle is the androgen-AR complex, and this signaling pathway likely plays a key role in regulating the hypertrophic response to resistance training (18).
In regard to AR signaling, muscle hypertrophy induced by electrical stimulation in rodents administered an AR antagonist (16) seems to suggest that the androgen-AR signaling pathway has a significant effect in exercise-induced muscle hypertrophy and emphasizes the importance of the AR in exercised muscle. A single bout of lower-body resistance exercise has been shown to significantly increase AR mRNA expression in humans (4); however, the responsiveness of the AR gene to sequential resistance exercise bouts characteristic of a typical training program along with the specific role that an up-regulation in AR expression may have on myofibrillar protein content is not well known. Therefore, rather than employ a single bout of resistance exercise, we attempted to determine the effects of three sequential bouts of heavy lower-body resistance training within a 7-d period (characteristic of a typical training regimen) on serum TST and SHBG levels, AR mRNA and protein expression, and myofibrillar protein. We hypothesized that heavy resistance exercise would increase serum TST and correspond to an increase in AR expression and myofibrillar protein content.
Approach to the problem.
As previously mentioned, many studies have shown elevated TST responses to heavy resistance exercise that vary in terms of the intensity, duration, volume, and types of muscular contractions. However, in lieu of the increased TST levels in these aforementioned studies, the critical element for AR signaling, the expression of the AR, has chiefly gone undetermined in human skeletal muscle in response to heavy resistance exercise. Because any TST-induced effects on skeletal muscle hypertrophy resulting from resistance exercise will likely occur by way of its AR interaction and subsequent transcription activation of muscle-specific genes, of primary purpose in relation to the AR was an attempt to determine the relative effects on AR mRNA and protein expression in response to sequential bouts of heavy-resistance exercise.
The present study included 18 apparently healthy, untrained (had not participated in any consistent resistance training involving the lower body or ingested any nutritional supplements for at least 6 months before the study) males comprising either a resistance exercise [RST (N = 9)] or a control [CON (N = 9)] group. Participants within the RST group had and average (±SD) age of 19.36 ± 2.17 yr, height of 181.60 ± 9.88 cm, and total body mass of 87.65 ± 20.24 kg, whereas the CON group was 20.66 ± 1.82 yr, 181.45 ± 2.16 cm, and 91.83 ± 13.91 kg. Before being considered eligible for the study, all participants were to be nonsmokers who agreed to abstain from using any caffeine and alcohol for at least 48 h before testing or the exercise trials, to limit their participation to any resistance training to only that involved in the study, and to abstain from ingesting any nutritional supplements for the duration of the study. Participants with contraindications to exercise as outlined by the American College of Sports Medicine (2) were not allowed to participate. All eligible participants signed university-approved informed consent documents, and approval was granted by the Institutional Review Board for Human Subjects. Additionally, all experimental procedures involved in the study conformed to the ethical consideration of the Helsinki Code. The subjects were explained the purpose of the training program, the protocol to be followed, and the experimental procedures to be used.
Approximately 12 d before the first resistance exercise session, all participants were subjected to a testing session in which each subject’s lower-body maximum strength [one-repetition maximum (1-RM)] was determined using the free-weight barbell squat, bilateral leg press, and leg-extension exercises (Cybex-Eagle, Owatonna, MN). For the three exercises, subjects were required to lower the resistance to approximately 90° of knee flexion. A rest interval of 3 min was required between attempts. However, due to the possibility of fatigue as a result of excessive trials (i.e., > five trials) during 1-RM testing, based on our previous work, a goal of no more than five trials was set for all 1-RM testing sessions throughout the study (30). All participants were able to obtain their 1-RM within five, and the average (±SD) trials for all subjects over the four 1-RM testing sessions was 4.17 (0.82). Upon arrival to the laboratory for testing, all participants were instructed perform a slow jog for 5 min as a warm-up. All participants were then lead through a stretching protocol to stretch the leg and lower-back areas. The stretching protocol involved static stretching of three sets for a 10-s count for each stretch. The stretches that were used were the standing quad stretch, toe touches, the adductor stretch, sit-and-reach stretch, and the knee to chest stretch. After stretching, participants were instructed to do two warm-up sets of 10 repetitions at 55% and 65% of their perceived 1-RM separated by 3 min. The 12-d time lapse between the 1-RM testing session and the first exercise session was chosen to allow for any up-regulation in mRNA and/or for AR protein expression to return to baseline levels. Also, this period of time would not likely result in significant strength decrements to occur from strength testing before the first session was conducted (21).
Resistance exercise protocol.
Only the RST group participated in the three resistance exercise sessions. However, the CON group was required to be in attendance at each exercise session and also underwent muscle biopsies and blood sampling at the same time intervals as the RST group. The exercise sessions utilized the same warm-up and stretching protocol that was used before 1-RM testing (walk/jog, stretch, submax sets). The exercise sessions also occurred at the same approximate time of day as the 1-RM testing for each participant. The training sessions were separated by 48 h for rest and recovery. Using the order principle and performing multi-joint before uni-joint exercises (3), the protocol involved three sets of 8–10 repetitions at 75–80% of the subjects 1-RM on each of the three exercises (squat, leg press, and leg extension). For every repetition of each exercise, subjects were required to lower the resistance to approximately 90° of knee flexion. Resistances were adjusted accordingly during the exercise bouts if a participant could not complete the exercise so that each set of each exercise was performed within the desired range of 8–10 repetitions. Rest intervals of 3 min were required between each set and exercise. The duration of each bout was approximately 50 min from warm-up to completion. At the conclusion of the study, it was shown that for all three exercise bouts the RST group completed all three exercises contained within each bout for an average (±SD) of 8.98 (0.08) repetitions and a relative intensity of 75.4% (5.66).
Muscle biopsies and venous blood sampling.
Consistent with previous work, we elected to biopsy the vastus lateralis muscle 48 h after the exercise bouts (4) so that the sample time coincided with data suggesting increased mixed muscle protein synthesis after an acute bout of heavy resistance exercise (24). Percutaneous muscle biopsies were obtained immediately before each exercise session (48 h after session 1 and 2) and 48 h after the third session. Upon receiving a local anesthetic (2% Xylocaine with epinephrine), muscle samples were obtained using the fine needle aspiration procedure and a 16-gauge biopsy needle (Medical Device Technologies, Gainesville, FL). An average (±SD) of 6.32 (2.94) mg of muscle was obtained from the middle portion of the right vastus lateralis muscle at the midpoint between the patella and the greater trochanter of the femur at a depth between 1 and 2 cm. For biopsies 2–4, attempts were made to extract tissue from approximately the same location by using the previous biopsy puncture scar, depth markings on the needle, and a successive puncture that was made approximately 0.5 cm to the former from medial to lateral (30). After removal, muscle specimens were immediately frozen in liquid nitrogen and then stored at −80°C for later analysis.
There was a total of nine blood draws over a 7-d period. The blood draws took place immediately before, immediately after, and 30 min after each exercise session. Venous blood samples were obtained from the antecubital vein into a 10-mL collection tube using a standard Vacutainer apparatus standardized to the same time of day for each sample. Blood samples were allowed to stand at room temperature for 10 min and then aliquots were used to assess plasma volume (10) while the remaining blood was centrifuged at 800 × g for 10 min. The serum was removed and frozen at −80°C for later analysis.
Total RNA isolation.
Total cellular RNA was extracted from the homogenate of biopsy samples with a monophasic solution of phenol and guanidine isothiocyanate (7) contained within the TRI-reagent (Sigma Chemical Co., St. Louis, MO). The RNA concentration was determined by optical density (OD) at 260 nm (by using an OD260 equivalent to 40 μg·μL−1) (8), and the final concentration was adjusted to 1 μg·μL−1. Aliquots (5 μL) of total RNA samples were then separated with 1% agarose gel electrophoresis, ethidium bromide stained, and monitored under an ultraviolet light (Chemi-Doc, Bio-Rad, Hercules, CA) to verify RNA integrity and absence of RNA degradation. This procedure yielded undegraded RNA, free of DNA and proteins as indicated by prominent 28s and 18s ribosomal RNAbands (Fig. 1), as well as an OD260/OD280 ratio of approximately 2.0 (8). The RNA samples were stored at 380°C until later analysis.
Reverse transcription and cDNA synthesis.
Two micrograms of total skeletal muscle RNA were reverse transcribed to synthesize cDNA. A reverse transcription (RT) reaction mixture [2 μg of cellular RNA, 10× reverse transcription buffer (20 mM Tris-HCL, pH 8.3; 50 mM KCl; 2.5 mM MgCL2; 100 μg of bovine serum albumin per milliliter), a dNTP mixture containing 0.2 mM each of dATP, dCTP, dGTP, and dTTP, 0.8 μM MgCl2, 1.0 u·μL−1 of rRNasin (ribonuclease inhibitor), 0.5 μg·μL−1 of oligo(dT)15 primer, and 25 u·μg−1of AMV reverse transcriptase enzyme (Promega, Madison, WI)] was incubated at 42°C for 60 min, heated to 95°C for 10 min, and then quick-chilled on ice. Starting template concentration was standardized by adjusting the RT reactions for all samples to 200 ng before PCR amplification (30).
Oligonucleotide primers for PCR.
The following 5′ sense and 3′ antisense oligonucleotide primers were constructed using Primer 3 online software (http://www.broad-.mit.edu/cgi-bin/primer/primer3_www.cgi) and used to isolate the mRNA expression of the skeletal muscle AR (5′ primer: bases 2804–2824, GC% = 60, Tm = 59.99°C; 3′ primer: bases 3272–3252, GC% = 50, Tm = 59.89°C, GenEMBL M34233). These primers were shown to amplify a PCR fragment of 469 bp. Due to its consideration as a constitutively expressed “housekeeping gene,” glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an external reference standard for detecting the relative change in the quantity of AR mRNA using PCR. For GAPDH mRNA (5′ primer: bases 616–636, GC% = 55, Tm = 60.12°C; 3′ primer: bases 1189–1169, GC% = 55, Tm = 59.96°C, GenEMBL AC NM 002046), we have previously shown these primers to amplify a PCR fragment of 574 bp (30).
A total of 200 ng of cDNA were added to each of the 25-μL PCR reactions for GAPDH and AR. Specifically, each PCR reaction contained the following mixtures: [10× PCR buffer, 0.2 μM dNTP mixture, 1.0 μM of a cocktail containing both the sense and antisense RNA oligonucleotide primers (Ransom Hill Biosciences, Ramona, CA), 2 mM MgCL2, 1.0 u·μL−1 of Taq DNA polymerase (Sigma, St. Louis, MO), and nuclease-free dH2O]. Each PCR reaction was amplified with a thermal cycler (Bio Rad). The amplification profile involved a denaturation step at 95°C for 30 s, primer annealing at 55°C for 30 s, and extension at 72°C for 60 s (30). To help control for differences in amplification efficiency during thermocycling, all PCR reactions were prepared from the same stock solution. Also, the number of cycles was optimized at 25 so that the amplified signal was still on the linear portion of a plot with the yield expressed as a function of the absorbance at OD260 and the number of cycles for the two target amplifications (Fig. 2A). The specificity of the PCR was demonstrated with an absolute negative control using a separate PCR reaction containing no cDNA. To assess reliability between amplifications, two separate PCR amplifications were performed for each sample to control for systemic differences between samples that could affect amplification efficiencies. Intra-assay coefficients of variation in mRNA concentrations for the two PCR runs for all participants were performed and resulted coefficients of variation of 3.85% and 4.12%, respectively, for GAPDH and AR mRNA. Additionally, the external control standard GAPDH displayed only a small amount of variation in expression from one sampling point to the next. Considering sampling point 1 as the baseline, the variation between sampling points 1–2, 1–3, and 1–4 were 5.12%, 7.34%, and 6.88%, respectively, for CON; and 5.48%, 6.34%, and 6.26% for RST, respectively (Fig. 2B).
The DNA within each amplified PCR reaction was purified of contaminants such as primer dimers and amplification primers using the Wizard PCR Preps DNA Purification System (Promega, Madison, WI). Aliquots (20 μL) of the purified PCR reaction mixtures were electrophoresed in 1.5% agarose gels in 1X Tris-Acetate-EDTA (TAE) buffer to verify positive amplification of AR mRNA and the gel stained with ethidium bromide (present in the TAE buffer at 1 μg·mL−1) and illuminated with UV transillumination (Chemi-Doc, Bio-Rad). Aliquots of each remaining purified PCR reaction were used to quantify mRNA spectrophotometrically at a wavelength of OD260, at which point the mRNA concentration of AR was calculated and normalized relative to GAPDH (25). It should be noted, however, that this method of PCR quantitation determines relative mRNA concentration only and should not be interpreted as absolute concentration values.
Serum testosterone and SHBG quantification.
The concentrations of serum total TST and SHBG were determined in duplicate and the average concentrations reported using an enzyme-linked immunoabsorbent assay (ELISA) (30) incorporating human-specific monoclonal antibodies against total TST and SHBG (Research Diagnostics Inc., Flanders, NJ). The secondary antibody immunoglobulin-G (IgG) was conjugated to the enzyme horseradish per-oxidase (ICN Biomedical, Aurora, OH). Standard curves were generated for TST (r2 = 0.95, P = 0.004) and SHBG (r2 = 0.91, P = 0.01) using specific control antigens for TST and SHBG (Research Diagnostics Inc., Flanders, NJ). The concentrations of TST and SHBG were determined at an optical density of 450 nm with a microplate reader (Bio Rad) and expressed relative to plasma volume. Intra-assay coefficients of variation were determined for each duplicate for all subjects and resulted in coefficients of 3.87% and 4.06% for TST and SHBG.
Free androgen index.
Based on previous work (22), the free androgen index (FAI) was determined as an indirect measurement of biologically active TST. The FAI (total TST/SHBG) is an indicator of the ratio of total TST concentration to the concentration (or binding capacity) of SHBG. In males, the central range encompassing the 95th percentile is 15% to 95%, with a corresponding median value of 35% (29).
Skeletal muscle androgen receptor quantification.
Muscle protein was isolated from the organic phase of the total RNA isolation using ethanol, isopropanol, and a 95% ethanol solution containing 0.3 M guanidine hydrochloride (30). The binding affinity of the anti-AR and selected purified protein samples was qualitatively verified with dot blotting (Fig. 4A) using a standard immuno-blotting protocol (Immuno-Blot Colorimetric Assay Kit, Bio-Rad). Briefly, equal amounts of myofibrillar protein extract (20 μg) were blotted onto a nitrocellulose membrane, blocked with 2% gelatin in TBS buffer, incubated with the a polyclonal anti-AR antibody (5 μg·mL−1) (Santa Cruz Biotech, Santa Cruz, CA), incubated with a secondary IgG antibody conjugated to alkaline phosphatase, and the color developed with BCIP/NBT. The blot was then illuminated with white light transillumination (Chemi-Doc, Bio-Rad).
Based on our previous procedures (30), the AR concentrations were then quantified in duplicate and the average concentrations reported using an ELISA (Fig. 4B). This incorporated the same human-specific poly-clonal anti-AR antibody (5 μg·mL−1) we used for immuno-blotting. The secondary antibody immunoglobulin-G (IgG) was conjugated to the enzyme horseradish peroxidase (ICN Biomedical, Aurora, OH). A standard curve was generated for AR (r2 = 0.92, P = 0.003) using a specific control antigen for AR (Research Diagnostics Inc., Flanders, NJ). Skeletal muscle AR concentrations were determined at an optical density of 450 nm with a microplate reader (Bio-Rad) and expressed relative to myofibrillar protein content. An intra-assay coefficient of variation was determined for each duplicate for all subjects and resulted in a coefficient of 4.67% for AR.
Myofibrillar protein quantitation.
Total protein remaining from the total RNA isolation procedure was isolated with isopropanol, ethanol, and 0.3 M guanidine hydrochloride. Myofibrillar protein was further isolated with repeated incubations in 1% SDS at 50°C. Based on our previous work (30), myofibrillar protein content was determined spectrophotometrically based on the Bradford method (6) at a wavelength of 595 nm. A standard curve was generated (r2 = 0.96, P = 0.001) using bovine serum albumin (Bio-Rad) and myofibrillar protein was quantified relative to muscle wet-weight. All assays were performed in duplicate with a coefficient of variation of 3.15%.
Using SPSS (version 11.1, SPSS Inc., Chicago, IL), for AR mRNA and protein expression statistical analyses were performed using separate 2 × 4 [Group (CON, RST)] × Test (4 muscle samples) factorial ANOVA with repeated measures. Serum TST and SHBG were analyzed with separate 2 × 9 [Group × Test (9 blood samples)] ANOVA with repeated measures. In the event of a significant F-ratio, between-group differences were determined involving the Newman-Keuls post hoc test. However, to protect against Type II error, the conservative Hunyh-Feldt epsilon correction factor was used to evaluate observed within-group F-ratios. Bivariate correlations between AR mRNA and protein and serum hormone levels were determined using the Pearson product moment correlation procedure. Statistical power and the effect size were determined using the partial eta2 statistic. Statistical power was also determined. For the standard curves generated from the spectrophotometric procedures (ELISA and Bradford), linear regression analysis was used. A probability level of ≤0.05 was adopted throughout to determine statistical significance, and statistical power was estimated a priori at 0.48 for a large effect size of 0.80.
Serum testosterone, SHBG, and free androgen index.
A significant Group × Test interaction was observed for serum TST (P = 0.001). However, no significant differences were located for SHBG (P > 0.05). For TST, post hoc analysis for the Test factor indicated that RST experienced significantly greater increases compared with CON at each of the three blood sampling points occurring immediately postexercise (samples 2, 5, and 8) when compared with each of the corresponding preexercise values (Fig. 3A). Significant main effects for Group (P = 0.029) were located for FAI indicating greater increases in estimated free TST for RST when compared with CON. In addition, significant main effects for Test (P = 0.043) were located for FAI. Post hoc analysis for the Test factor indicated that RST also experienced significantly greater increases in FAI at each of the three blood sampling points occurring immediately postexercise (samples 2, 5, and 8) when compared with each of the corresponding preexercise values (Fig. 3C). Plasma volume shifts between exercise bouts were not significantly different (P > 0.05).
Skeletal muscle androgen receptor mRNA and protein expression.
Significant Group × Test interactions were observed for skeletal muscle AR mRNA (P = 0.003) and protein (P = 0.039), indicating RST to be significantly different from CON. Post hoc analyses for the Test factor indicated that RST experienced significantly greater increases in skeletal muscle AR mRNA occurring 48 h after all three exercise bouts (muscle sampling points 2, 3, and 4) compared with the initial preexercise value for bout 1 (sample 1) (Fig. 4). The AR mRNA expression occurring 48 h after bout 1 (sampling point 2) was significantly correlated to the serum TST occurring immediately after bout 1 [sampling point 2 (r = 0.890, P = 0.023)], immediately after bout 2 [sampling point 5 (r = 0.846, P = 0.028)], and immediately after bout 3 [sampling point 8 (r = 0.790, P = 0.035)]. The AR mRNA expression occurring 48 h after bout 2 (sampling point 3) was significantly correlated to the FAI occurring immediately after bout 1 [sampling point 2 (r = 0.911, P = 0.004)] and immediately after bout 2 [sampling point 5 (r = 0.853, P = 0.015)], serum TST occurring immediately after [sampling point 5 (r = 0.757, P = 0.047)] and 30 min after [sampling point 6 (r = 0.938, P = 0.002)] after bout 2, and serum SHBG occurring immediately after bout 1 [sampling point 2 (r = −0.814, P = 0.026)], 30 min after bout 1 [sampling point 3 (r = −0.768, P = 0.044)], and 30 min after bout 3 [sampling point 9 (r =− 0.767, P = 0.044)]. The AR mRNA expression occurring 48 h after bout 3 (sampling point 4) was significantly correlated to the FAI occurring immediately after bout 1 [sampling point 2 (r = 0.799, P = 0.030)] and immediately after bout 2 [sampling point 5 (r = −0.832, P = 0.020)], and the serum TST occurring immediately after bout 3 [sampling point 8 (r = 0.776, P = 0.040)].
For AR protein expression, significantly greater increases occurred 48 h after the second and third exercise bouts (muscle sampling points 3 and 4) compared with the initial preexercise value for bout 1 (sample 1) (Fig. 5). The AR protein expression occurring 48 h after bout 1 (sampling point 2) was significantly correlated to the serum TST occurring immediately after bout 1 [sampling point 2 (r = 0.821, P = 0.024)]. The AR mRNA expression occurring 48 h after bout 3 (sampling point 4) was significantly correlated to the serum TST occurring immediately after bout 1 [sampling point 2 (r = −0.833, P = 0.020)] and immediately after bout 2 [sampling point 5 (r = 0.901, P = 0.014)].
A significant Group × Test interaction was observed for myofibrillar protein (P = 0.002) indicating RST to be significantly different from CON. Post hoc analysis of the Test factor indicated that RST experienced significantly greater increases in myofibrillar protein content 48 h after the third exercise bout (muscle sampling point 4) compared with sampling points 1, 2, and 3 (Fig. 6).
The results of the present study demonstrate that the mRNA and protein expression of the AR receptor to be significantly correlated to serum TST levels and also up-regulated after only three sequential bouts of heavy lower-body resistance exercise (P < 0.05). This increase in skeletal muscle AR expression likely occurs pretranslationally and is governed by androgen-AR signaling mechanisms (i.e., myogenic regulatory factors and steroid receptor co-activators). The AR mRNA results presented herein coincide with other data in humans, demonstrating AR mRNA concentration to be elevated 63% and 102%, respectively, 48 h after a single bout of either eccentric or concentric resistance exercise (4). Herein, we also show 35% and 43% increases, respectively, in AR mRNA levels 48 h after the first and third resistance exercise bouts; however, a peak of 68% (P < 0.05) was observed 48 h after the second bout compared with 2% for the control.
Up-regulation of the androgen-AR signaling pathway increases lean muscle mass, muscle strength, and muscle protein synthesis in rodents (5,26) and emphasizes the importance of the increase in the number of androgen receptors in exercised muscle (16). The AR content has been shown to preferentially increase in Type II (extensor digitorum longus) muscle after resistance training in rodents (9). Also in rodents, hypertrophy of the gastrocnemius (a mixed fiber muscle composed of red, white, and mixed portions contains approximately 88% Type II fibers) by electrical stimulation was shown to be associated with an increase in muscle AR content (17). Our present results illustrate that heavy resistance exercise in humans resulted in increases in AR protein expression of 40% and 100% 48 h after the first and second exercise bouts, respectively. However, a peak increase in AR protein expression of 202% (P < 0.05) occurred 48 h after the third exercise bout compared with 6% for the control. This, in turn, also corresponded to a peak increase in myofibrillar protein content of 79% (P < 0.05) occurring at the same sampling point. However, the human vastus lateralis only contains approximately 57% Type II muscle fibers (14). Therefore, irrespective of a predominance of Type II fibers, our results highlight preferential increases in skeletal muscle AR protein expression that occur as a result of three sequential bouts of lower-body heavy resistance exercise.
Much work has been done to determine the TST responsiveness to a single bout (1,4) or several bouts of resistance exercise (21); however, little has been done to determine any corresponding responsiveness of the AR to elevated serum TST that accompanies sequential bouts of resistance exercise. This issue is of importance because load-mediated modulation of AR receptor expression via resistance exercise may explain the increases in myofibrillar protein content that apparently occurs with elevated serum TST. By way of the FAI, we observed a resistance exercise-induced peak increase of 42% (P < 0.05) in the percent of unbound TST occurring immediately after the first exercise bout (sampling point 2) that was significantly correlated to both AR mRNA and protein expression (P < 0.05). We observed modest exercise-induced increases in SHBG following each bout, with a peak increase of 15% (P > 0.05) also occurring immediately after the first bout. Although, any increases that occur for SHBG typically mimic changes in serum total TST (13) in an attempt to maintain the level of free TST. Assuming the level of SHBG remains constant, and increase in total TST should lead to an increase in free TST (11). Therefore, our results for SHBG were not unexpected since the androgen binding affinity of SHBG does not appear to significantly change after acute exercise (12).
The TST response to resistance exercise is highly variable (20); however, it is the unbound (biologically active) fraction of TST that is able to serve as the ligand for AR binding. Furthermore, consistent with animal studies, it has also been suggested that consistent elevations in serum TST enhance AR expression, thereby likely mediating increased muscle protein synthesis (5) and hypertrophy (15), resulting from enhanced trans-activation and subsequent interaction of the androgen-AR DNA binding complex (ligand-activated AR) within the regulatory androgen response element present in the promoter of muscle-specific genes (16). However, because other hormones and growth factors such as T3 and IGF-1 are also involved in stimulating myofibrillar protein synthesis in response to resistance exercise, their possible involvement here cannot be discounted. In the present study, however, it is conceivable that the consistent elevations in serum TST after each exercise bout mediated the up-regulation in AR expression, which subsequently led to an enhanced ligand-binding capacity and a concomitant increase in myofibrillar protein.
Herein, we have presented evidence suggesting heavy resistance training to possibly increase myofibrillar protein content by way of the androgen-AR signaling pathway. Specifically, we demonstrated that both total and free TST (by order of the FAI) levels are subject to increases in response to individual sequential bouts of heavy lower-body resistance exercise. In addition, the mRNA and protein expression of the AR were significantly up-regulated after the second and third exercise bouts, respectively, correlated to serum TST, and corresponded to significant increases in myofibrillar protein by the third exercise bout. Therefore, we conclude that three sequential bouts of lower-body heavy resistance exercise to be effective in up-regulating AR expression, likely by way of a pretranslational mechanism that may be contingent on increases in free TST. Furthermore, the increased AR expression may have increased myofibrillar protein content by way of the androgen-AR signaling pathway.
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