EXG-09641; No of Pages 11 Experimental Gerontology xxx (2015) xxx–xxx

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Article history: Received 24 February 2015 Received in revised form 5 June 2015 Accepted 6 June 2015 Available online xxxx

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Keywords: Androgen receptor Luteinizing hormone GnRH Androsterone Etiocholanolone

Department of Biology of Physical Activity, University of Jyväskylä, Finland Department of Cardiology, Central Hospital, Jyväskylä, Finland Department of Physiology, University of Turku, Finland d Department of Surgery and Cancer, Imperial College London, Hammersmith Campus, London, UK e Department of Clinical Physiology, Central Hospital, Jyväskylä, Finland f Department of Gynecology, Central Hospital, Jyväskylä, Finland g Department of Urology, University of Helsinki, Finland h The Department of Human Sciences, The Ohio State University, USA c

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This study investigated the effects of resistance training (RT) on the metabolism of testosterone (T) in younger (n = 5, 28 ± 3 yrs.) and older (n = 8, 70 ± 2 yrs.) men. Experimental heavy resistance exercises (5 × 10RM leg presses) were performed before and after a 12-month of RT. No age differences were found in the production or metabolic clearance rate of T (determined by stable isotope dilution method), skeletal muscle androgen receptor content or serum LH concentrations due to acute or chronic RT. The T production capacity response to gonadotropin stimulation and the concentrations of the urinary T metabolites (androsterone and etiocholanolone) were lower in the older compared to younger men (p b 0.05–0.01). This study further showed that RT may have acute effect on T production and clearance rates, while the exercise-induced increases in serum T appeared to be induced by decreased metabolic clearance rate of T. Attenuated T production capacity and urinary excretion of T metabolites in older men may reflect the known reduction in testicular steroidogenesis upon aging. No changes were observed in T metabolism due to RT indicating a homeostatic stability for this hormone in men of different ages. © 2015 Published by Elsevier Inc.

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1. Introduction

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In men, levels of testosterone (T) gradually decrease from age 30 (Feldman et al., 2002) and low T concentrations are associated with low skeletal muscle mass (Baumgartner et al., 1999; Iannuzzi-Sucich et al., 2002). The age-related loss of skeletal muscle mass and function represents increasing risks for physical disability and metabolic disorders in older persons. Resistance training (RT) is one of the most promising interventions to counteract the age-related muscle decline (Dela and Kjaer, 2006). We have previously shown that the acute resistance exercise-induced responses in serum T may change during long-term RT, and the changes could be related to size changes of the trained muscles (Ahtiainen et al., 2003). This finding suggests that acute changes in

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Juha P. Ahtiainen a,⁎, Kai Nyman b, Ilpo Huhtaniemi c,d, Tapani Parviainen e, Mika Helste f, Antti Rannikko g, William J. Kraemer h, Keijo Häkkinen a

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Effects of resistance training on testosterone metabolism in younger and older men

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Abbreviations: T, testosterone; PRT, production rate of testosterone; MCRT, metabolic clearance rate of testosterone; HPT-axis, hypothalamic–pituitary–testicular axis; GnRH, gonadotrophin-releasing hormone; LH, luteinizing hormone; SHBG, serum sex-hormone binding globulin. ⁎ Corresponding author at: University of Jyvaskyla, Department of Biology of Physical Activity, P.O. Box 35, FI-40014 University of Jyvaskyla, Finland. E-mail address: [email protected]fi (J.P. Ahtiainen).

serum T may have a role in the RT-induced adaptations of skeletal muscles and that T metabolism may change during long-term RT, possibly by transient exercise-induced alterations in T metabolism. However, the effects of RT on T metabolism in aged men have not yet been investigated in detail. Previous studies have shown that constant infusion of stable isotope-labeled T, combined with T analysis with liquid or gas chromatography–tandem mass spectrometry provides valid measurements of the production rate (PRT) and metabolic clearance rate of T (MCRT) upon physiological interventions (Vierhapper et al., 1997; Wang et al., 2004). In men, PRT is maintained by the feedback regulatory system of the hypothalamic–pituitary–testicular (HPT) axis. The hypothalamus releases in pulsatile fashion gonadotrophin-releasing hormone (GnRH), which in turn stimulates the synthesis and release of luteinizing hormone (LH) from the anterior pituitary gland, which subsequently stimulates T production in testicular Leydig cells. With aging, serum T concentrations progressively decline, by about 1% per year (Harman et al., 2001), mainly due to a decline in the capacity of aging Leydig cells to produce T in response to LH stimulation (Chen et al., 2009). The functional capacity of the HPT axis can be assessed by exogenous

http://dx.doi.org/10.1016/j.exger.2015.06.010 0531-5565/© 2015 Published by Elsevier Inc.

Please cite this article as: Ahtiainen, J.P., et al., Effects of resistance training on testosterone metabolism in younger and older men, Exp. Gerontol. (2015), http://dx.doi.org/10.1016/j.exger.2015.06.010

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2. Materials and methods

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2.1. Subjects

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The intervention group consisted of healthy untrained young adult (~ 25–30 yrs) and older men volunteers (~ 70–75 yrs). The present study was a part of larger research project whereof the subjects were randomized to the current investigation. The subjects were recruited by advertisements in the local newspaper. Subjects with a background in systematic physical training during the year before the study were excluded. The subject's health status was screened by a physician before inclusion in this study. More detailed medical screening was used for the older men including resting electrocardiogram. Exclusion criteria included cardiovascular and pulmonary diseases, malfunctions of the thyroid gland, diabetes, obesity (body mass index N 30), or any other disease that may have precluded the ability to perform the exercise training and testing. Exclusion criteria included also medications known to influence the endocrine system, heart rate and cardiovascular or neuromuscular performance. The subjects who passed the baseline physical examination were accepted to the study. In accordance with the Declaration of Helsinki, all subjects were carefully verbally informed about the possible risks and benefits of the study and all subjects signed a written consent form before participation in the study. The ethics committees of the University of Jyväskylä (October 24th 2006) and the Central Finland Health Care District approved the study (K-S shp:n Dnro 58/2006).

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2.2. Experimental design

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The study groups consisted of 9 younger and 9 older men. After the pre-measurements, four younger men withdrawn from the study due to low back pain (n = 1), work-related injury (n = 1), an unknown reason (n = 1) and change of residence (n = 1), and one older man deceased

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2.3. Resistance training program

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of natural cause. Thus, the final experimental groups consisted of 5 younger (YM; age 28 ± 3 yrs) and 8 older men (OM; age 70 ± 2 yrs). The subjects were familiarized with all physical performance testing procedures prior to the study. The experimental variables were measured in four separate measurement sessions before and after the 12-month experimental resistance training (RT) period; 1) basal total and free testosterone concentration and anthropometrics in fasting conditions, 2) muscle strength and size, 3) T production and clearance rates, urine excretion of T metabolites, serum LH concentration, and muscle AR protein concentration and 4) HPT-axis stimulation tests. Timetable of the measurements during experimental heavy resistance exercise performed before and after RT is presented in Fig. 1. All premeasurements were performed 1–2 weeks before the RT and postmeasurements were performed within a week following the last training session. To minimize variability in the measures, the subjects were asked to refrain from any strenuous physical activity for at least three days before the measurements and maintain a similar activity and dietary behavior pattern each time. Furthermore, all measurements were always performed at the same time of day to exclude the effects of diurnal variations.

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Subjects participated in a supervised progressive RT program for 12months. The present experimental RT program was designed to increase muscle mass and strength extensively throughout the training period according to the recommendations of RT programming for healthy adults (American College of Sports Medicine, 2009; Garber et al., 2011). Leg press, squat and knee extension and flexion exercises were performed for the lower extremity muscles. Four to five other exercises were performed for the other main muscle groups of the body (e.g. bench press, triceps pushdown, lateral pull-down, sit-up, elbow flexion). The training program consisted of whole body RT sessions two times a week during months 1–6. During the first month the training was carried out with light loads (40–60% of one repetition maximum; 1RM) but with multiple 10–20RM sets and with short rest periods between the sets to familiarize to resistance exercises and to improve strength endurance of trained muscles. Then the loads increased progressively up to 60–80% of the 1RM (8–12RM per sets) with a relatively short recovery time between the sets to increase muscle mass. After three months the training program included also higher loads (70–90% of the 1RM) with a longer recovery time between the sets using 5–10RM loads to optimize gains in maximal strength of trained muscles while still increasing muscular hypertrophy. The training program included also two sets performed with lower loads (40–50% of the 1RM with 8–12 repetitions) and higher movement velocities to improve muscle power. At months 7–12, the training program consisted of RT sessions three times per week, with upper and lower body training sessions in turn. All training sessions were supervised by the research team to make sure that proper techniques and progression in the training loads were used in each exercise.

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2.4. Experimental heavy resistance exercise

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To examine possible changes in acute exercise-induced responses due to long-term RT, the experimental heavy resistance exercise (HRE) sessions were performed before and after the 12-month RT. After the training period, the HRE was performed 3–4 days after the last training session to adapt the recovery time similar to the exercise frequency used during RT. The HRE protocol was hypertrophic in nature and comprised five sets of 10 repetition maximum (10RM) sets of bilateral leg presses (David 210, David Fitness and Medical Ltd., Finland) from a knee angle of 70° to 180° (= leg straight) with a two-minute recovery between the sets.

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GnRH or human chorionic gonadotropin (hCG; an analogue of pituitary LH) stimulation tests. Previously it has been shown that acute and 78 chronic endurance training suppresses T responses to GnRH and hCG 79 (Hackney et al., 2003; Kujala et al., 1990; Safarinejad et al., 2009; 80 Vasankari et al., 1993). However, the effects of aging and RT on the 81 HPT axis have not been examined. 82 The main component of MCRT is the extensive enzymatic conver83 sions of T in the liver and elimination of T metabolites, such as andros84 terone and etiocholanolone, by the kidney (Pozo et al., 2010). Thus, 85 urinary androsterone and etiocholanolone can be considered the final 86 end products in the T degradation pathway. The MCRT processes include 87 also, to a smaller extent, aromatization of T to estradiol (Longcope et al., 88 1969) and interaction of T with target tissues via androgen receptors 89 Q14 (ARs) (Heemers and Tindall, 2007). Changes in AR content determine 90 for the most part the magnitude of the target cell response to T, and 91 therefore down- or up-regulation of AR may be crucial in determining 92 the effects of T upon target tissues. The effects of aging and RT for T me93 tabolism and skeletal muscle AR content are largely unknown. 94 We hypothesize that regular resistance exercises with possible 95 chronic stimulation of the HPT axis during the long-term RT may induce 96 adaptations in T synthesis and metabolism. Since aging affects serum T 97 concentrations (Vermeulen, 2000), we also hypothesized that the ex98 pected RT-induced acute and chronic responses in T metabolism may 99 be attenuated in older compared to younger men and, therefore, may 100 explain possible aging-induced interference in muscular adaptations 101 to long-term RT (Welle et al., 1996). Thus, the purpose of the present 102 study was to examine acute and chronic RT-induced responses on 103 serum T and LH, MCRT and PRT, urine androsterone and etiocholanol104 one, skeletal muscle ARs and testicular production capacity of T (deter105 mined by stimulation tests of the HPT-axis with GnRH and hCG), as well 106 as their associations with the muscular adaptations to RT, in younger 107 and older men.

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Please cite this article as: Ahtiainen, J.P., et al., Effects of resistance training on testosterone metabolism in younger and older men, Exp. Gerontol. (2015), http://dx.doi.org/10.1016/j.exger.2015.06.010

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Fig. 1. Overall study design (A) and detailed design (B) to study experimental heavy resistance exercise-induced responses in serum testosterone (T) and luteinizing hormone (LH) concentrations, metabolic clearance and production rate of T (from the pooled blood samples, urinary excretion of metabolites of T, and skeletal muscle androgen receptor (AR) concentration.

2.5. Laboratory procedures

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2.5.1. Anthropometry Body height and mass were measured. Body mass index (BMI) was calculated by dividing weight in kilograms by the square of height in meters (m− 2·kg). Body composition was estimated by DXA (LUNAR Prodigy, GE Healthcare with enCORE 2005, version 9.30). Total body mass, total lean mass, legs lean mass and tissue fat% were utilized for subsequent analyses.

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2.5.2. Muscle strength A horizontal leg press device (David 210, David Sports Ltd., Helsinki, Finland) was used to measure maximal bilateral concentric strength (i.e. one repetition maximum, 1RM) of the le extensors. Muscle strength is proportional to its mass and composition that is typically compromised in aged people (Hairi et al., 2010; Visser et al., 2002). To study

muscle's functional properties, the 1RM results were also normalized 211 to legs lean mass to determine specific muscle force (i.e. muscle 212 quality). 213 2.5.3. Cross-sectional area of m. vastus lateralis An ultrasonographic (US) device (ProSound alpha-10; 7.5 MHz probe; Aloka, Japan) was applied to obtain axial-plane images of the vastus lateralis (VL) muscle and to generate cross-sectional area (CSA) images from the VL. Three CSA images from two levels of the right thigh, midpoint and 2 cm distally, were scanned. Orientated in the axial-plane, the transducer was moved manually with slow and continuous movement from a lateral to medial along a marked line on the skin. Image-J (National institute of health, USA, version 1.37) software was used for analyzing the US images. CSA was determined by tracing manually along the border of VL muscle, and the mean of two closest values of each other was taken as the CSA result of the corresponding level. The average of the two levels was used as CSA result.

Please cite this article as: Ahtiainen, J.P., et al., Effects of resistance training on testosterone metabolism in younger and older men, Exp. Gerontol. (2015), http://dx.doi.org/10.1016/j.exger.2015.06.010

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253 Q19 solution of GnRH (Synarela 200 mikrog) or hCG injection (Pregnyl 254 5000 IU) was dosed in a randomized order with a two week interval.

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Basal venous blood samples were drawn (after 12 h of fasting and 8 h of sleep) before and 24 h (i.e. next morning) after the administration of GnRH as well as before and 48 h, 72 h and 96 h after the administration of hCG.

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2.5.4.4. Determination of testosterone metabolic clearance and production rates. Testosterone isotope 1,2-D2 (D2T) was obtained from Cambridge Isotope Laboratories (Andover, MA, USA). The infusion solutions (0.25 mg per 500 ml) were made by a pharmacist of Jyväskylä Central Hospital (Jyväskylä, Finland), and they were tested for sterility and pyrogenicity. The solution was protected from light and infused constantly over 10.5 h between 6 AM and 4:30 PM at approximately 42 ml·h−1. The subjects remained in a recumbent position throughout the infusion period, except during the HREs. The rate of the infusion was checked by weighing the infusion bag and the tubing before and immediately after the completion of each infusion. The average amount of infusion was 408.6 ± 42.2 ml with no differences between the pre- and posttraining values. Aliquots of the infusate were collected before and at the end of the infusion for the determination of D2T concentrations. The data revealed no changes in D2T indicating constant D2T infusion (0.0005 mg⋅ml−1) throughout the 10.5 h experiment. Thus, an average of 0.20 mg of T infused in 10.5 h was less than 3% of the estimated daily T production in a healthy adult male (Vierhapper et al., 1997). Analyses for unlabeled T (D0T) and D2T determinations were performed in duplicate by the liquid chromatography (LC) system (Waters 2795 Alliance HT, Milford, Massachusetts, USA) connected with a tandem mass spectrometry (MS–MS) (Waters Quattro Micro, Milford, Massachusetts, USA). All samples from each subject were analyzed in the same assay. 50 μl of internal standard 19-nortestosterone (no. 74640, SigmaAldrich, St. Louis, MO, USA) in methanol (0.05 μg·μl−1), and 1 ml sodium acetate buffer at pH 5.5 were added to 500 μl of serum, and vortexed. After that 5 ml of hexane was added to mixture and vortexed, mixed for 25 min in Sarmix GM1 rotating mixer (Sarstedt, Nümbrecht, Germany), and centrifuged using Heraeus Megafuge 1.0 (Buckinghamshire, England) for 5 min at 4000 rpm. After freezing the sample overnight, the upper layer was transferred to a clean tube and evaporated to dryness under nitrogen. The extract was reconstituted in 200 μl methanol with 0.1 % formic acid, moved to the vials, and stored in deep freeze before the LC–MS–MS analysis. The samples were injected on the XBridge C18 (Waters) (100 × 2.1 mm 3.5 μm) analytical column. Gradient elution was used at 28 °C. Solvent A was methanol and solvent B 0.1% formic acid. The flow rate was 0.2 ml·min−1. The gradient program began with 50% A, ramped to 90% A at 10 min and was held 2 min and returned to 50% of A in 4 min. MS–MS spectra were recorded in the positive mode using electro-spray interface. The desolvation gas and temperature were nitrogen and 200 °C respectively. The source temperature was 100 °C, capillary voltage 3.25 kV and cone 30 eV and collision energy 23 V with 0.2 s dwell time. Testosterone was monitored by using transition m/z 289.2 to m/z 96.9, dideuterated testosterone m/z 291.2 to m/z 98.9 and nortestosterone transition m/z 275.2 to m/z 109.0. Integration of peaks of analytes and internal standards, calibration curves, and unknown sample concentration computation were performed by MassLynx version 4.1 software from Waters, Micromass. The recoveries for D0T and D2T were 93.7 and 88.1 %, respectively. Inter-assay precision was 14.1 % and 15.9 % in D0T and D2T, respectively. No changes were observed between the Control and Pre-samples in D0T and D2T, indicating that equilibration was achieved before the exercise intervention.

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2.5.4.1. Basal blood samples. Venous blood samples were obtained four times, at 30 min intervals, within 8.00–9.30 AM after 12 h of fasting 230 and 8 h of sleep. The samples were held for 15 min at room temperature 231 Q16 before being centrifuged for 10 min at 3500 rpm. Once the serum had 232 been separated from red blood cells, it was transferred into cryotubes 233 and kept frozen at −80 °C until assayed. Serum total testosterone con234 centrations were analyzed by the immunometric chemiluminescence 235 method with the Immulite 1000 device and hormone-specific immuno236 assay kits (DPC, Los Angeles, USA). The sensitivity of the assay for total 237 testosterone was 0.5 nmol·L−1 and intra-assay coefficient of variation 238 Q17 was 5.7%. All samples for each test subject were analyzed in the same 239 assay. Serum sex-hormone binding globulin (SHBG) concentrations 240 were analyzed by the immunometric chemiluminescence method 241 with the Immulite 1000 device and hormone-specific immunoassay 242 kits (DPC, Los Angeles, USA). The sensitivity of the assay for SHBG was −1 243 Q18 5.5 nmol·L and intra-assay coefficient of variation was 2.4%. All sam244 ples for each test subject were analyzed in the same assay. Free testos245 terone (FT) concentrations were derived from measurements of total 246 testosterone, sex hormone-binding globulin, and albumin (Vermeulen 247 et al., 1999). 248 A complete blood count was determined by Sysmex KX 21N249 analyzer (Sysmex Co., Kobe, Japan) from basal blood samples before 250 and after RT. The data showed that values were within the normal 251 ranges in all subjects.

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immediately before the HRE (30–0 min pre-exercise), Post0-sample during and immediately after (mid-exercise and 0 min post-exercise) and Post1h-sample (30–60 min post-exercise) after the HRE. MCRT was calculated by the formula: MCRT = amount of D2T infused per hour divided by concentration of D2T in the serum and multiplied by 24 h to express as liters per day (per body surface area as liters per day per meter2). PRT was then calculated from the formula: PRT = MCRT multiplied by serum D0T concentration, expressed in milligrams per meter2 per day (mg/m2/d).

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2.5.4.3. Exercise blood samples. Fingertip blood samples were collected before and immediately after exercises to determinate blood lactate 261 (EKF diagnostic, Biosen, Barleben, Germany). 262 Hemoglobin and hematocrit were measured using the automated 263 analyzer (Sysmex KX 21N, Kobe, Japan) and plasma volume shifts 264 were calculated (Dill and Costill, 1974). The plasma volume shift from 265 Q20 pre- to post-exercise was − 10.7 ± 2.9% and − 9.5 ± 1.8% in YM and 266 −9.6 ± 1.4% and − 9.9 ± 1.3% in OM before and after the RT, respec267 tively. Because no significant differences were observed between the 268 groups or in the pre- and post-RT values and to study molar exposure 269 of hormones at the tissue level, the circulating hormone concentrations 270 are presented as uncorrected to plasma volume changes. 271 Serum testosterone concentrations and testosterone metabolic 272 clearance and production rates by D2T method during HREs were deter273 mined by the liquid chromatography (LC) connected with a tandem 274 mass spectrometry (MS–MS). Testosterone isotope 1,2-D2 infusion 275 was started at 6 am and after an equilibration period of 6 h, blood sam276 ples were collected in 15 min intervals during 2 h before (12 am– 277 14 pm) and after (14:30–16:30 pm) the HREs, as well as after the 278 third set of the leg presses by an indwelling Teflon cannula that was 279 inserted into an antecubital forearm vein to the arm not receiving the 280 infusion. The cannula was kept patent with a 10% heparin lock/saline so281 lution. Prior to each blood draw, 3 ml of blood was extracted and 282 discarded to avoid inadvertent saline dilution of the blood sample. 283 Serum was obtained, and individual samples were stored at − 80 °C 284 until assayed. Serum LH concentrations were analyzed using the 285 immunometric chemiluminescence method (Immulite® 1000, DPC, 286 Los Angeles, USA). The sensitivity of the assay for LH was 0.1 UI·L− 1 287 and intra-assay coefficient of variation was 4.8%. Before analyses of 288 unlabeled T and D2T the samples were pooled as follows (see Fig. 1); 289 Q21 Control sample 2 h before (120–90 min pre-exercise), Pre-sample

Please cite this article as: Ahtiainen, J.P., et al., Effects of resistance training on testosterone metabolism in younger and older men, Exp. Gerontol. (2015), http://dx.doi.org/10.1016/j.exger.2015.06.010

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Table 1 Body mass and fat%, BMI, lean body mass, cross-sectional area of m. vastus lateralis, one repetition maximum and the total volume of the work performed in the HRE (loads ×

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sets × repetitions) (mean ± SD), and their relative changes with 12-month resistance training. Baseline Group Mean ± SD Body mass (kg) Body fat% BMI LBM (kg) CSA of VL (cm2) 1RM (kg) 1RM/LBM (kg) Total work during HRE (kg)

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97.1 ± 13.8 102.8 ± 19.6 5.5 ± 6.5 80.0 ± 8.3## 80.0 ± 9.1# 0.0 ± 2.0 29.0 ± 6.1 30.4 ± 6.3 5.7 ± 15.2 25.5 ± 4.1 23.4 ± 5.1⁎ −8.8 ± 10.0 27.8 ± 3.4 30.3 ± 8.4 8.4 ± 10.4 25.2 ± 1.8 25.2 ± 1.8 −0.2 ± 2.2 67.1 ± 14.4 69.3 ± 14.5⁎ 3.3 ± 2.3 56.8 ± 4.4 58.3 ± 4.5⁎ 2.7 ± 2.6 24.7 ± 3.4 31.2 ± 4.3 26.6 ± 8.6 18.6 ± 2.6# 22.0 ± 2.9⁎,## 18.6 ± 8.8 179.5 ± 12.0 211.0 ± 15.6⁎ 17.8 ± 8.5 ## ⁎,## 118.4 ± 18.2 133.8 ± 18.4 13.3 ± 5.3 ⁎ 2.5 ± 0.3 2.8 ± 0.4 11.5 ± 8.2 2.0 ± 0.3## 2.2 ± 0.3⁎,# 10.5 ± 5.3 9110 ± 641 11500 ± 723⁎ 26.4 ± 4.9 6119 ± 1156## 7256 ± 1231⁎,## 19.2 ± 7.3

2.5.6. Muscle biopsy sampling and analyses The muscle biopsies were obtained before HREs (Pre), as well as immediately after (Post0) and 2 h after (Post2h) the HREs. Muscle samples were obtained from the middle portion of the vastus lateralis muscle by the use of the percutaneous needle biopsy technique. The first biopsy was taken from the left leg, and the subsequent biopsies were obtained from the right leg (Post2h biopsy proximally from the preceding biopsy). The surrounding area was cleaned with an antiseptic solution and local anesthetics (2 ml lidocaine–adrenalin, 1%) were administered subcutaneously prior to incision of the skin. A needle (5 mm) was inserted into the muscle belly and, with the aid of suction, approximately 100 mg of muscle tissue was extracted. The muscle sample was cleaned of any visible connective and adipose tissues, as well as blood, and the sample was frozen within 1 min in liquid nitrogen (− 180 °C). Samples were stored at −80 °C until the analysis. To determinate androgen receptor (AR) protein concentration in each muscle biopsy specimen, western blot was performed as previously described (Ahtiainen et al., 2011). Antibodies against AR (1:2000; A9853, Sigma-Aldrich, St. Louis, MO, USA) and GAPDH (1:30000; G9545, Sigma-Aldrich, St. Louis, MO, USA) were used. Proteins were visualized by chemiluminescent method (SuperSignal West femto maximum sensitivity substrate, Pierce Biotechnology, Rockford, USA) and quantified (band intensity multiplied

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437.93 nmol of 1,2-D2 as internal standard, moved to the vials, and stored in deep freeze before the LC–MS–MS analysis. The samples were injected on the XBridge C18 (Waters) (100 × 2.1 mm 3.5 μm) column. Gradient elution was used at 28 °C. Solvent A was methanol and solvent B 0.1% formic acid. The flow rate was 0.2 ml·min− 1. The gradient program began with 35% A for 4 min, ramped to 45% A at 10 min, 65% A at 50 min and 90% A at 65 min held for 10 min, then return to 35% in 5 min. MS–MS spectra were recorded in the positive mode using electro-spray interface. The desolvation gas and temperature were nitrogen and 200 °C respectively. The source temperature was 100 °C, capillary voltage 3.25 kV and cone 30 eV and collision energy 23 V with 0.2 s dwell time. Androsterone and etiocholanolone were monitored by using transitions m/z 273.2 to m/z 109.0 and m/z 147.0. The internal standard deuterated testosterone m/z 291.2 to m/z 98.9. Integration of peaks of analytes and internal standards, calibration curves, and unknown sample concentration computation was performed by MassLynx version 4.1 software from Waters, Micromass. Urinary steroid metabolite concentrations were reported relative to urine creatinine levels determined by enzymatic photometric method (Konelab 20 XTi, Thermo Fisher Scientific, Vantaa, Finland; Konelab Creatinine Kit no. 981845).

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BMI, body mass index; LBM, lean body mass; CSA of VL, cross-sectional area of m. vastus lateralis; 1RM, one repetition maximum; HRE, heavy resistance exercise; YM, younger men (n = 5); OM, older men (n = 8). ⁎ Statistically significant change (p b 0.05) within group from corresponding baseline value. # Statistically significant difference (p b 0.05) compared to younger men. ## Statistically significant difference (p b 0.01) compared to younger men.

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2.5.5. Analysis of urinary T metabolites Subjects voided at 6 am and urine samples were collected for each subject before (10–12 am) and 1 h after the HREs. All urine samples were frozen at −80 °C until analysis. Urinary concentrations of the androgen metabolites androsterone and etiocholanolone were determined by the same LC–MS–MS as blood samples. 1 ml of K-phosphate buffer (pH 7) and 10 μl of β-glucuronidase enzyme (no. G7017, Sigma-Aldrich, St. Louis, MO, USA) were added to a 3 ml aliquot of urine, vortexed, and incubated at 50 °C for 80 min. Then samples were cooled and 750 μl of K2CO3:KHCO3 solution (1:1, 10 %) and 5 ml of methyl tert-butyl ether (MTBE) (no. C39C11X, Lab-Scan, Gliwice, Poland) were added and shaken for 40 min. After that, samples were centrifuged for 4 min at 4000 rpm. The organic phase was then transferred to a clean tube and evaporated to dryness under a gentle nitrogen stream. The extract was reconstituted in 250 μl of methanol with 0.1% formic acid (no. 33015, Sigma-Aldrich, St. Louis, MO, USA), including

t2:1 t2:2 t2:3

Table 2 Serum testosterone (nmol·L−1), LH (IU·L−1), SHBG (nmol·L−1) and free testosterone concentrations (nmol·L−1) (mean ± SD) in HREs before and after 12-month resistance training in younger and older men.

364 365 366 367

t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 Q4 t2:17 t2:18 Q5 t2:19 t2:20 t2:21

D

E

T

C

E

362 363

R

361

R

359 360

N C O

357 358

YM

U

355 356

P

t1:25 t1:26 t1:27 Q3 t1:28 t1:29 t1:30 t1:31

Pre-RT

Post-RT

OM

Pre-RT

Post-RT

Pre Post Post1h Pre Post Post1h Pre Post Post1h Pre Post Post1h

LH

Testosterone

SHBG

FT

3.2 ± 1.8 5.0 ± 1.8 4.6 ± 2.3 3.5 ± 2.0 3.5 ± 2.1 3.0 ± 1.4 3.9 ± 1.5 4.2 ± 1.9 3.9 ± 1.5 4.8 ± 1.9 6.9 ± 3.2 5.0 ± 1.8

12.1 ± 2.8 11.8 ± 3.3 11.9 ± 2.8 11.0 ± 1.8 10.8 ± 1.5 10.8 ± 2.0 11.9 ± 1.3 12.5 ± 1.5 12.3 ± 1.6 12.9 ± 0.9 14.9 ± 1.8 14.8 ± 1.6

25.7 ± 11.0 29.0 ± 12.8 24.1 ± 11.7 25.4 ± 10.8 27.8 ± 11.3 25.2 ± 10.6⁎ 40.5 ± 8.2# 44.8 ± 9.3# 36.8 ± 7.8⁎ 43.1 ± 11.2# 48.6 ± 13.0# 43.0 ± 11.5#,⁎

0.287 ± 0.156 0.351 ± 0.365 0.363 ± 0.310 0.246 ± 0.067 0.233 ± 0.054 0.239 ± 0.078 0.211 ± 0.071 0.210 ± 0.083 0.234 ± 0.096 0.221 ± 0.040 0.244 ± 0.088 0.257 ± 0.073##

LH, luteinizing hormone; SHBG, sex-hormone binding globulin; FT, free testosterone; HRE, heavy resistance exercise; RT, resistance training; YM, younger men (n = 5); OM, older men (n = 8). ⁎ Statistically significant change (p b 0.05) within group from preceding Post-value. # Statistically significant difference (p b 0.05) compared to younger men. ## Statistically significant difference (p b 0.01) compared to younger men.

Please cite this article as: Ahtiainen, J.P., et al., Effects of resistance training on testosterone metabolism in younger and older men, Exp. Gerontol. (2015), http://dx.doi.org/10.1016/j.exger.2015.06.010

372 373 374 Q31 375 376 377 378 379 Q32 380 381 382 383 384 385 386 Q33 387 388 389 390

393 394 395 396 397 398 399 Q34 400 401 402 403 404 405 406 407 408 409 410 411 412

6

424 425 426 427 428 429 430 431 432 433 434 435 436 437

450

3. Results

451

3.1. Body composition

452 453 454

Body mass was greater (p b 0.01) in YM compared to OM before the RT (Table 1). Body fat% decreased in OM (p b 0.05) following RT. Lean body mass increased (p b 0.05) both in YM and OM following RT.

455

3.2. Muscle cross-sectional area of m. vastus lateralis (CSA of VL)

456 457

CSA of VL was greater (p b 0.05) in YM compared to OM before RT. CSA of VL increased (p b 0.05) in OM following RT (Table 1).

458

3.3. Lower-extremity muscle strength

459

3.3.1. One repetition maximum (1RM) leg press 1RM was greater (p b 0.01) in YM compared to OM before RT. 1RM increased (p b 0.05) in YM and OM following RT (Table 1). 1RM per CSA of VL or lean body mass did not differ significantly between YM and OM.

460 461 462 463 464 465 466

C

E

R

R O

447 448

C

445 446

N

443 444

U

441 442

T

449

439 440

3.3.2. Specific force 1RM to LBM ratio was greater in YM compared to OM before (p b 0.01) and after (p b 0.05) RT (Table 1). The 1RM/LBM ratio increased (p b 0.05) in YM and OM following RT.

469 470 471

3.4.1. Basal blood samples 473 No statistically significant differences or changes were observed in 474 serum basal total testosterone concentrations before or following RT 475

2.5.7. Standard meal and dietary analyses The subjects came to the laboratory at 6 am in fasted conditions (12 h of fasting and 8 h of sleep). After the blood sampling, subjects consumed a meal replacement drink and bar (Nutrilett, Nutri Pharma, Oslo, Norway) including totally 434 kcal of energy, 31 g of protein, 54 g of carbohydrates and 10 g of fat. The same meal was consumed at 12 am before the blood sampling. Subject also drank 1.5 l of water bit by bit between the blood samplings at 6 am and 12 am. After 12 am, drinking was not allowed. Dietary intake of the subjects was registered by dietary diaries for three preceding days prior to the HREs before and after the RT, and analyzed using Micro Nutrica nutrient analysis software Version 3.11 (The Social Insurance Institution of Finland). 2.5.8. Statistics Data are presented as mean values ± SD in text and tables. For simplicity of presentation, all data in the figures are given as mean ± sem. Due to the low number of the subjects in the experimental groups, and partly non-normally distributed data (revealed by the Shapiro– Wilk test), non-parametric statistics were utilized. Mann–Whitney U test was applied to analyze the differences between the groups. The comparisons between paired data were made using the Wilcoxon's signed rank test. To analyze changes over time with 3 or more variables, a Friedman test was applied. When the Friedman test revealed significant F-ratios, pairwise comparisons with Wilcoxon test were utilized to localize significant differences. Linear regression analysis (Spearman's product moment) was used to compare association between variables. The level of significance was set at p ≤ 0.05.

438

472

F

422 423

3.4. Serum testosterone concentrations

O

420 421

R O

419

467 468

P

417 418

3.3.3. Acute heavy resistance exercise (HRE) Total work of the HREs (sets × repetitions × loads; i.e. weight stack lifted) was greater (p b 0.01) in YM compared to OM before RT (Table 1). Total work increased significantly (p b 0.05) following RT in both YM and OM.

D

415 416

by area) using a ChemiDoc XRS device in combination with Quantity One software (version 4.6.3; Bio-Rad Laboratories Inc., Hercules, CA, USA). The AR bands were identified with a positive control of AR (cat# sc-2241, ZR-75-1 Cell Lysate, Santa Cruz BioTechnology, CA, USA) and a colored molecular weight standard (Precision Plus Protein™ Dual Colour Standards, Bio-Rad Laboratories, Richmond, CA, USA). The AR results were normalized to GAPDH. All six samples of each subject were loaded in the same gel and, in addition to that, the pre-training samples were analyzed with a separate run to compare the differences between the groups. The pre-exercise samples before RT were analyzed in duplicate and the intra-assay CV was 13.4%.

E

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J.P. Ahtiainen et al. / Experimental Gerontology xxx (2015) xxx–xxx

Fig. 2. Changes in serum testosterone concentrations, and testosterone clearance (MCRT) and production rate (PRT) (mean ± sem) due to the heavy resistance exercise bout (HRE; 5 × 10RM leg presses) in younger (n = 5) and older men (n = 8) before and after the 12month experimental heavy resistance training period. *Statistically significant change (* = p b 0.05). #Statistically significant difference compared to older men (# = p b 0.05).

Please cite this article as: Ahtiainen, J.P., et al., Effects of resistance training on testosterone metabolism in younger and older men, Exp. Gerontol. (2015), http://dx.doi.org/10.1016/j.exger.2015.06.010

7

R O

O

F

J.P. Ahtiainen et al. / Experimental Gerontology xxx (2015) xxx–xxx

485 486 487 488 489 490

492

D

MCRT and PRT did not change statistically significantly between the Control- and Pre-samples. In MCRT greater values (p b 0.05) were observed in YM compared to OM at Post0 before RT (YM: 493 ± 48 l/m2/d, OM: 364 ± 22 l/m2/d) and at Pre after RT (YM: 542 ± 47 l/m2 /d, OM: 379 ± 21 l/m2/d) (Fig. 2). MCR T decreased from Post0 (493 ± 48 l/m2/d) to Post1h (429 ± 41 l/m2/d) in YM before RT (p b 0.05) and from Post0 (388 ± 39 l/m2/d) to Post1h (351 ± 38 l/m2/d) in OM after RT (p b 0.05). No statistically significant differences or changes were observed in PRT during HREs before or following RT within or between the groups, except of a decrease in PRT from Post0 (2.0 ± 0.5 mg/m2/d) to Post1h (1.4 ± 0.3 mg/m2/d, p b 0.05) in YM after RT. Since only a few differences were observed between the age groups, the data were also analyzed with the combined groups of YM and OM to increase statistical power. When the groups of YM and OM were

E

T

C

483 484

E

481 482

R

479 480

3.4.2. Exercise blood samples Blood lactate increased (all p b 0.05) before RT up to 8.4 ± 2.6 mmol·l−1 and 5.4 ± 1.9 mmol·l−1, and following RT up to 5.6 ± 1.8 mmol·l−1 and 4.7 ± 1.2 mmol·l−1 in YM and OM, respectively. No statistically significant differences or changes were observed in LH or T concentrations during HREs before or following RT within or between the groups (Table 2). Furthermore, no statistically significant differences or changes were observed in LH concentrations or pulse analysis (pulse frequency, mean pulse amplitude, and mean area under the LH curve) before or following RT within or between the groups. SHBG concentrations were lower in OM compared to YM (p b 0.05) and decreased from Post to Post1h (p b 0.05), except in YM before RT (Table 2).

491

R

478

3.5. T metabolic clearance and production rates

N C O

477

within or between the groups; from 17.2 ± 3.3 to 14.2 ± 2.3 nmol·l−1 in YM, and from 15.9 ± 3.4 to 16.2 ± 3.8 nmol·l−1 in OM.

U

476

P

Fig. 3. A relationship between the relative changes from pre- to post-exercise in serum testosterone concentration and metabolic clearance rate of testosterone (MCRT) in the combined group of younger (n = 5) and older men (n = 8) after the resistance training period.

Fig. 4. Changes in urine androsterone and etiocholanolone concentrations (mean ± sem) due to the heavy resistance exercise bout (5 × 10RM leg presses) in younger (n = 5) and older men (n = 8) before and after the 12-month experimental heavy resistance training period. #Statistically significant difference (# = p b 0.05, ## = p b 0.01) between the experimental groups.

Please cite this article as: Ahtiainen, J.P., et al., Effects of resistance training on testosterone metabolism in younger and older men, Exp. Gerontol. (2015), http://dx.doi.org/10.1016/j.exger.2015.06.010

493 494 495 496 497 498 499 500 501 502 503 504 505 506

J.P. Ahtiainen et al. / Experimental Gerontology xxx (2015) xxx–xxx

3.6. Urinary T metabolites

517 518

No statistically significant changes were observed in urine androsterone and etiocholanolone concentrations due to HREs before or following RT within the groups, except a trend of decrease in androsterone (from 3.0 ± 1.4 μg·ml−1 to 1.7 ± 0.5 μg·ml−1, p = 0.08) before RT

526

Serum free testosterone (FT) was greater (p b 0.05) in YM than OM before the hCG and GnRH tests (Fig. 5). The hCG tests induced significant increases (p b 0.05) in T and FT before and after the training period in both YM and OM. The GnRH tests induced increases (p b 0.05) in T and FT before the training period in YM and OM, but after the RT, T and FT increased significantly (p b 0.05) only in OM. The relative T and FT responses (mean of 48 h, 72 h and 96 h) to hCG were greater (p b 0.05) in YM than OM after RT (data not shown). No significant

527 528

P D E T C E R R O C

519 520

3.7. GnRH and hCG stimulation tests

N

513

U

511 512

521 522

F

516

509 510

in YM (Fig. 4). Urinary etiocholanolone concentration was lower (p b 0.01) in OM compared to YM before RT. Urinary androsterone concentrations (p b 0.05–0.01) as well as the urine androsterone/etiocholanolone ratio were lower (p b 0.05) in OM compared to YM in every time point.

R O

514 515

combined, MCRT decreased from Post0 (474 ± 58 l/m2/d) to Post1h (394 ± 34 l/m2/d, p b 0.05) and PRT increased from Pre (1.5 ± 0.1 mg/m2/d) to Post0 (1.7 ± 0.2 mg/m2/d, p b 0.05) and decreased from Post0 to Post1h (1.4 ± 0.1 mg/m2/d, p b 0.01) after RT. In the combined group of YM and OM, the relative changes from Pre to Post0 in serum T and MCRT after the RT correlated (r = − 0.70, p b 0.01) with each other (Fig. 3). Also in the combined group of YM and OM, the Pre-values in serum SHBG and MCRT after the RT correlated (r = −0.73, p b 0.01) with each other.

507 508

O

8

Fig. 5. Responses of serum basal total testosterone and free testosterone (mean ± sem) to (a) hCG and (b) GnRH stimulation tests in younger (n = 5) and older men (n = 8) before and after the 12-month resistance training period. The hCG stimulation and GnRH stimulation were administered following pre-samples. *Statistically significant change (* = p b 0.05) from the corresponding pre-stimulation value. #Statistically significant difference (# = p b 0.05, ## = p b 0.01) between the experimental groups.

Please cite this article as: Ahtiainen, J.P., et al., Effects of resistance training on testosterone metabolism in younger and older men, Exp. Gerontol. (2015), http://dx.doi.org/10.1016/j.exger.2015.06.010

523 524 525

529 530 531 532 533 534

J.P. Ahtiainen et al. / Experimental Gerontology xxx (2015) xxx–xxx

3.9. Dietary intake

542 543 544

No statistically significant differences or changes were observed in total energy, protein, fat and carbohydrate intake normalized to the body weight within or between the groups (Table 3).

545

4. Discussion

546

In this study, RT-induced responses to the several aspects of T metabolism were thoroughly examined for the first time. We found the attenuated responses of T to gonadotropin stimulation and the lower urinary excretion of T metabolites in older men indicating that aging could affect T metabolism. However, the present long-term RT program did not appear to have an impact on T metabolism determined in the present study. Although RT increased muscle strength and mass in both YM and OM, these changes were not associated with changes in any of the measured variables of T production or metabolism. Thus, age related changes in T metabolism do not appear to have an effect on RT-induced adaptations of muscular characteristics. These findings do not support the assumption of possible endocrine system adaptations of T due to long-term RT as hypothesized in the present study. The present RT program was designed to optimize muscle strength and mass gains safely and effectively for previously untrained younger and older men. As expected, muscle strength (1RM leg press and total work of the HREs) increased significantly in both YM and OM following RT. Also lean body mass increased in both groups. Muscle size (CSA of VL) as well as muscle “quality” (1RM to LBM ratio) were lower in OM

559 560 561 562 563 564

C

557 558

E

555 556

R

553 554

R

551 552

N C O

549 550

U

547 548

F

541

O

540

No statistically significant differences or changes were observed in skeletal muscle androgen receptor concentrations within or between the groups (Fig. 6).

R O

538 539

P

3.8. AR protein concentration

D

537

indicating age-related decreases in muscle strength (i.e. sarcopenia). However, both age groups showed RT-induced muscular adaptations approximately to the same extent. Dietary intake did not differ between YM and OM and did not change during the study (see Table 3). The daily protein intake (~1.1–1.4 g per kg of body mass per day) of the subjects should adequately and safely meet the needs of young and older adults engaged in RT (Campbell and Leidy, 2007). The nutritional status and body composition may have influence on T production, possibly through the effect of leptin on testicular Leydig cells and hypothalamic GnRH secretion (Mah and Wittert, 2010; Saad, 2009). Also the metabolic health status, such as insulin resistance, may have effect on Leydig cell function (Pitteloud et al., 2005). In the present study the diet, body composition or health status of the subjects did not change drastically and, therefore, they may have only minimal role explaining the observed findings. In this study, GnRH and hCG stimulation tests were used to investigate the possible training-induced changes in testicular T production capacity before and after long-term systematic RT. We found that the gonadotropin-stimulated testicular production of T was lower in OM than YM, which confirms the findings that the aging-related suppression of T production is mainly due to primary testicular, rather than secondary hypothalamic–pituitary failure (Wu et al., 2008). The significant differences in FT between YM and OM can be, at least in partly, explained by the aging-related increase in serum SHBG concentrations. It could be speculated that the age related attenuation in T production capacity has effects on muscular characteristics and training-induced muscular adaptations. However, the present results showed no differences between the two age groups in the responses of muscle strength and mass, and no training-induced adaptations occurred in testicular capacity to secrete T. Exercise can impact on serum T concentrations by altering MCRT, especially through exercise-induced decreases in the hepatic blood flow (Cadoux-Hudson et al., 1985), while increases in PRT may be caused by increases in LH pulsatility or production (Metivier et al., 1980; Vermeulen et al., 1972). In the present study, however, serum LH that induces T production in Leydig cells did not change systematically due

E

changes were observed in hCG and GnRH induced T and FT responses due to RT in YM or OM.

T

535 536

9

Fig. 6. Changes in skeletal muscle androgen receptor content (mean ± sem) due to the heavy resistance exercise bout (5 × 10RM leg presses) in younger (n = 5) and older men (n = 8) before and after the 12-month experimental heavy resistance training period.

Please cite this article as: Ahtiainen, J.P., et al., Effects of resistance training on testosterone metabolism in younger and older men, Exp. Gerontol. (2015), http://dx.doi.org/10.1016/j.exger.2015.06.010

565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 Q35 587 588 589 590 591 592 593 594 595 596 597 598 599 600

10 t3:1 t3:2

J.P. Ahtiainen et al. / Experimental Gerontology xxx (2015) xxx–xxx

Table 3 Total energy and macronutrient intake per day (mean of 3 days before the heavy resistance exercise session ± SD) before and after the 12-month resistance training period.

t3:3

Younger men (n = 5)

t3:4

Month-0

Month-12

Month-0

Month-12

100.2 ± 32.7 1.4 ± 0.2 0.9 ± 0.4 2.6 ± 1.1

97.2 ± 17.7 1.2 ± 0.3 0.7 ± 0.1 2.5 ± 0.4

98.1 ± 13.8 1.2 ± 0.2 0.8 ± 0.2 2.8 ± 0.3

104.17 ± 12.4 1.1 ± 0.1 0.8 ± 0.2 3.0 ± 0.6

O

F

LH or SHBG concentrations, MCRT and PRT, urine androsterone and etiocholanolone concentrations, testicular production capacity of T, and skeletal muscle ARs) changed during a 12-month resistance training period in YM or OM. Moreover, none of the present variables were related to the resistance training-induced changes in muscle strength and mass. These findings suggest that biological age-related changes in T metabolism may not markedly limit the resistance training-induced adaptations in muscular function in healthy older men.

R O

to exercise and it may not, therefore, explain the observed changes in PRT. Metabolic processes of hormones in the liver are primarily a function of hepatic blood flow and, hence, any changes in the hepatic 604 blood flow can result in concomitant changes in the elimination rate 605 of T. Accordingly, in the present study, a statistically significant negative 606 correlation was found between the changes in MCRT and the changes in 607 serum T concentrations when the data of YM and OM groups were com608 bined. This finding supports the assumption that acute resistance 609 exercise-induced changes in serum T are probably explained by alter610 ations in MCRT. The hepatic extraction of T is presumed to be occurring 611 from the non-SHBG-bound fraction of T (Coviello et al., 2006). This as612 sumption is supported in the present study by the negative correlation 613 observed between serum SHBG concentrations and MCRT at rest. Thus, 614 higher SHBG levels may be a contributing factor to the observed lower 615 MCRT in OM compared to YM. 616 The main site of T clearance following its metabolism and conjuga617 tion in the liver is the kidney. Although no statistical significances 618 were reached, the urinary androsterone and etiocholanolone concen619 trations appeared to be lower following exercise in YM suggesting 620 Q36 that the hepatic clearance of T has been decreased due to exercise. Inter621 estingly, the urinary concentrations of these two T metabolites were 622 lower in OM compared to YM, with no obvious acute responses to exer623 cise. The decrease in hepatic extraction of T with aging is presumably re624 Q37 lated to the increase in SHBG seen in OM. The reduction of the urinary 625 androsterone/etiocholanolone ratio in OM compared to YM could re626 flect the age-related decrease of the metabolic conversions involving 627 5α-reductase (Matzkin et al., 1992). However, the physiological signif628 icance of these findings remains unclear. 629 Skeletal muscles have a role in the extra-hepatic metabolic removal 630 of T from plasma, although the precise contributions of the skin, muscle 631 and other tissues to T clearance have not been determined. Variable 632 findings have been made in previous studies on acute heavy resistance 633 exercise, showing increases (Kraemer et al., 2006), decreases (Ratamess 634 et al., 2005; Vingren et al., 2009) and no changes (Hulmi et al., 2008; 635 Spiering et al., 2009; Ahtiainen et al., 2011) in AR protein concentrations 636 in exercised muscle. Chronically RT has not induced changes in skeletal 637 muscle AR content (Ahtiainen et al., 2009, 2011). In the present study, 638 no age differences or exercise training responses were observed in skel639 etal muscle AR concentrations. Thus, skeletal muscle AR content does 640 not appear to play an important role in the present changes of T metab641 olism acutely or chronically in YM or OM. Otherwise skeletal muscle ste642 roidogenesis may have a role in RT-induced muscular adaptations since 643 recently Sato et al. (2014) concluded that RT restored age-related de644 cline of steroidogenic enzyme expressions and muscle sex steroid hor645 mone levels in older men and, moreover, these enzymes and hormone 646 Q38 levels were significantly correlated with RT-induced changes in muscle 647 size and strength.

Conflict of interest

C

E

R

R

O

C

N

U

648

5. Conclusions

649

Stimulated testicular production of T was lower in OM compared to YM. Age differences were also observed in urinary excretion of the T metabolites androsterone and etiocholanolone, whose concentrations were lower in OM compared to YM. In contrast to our hypothesis, none of the present variables determining various aspects of T metabolism (basal serum total T or FT, HRE-induced changes in serum T, serum

650 651 652 653 654

655 656 657 658 659 660 661 662 663

No conflicts of interest, financial or otherwise, are declared by the 664 authors. 665 Acknowledgments

666

The authors thank Mr. Risto Puurtinen, Mrs. Aila Ollikainen and Mrs. Mirja Lahtiperä for their help in data collection and analysis. We also thank the very dedicated group of subjects who made this project possible. The Juho Vainio Foundation, Miina Sillanpää Foundation and Jyväskylä Central Hospital, Jyväskylä, Finland supported this research.

667 668

T

602 603

P

601

D

Total energy intake/body weight (kJ·kg−1) Protein intake/body weight (g·kg−1) Fat intake/body weight (g·kg−1) Carbohydrate intake/body weight (g·kg−1)

E

t3:5 t3:6 t3:7 t3:8

Older men (n = 8)

669 670 671 Q39

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Please cite this article as: Ahtiainen, J.P., et al., Effects of resistance training on testosterone metabolism in younger and older men, Exp. Gerontol. (2015), http://dx.doi.org/10.1016/j.exger.2015.06.010

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Effects of resistance training on testosterone metabolism in younger and older men.

This study investigated the effects of resistance training (RT) on the metabolism of testosterone (T) in younger (n=5, 28±3yrs.) and older (n=8, 70±2y...
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