Estrogen Supplementation Reduces Whole Body Leucine and ...

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The Journal of Clinical Endocrinology & Metabolism 90(6):3592–3599 Copyright © 2005 by The Endocrine Society doi: 10.1210/jc.2004-1743

Estrogen Supplementation Reduces Whole Body Leucine and Carbohydrate Oxidation and Increases Lipid Oxidation in Men during Endurance Exercise Mazen J. Hamadeh, Michaela C. Devries, and Mark A. Tarnopolsky Departments of Pediatrics and Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5 Healthy active men exhibit higher rates of carbohydrate (CHO) and leucine oxidation and lower rates of lipid oxidation compared with their female counterparts both at rest and during moderate intensity endurance exercise. We postulated that this reduced dependence on amino acids as a fuel source in women was due to the female sex hormone estrogen. In a randomized, double-blind, placebo-controlled, cross-over design, we investigated the effect of supplementing 12 recreationally active men with estrogen on whole body substrate oxidation and leucine kinetics at rest and during moderate intensity endurance exercise. Subjects cycled for 90 min at an intensity of 65% maximum O2 consumption after 8 d of either estrogen supplementation (2 mg 17␤-estradiol/d) or placebo (polycose). After a 2-wk washout period, they repeated the test after 8 d of the alternate treatment. On the test day, after a primed continuous infusion of L-[13C]leucine, O2 consumption, CO2 production, steady-state breath 13CO2, and plasma ␣-[13C]ketoisocaproate enrichments were measured at rest

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ENDER DIFFERENCES IN substrate oxidation at rest and during endurance exercise have been reported (1–11). Women have higher rates of lipid oxidation and lower rates of carbohydrate (CHO) and leucine oxidation compared with men. In addition, women have higher rates of whole body protein synthesis, as assessed by nonoxidative leucine disposal during exercise when studied in the postabsorptive state (7). Women and men exhibit different temporal patterns in substrate oxidation during endurance exercise, insofar as men show increased plasma fatty acid uptake into the skeletal muscle over the duration of the exercise bout, mobilize skeletal muscle lipids later during exercise, and exhibit an earlier reliance on CHO as a fuel source compared with women (11, 12). However, after ingestion of a CHO drink, women oxidize more CHO in the latter part of a moderate intensity exercise bout compared with men (13). The effects observed in the postabsorptive state have been attributed to the influence of the female sex hormone estrogen on increasing the availability of lipids for substrate oxidation in the exercising muscle (14 –16). Estradiol suppleFirst Published Online March 8, 2005 Abbreviations: CHO, Carbohydrate; CV, coefficient of variation; ␣KIC, ␣-ketoisocaproate; LPL, lipoprotein lipase; NOLD, nonoxidative leucine disposal; Ra, rate of appearance; Rd, rate of disappearance; RER, respiratory exchange ratio; urea-N, urinary urea nitrogen; VCO2, CO2 production; VO2, O2 consumption; VO2max, maximum O2 consumption. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

and at 60, 75, and 90 min during exercise in the postabsorptive state. Exercise increased energy expenditure more than 5-fold, CHO oxidation more than 6-fold, lipid oxidation more than 4-fold, and leucine oxidation 2.2-fold (all P ⬍ 0.0001), whereas it decreased the ratio of lipid to CHO oxidation by 50 –70% (P ⫽ 0.003) compared with values at rest. Estrogen supplementation decreased respiratory exchange ratio during exercise (P ⫽ 0.03). Estrogen supplementation significantly decreased CHO oxidation by 5–16% (P ⫽ 0.04) and leucine oxidation by 16% (P ⫽ 0.01), whereas it significantly increased lipid oxidation by 22– 44% (P ⫽ 0.024) at rest and during exercise. We conclude that estrogen influences fuel source selection at rest and during endurance exercise in recreationally active men, characterized by a reduced dependence on amino acids and CHO and an increased reliance on lipids as a fuel source. (J Clin Endocrinol Metab 90: 3592–3599, 2005)

mentation in male and oophorectomized female rats helped elucidate the role of sex hormones in influencing substrate oxidation. Estradiol supplementation in male rats increases plasma free fatty acid and vastus lateralis triacylglycerol content at rest and after submaximal treadmill exercise compared with placebo (17). There is a parallel reduction in adipocyte lipoprotein lipase (LPL) activity and increases in myocardial and vastus lateralis LPL activity and the ratio of adipocyte to muscle (both vastus lateralis and myocardial) LPL activity at rest and after exercise (14). Together, these results suggest that estradiol increases the intramuscular oxidation of lipids derived from different sources, corroborated by a significant decrease in retroperitoneal fat pad mass (14, 17). In addition, the liver glycogen content was significantly higher in supplemented rats in the latter half of a 2-h treadmill exercise, whereas soleus and red and white vastus lateralis glycogen contents were significantly higher throughout the exercise bout. Increased availability of lipids for oxidation during exercise results in glycogen sparing, which, in turn, improves performance (17, 18). Supplemented rats run longer and perform more work during a run to exhaustion treadmill test (18). Similar results in glycogen sparing and performance are observed in estradiol-supplemented oophorectomized rats after submaximal treadmill exercise (19). Few studies involving estradiol supplementation in humans have been reported. Estradiol supplementation using medicated transdermal patches in amenorrheic women decreased glucose rates of appearance (Ra) and disappearance (Rd), but not glycerol kinetics (20). Whereas transdermal patches in men had no effect on substrate oxidation, results

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Hamadeh et al. • Estrogen Reduces Leucine Oxidation in Men

were confounded by the very low dose of estradiol (0.1– 0.3 mg/d) provided (21). Consequently, oral administration of estradiol in doses 10- to 30-fold higher in men elicited significant reductions in glucose Ra and Rd, with no change in the glycerol Ra to Rd ratio (22). It is possible that estradiol influences some, but not all, aspects of substrate oxidation, or that glucose metabolism is more sensitive to hormonal changes than lipid metabolism. In addition, the sample size was relatively low in the latter three studies, and a type II error could not be discounted. Substrate oxidation during exercise appears to be influenced by sympathoadrenergic modulation. ␤-Adrenergic receptor antagonists increase CHO oxidation, decrease lipolysis and lipid oxidation (23), and increase leucine oxidation during exercise (24). Conversely, ␤-adrenergic receptor stimulation increases lipolysis and plasma free fatty acid concentration, but decreases fatty acid oxidation, with a concomitant increase in glycogen oxidation (23, 25). It is possible that estradiol exerts its effect on substrate oxidation by influencing ␤- and ␣-adrenoreceptor signaling (26, 27). Unlike the effects on CHO and lipid metabolism, the influence of estradiol supplementation on whole body amino acid metabolism has not been studied. We therefore investigated the effect of supplementing healthy, recreationally active men with 17␤-estradiol on whole body substrate oxidation and leucine kinetics at rest and during 90 min of moderate intensity endurance exercise. Given the gender differences observed in protein metabolism, we hypothesized that estradiol supplementation would decrease leucine oxidation and that this would be consistent with a reduction in CHO oxidation and an increase in lipid oxidation during endurance exercise. Subjects and Methods Subjects Twelve healthy, recreationally active, young men participated in the study. The subject characteristics were: age, 23 ⫾ 2 yr; weight, 79 ⫾ 3 kg; height, 177 ⫾ 1 cm; body mass index, 25 ⫾ 1 kg/m2; fat-free mass, 63 ⫾ 2 kg (80 ⫾ 2% of body weight); and maximum O2 consumption (VO2max), 44 ⫾ 2 ml O2/kg body weight䡠min. They were informed of the study details and advised of the risks and benefits associated with the study before providing written consent. The study was approved by the McMaster University human research and ethics board and was conducted in accordance with the guidelines of the Declaration of Helsinki.

Study design The subjects were randomly assigned to one of two groups following a double-blind, crossover, placebo-controlled design. One group received 17␤-estradiol (Estrace, Shire Biochem Inc., St. Laurent, Quebec, Canada) for 8 d, whereas the other group received a placebo (400 mg/d Polycose; Abbott Laboratories, Ross Division, St. Laurent, Quebec, Canada). On d 9, the subjects cycled for 90 min at an intensity of 65% VO2max. After a minimum washout period of 2 wk, the groups received the alternate treatment for 8 d, with testing occurring on d 9. The 17␤-estradiol tablets were placed in gelatin capsules filled with Polycose and taken daily at a dose of 1 mg/d for 2 d, followed by 2 mg/d for 6 d. Subjects were instructed to take the capsules at the same time every day and to return unused capsules. All subjects reported 100% compliance. VO2max was determined at least 7 d before the first testing day using a progressive exercise test on an electronically braked cycle ergometer and a computerized open-circuit gas collection system (Moxus Modulator VO2 system with O2 analyzer S-3A/I and CO2 analyzer CD-3A, AEI

J Clin Endocrinol Metab, June 2005, 90(6):3592–3599

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Technologies Inc., Pittsburgh, PA). Subjects cycled (Lode cycle ergometer, Excalibur Sport, Lode, Groningen, The Netherlands) at 75 watts for 2 min, followed by 2 min at 150 watts, 2 min at 200 watts, and thereafter increasing in increments of 25 watts/min. VO2max was established when O2 consumption (VO2) values reached a plateau or was the highest value during the incremental ergometer protocol, pedal revolutions could not be maintained over 60 rpm despite vigorous encouragement, and the respiratory exchange ratio (RER) was more than 1.12. The VO2max was used to estimate the work intensity required to elicit 65% of the subject’s VO2max, and this was confirmed by measuring VO2 at the estimated work intensity for each subject approximately 30 min after the test. The intensity (65% VO2max) and length (90 min) of the cycling bout were established to maintain consistency with previous work conducted in our and other laboratories (2, 3, 5, 13, 21, 22). Moreover, these parameters were chosen to allow all subjects to complete the 90-min cycling bout without midexercise failure. All subjects completed prospective diet records for a minimum of 2 weekdays and 1 weekend day during the first trial. The diet records were returned to the subjects during the second trial, and they were instructed to consume similar foods. A subset of three subjects completed diet records during both trials of the study. Subjects were not tested during major holidays or when their diets on the different trials were not similar. They were also instructed to complete a physical activity record during both trials and to maintain their regular exercise throughout the study. Subjects consumed the same meal the evening preceding the testing day. On testing days, subjects arrived to the research center after an overnight fast. On arrival, the 24-h urine collection was completed, and body weight and fat-free mass (by bioelectric impedance analysis; BIA101A, RJL Systems, Mt. Clemens, MI) were measured (28). A 20-gauge plastic catheter (BD Biosciences, Franklin Park, NJ) was placed into the antecubital vein of the right arm for blood collection. A second catheter was placed into the antecubital vein of the left arm for the infusion of the tracers with the use of a constant rate infusion pump (model 74900, Cole-Parmer, Vernon Hills, IL). The right arm was placed in a heating pad (65 ⫾ 5 C; Dunlap, Kaz, Inc., New York, NY) 15 min before blood collection to arterialize the blood when the subjects were not cycling. Urine was collected before and immediately after the exercise session. Subjects were not allowed to eat until testing was completed. Water was provided ad libitum during the testing day, with a minimum intake of 250 ml/h.

Stable isotope infusions l-[1-13C]Leucine and [13C]NaHCO3 (both 99% enriched; Cambridge Isotope Laboratories, Inc., Cambridge, MA) were aseptically mixed with 0.9% saline and filtered through 0.2-␮m pore size filters (sterile, nonpyrogenic; Acrodisc, Pall Gelman Corp., Ann Arbor, MI) into single-use vials at the McMaster University Medical Center pharmacy. The l-[113 C]leucine vials were wet-sterilized at 121 C for 30 min, whereas the [13C]NaHCO3 vials were stored at 4 C. The solutions were cultured for 5 d at both room temperature and 35 C to test for the absence of bacteria. Subjects were infused for 2 h at rest before exercise and during the 90-min exercise session. A priming dose of 1 mg/kg l-[1-13C]leucine was administered, followed by a constant infusion of 1 mg/kg䡠h. A dose of 0.295 mg/kg [13C]NaHCO3 was infused with the l-[1-13C]leucine priming dose. Arterialized blood and breath samples were obtained before the primed-constant infusion to determine the natural background enrichment and at 0 (rest; immediately before the exercise session), 60, 75, and 90 min during exercise to determine leucine kinetics. Blood was collected into heparinized tubes placed on ice, centrifuged at 1750 ⫻ g at 4 C for 10 min, and stored at ⫺50 C until analyzed. Breath samples were collected in 10-ml sterile vacuum tubes (no additive; Vacutainer, BD Biosciences) after allowing 2 min for breath equilibration, and the tubes were stored at room temperature until analyzed. CO2 production (VCO2) and VO2 were measured over 5-min periods at 0, 30, 60, 75, and 90 min during exercise using a computerized, open-circuit, gas collection system to determine leucine kinetics and calculate substrate oxidation.

Analytical methods Plasma ␣-[13C]ketoisocaproate ([13C]␣KIC)/[12C]␣KIC enrichments were determined by gas chromatography-mass spectrometry (model

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6890, Hewlett-Packard, Palo Alto, CA; mass selective detector 5973 network, Agilent Technologies, Inc., Palo Alto, CA) using the o-trimethylsilyl quinoxalinol derivatives following a modified standard procedure (29). To precipitate proteins, 200 ␮l plasma were mixed well with 2 ml ice-cold absolute ethanol and centrifuged at 4000 ⫻ g for 25 min at 4 C. The protein-free supernatant was dried at 50 C under a gentle stream of nitrogen, resuspended in 1 ml ultrapure water (resistance ⬎18 Mohm/ cm; Milli-Q, Millipore Corp., Bedford, MA), and mixed well. To this, 1 ml freshly prepared o-phenylenediamine solution (2% in 4 m HCl) was added and gently mixed, and the container was capped and heated at 100 C for 1 h. After cooling at room temperature, methylene chloride (1 ml) was added, and the solution was mixed and centrifuged at 1200 ⫻ g for 3 min at 4 C. The bottom layer was transferred into a screw-cap tube, and the procedure was repeated. The solution was dried at room temperature under a gentle stream of nitrogen to which 75 ␮l N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (Pierce Chemical Co., Rockford, IL) was added, and the solution was heated at 100 C for 30 min for final derivatization. The derivatized sample was injected (0.3 ␮l) into the gas chromatograph under the following conditions: 15-m fused silica capillary column; initial temperature of 120 C; temperature increased from120 to 160 C at 8 C/min and to 290 C at 20 C/min, then held at 290 C for 3 min. Helium was used as the carrier gas (32 cm/sec). Mass spectrometry monitored molecules with a mass to charge ratio (m/z) of 232.1 for M ([12C]␣KIC) and 233.1 for M⫹1 ([13C]␣KIC) by selected ion monitoring. The intra- and interassay coefficients of variation (CVs) for this procedure are less than 1%. Breath 13CO2/12CO2 enrichments, expressed as ‰ (␦ 13C) PDB-1, were determined by isotope ratio-mass spectrometry (BreathMat Plus, Finnigan MAT GmbH, Bremen, Germany) as previously reported (13). Samples were corrected for natural background enrichments. The urea concentration was analyzed using a MOD P automated analyzer (Roche/Hitachi, Laval, Quebec, Canada) based on the urease/glutamate dehydrogenase enzyme system (Roche, Indianapolis, IN). The intra- and interassay CVs were less than 2%. For hormone analysis, clotted arterialized venous blood collected at rest was centrifuged at 1200 ⫻ g for 30 min at room temperature, and the serum was stored at ⫺50 C until analyzed. Serum total testosterone, estradiol, and progesterone concentrations were measured using solid phase RIA methods (kits TKTT1, TKE21 and TKPG1, respectively, Diagnostic Products Corp., Los Angeles, CA). The intra- and interassay CVs were less than 7% for total testosterone, 8% or less for estradiol, and 9% or less for progesterone. Diet records were analyzed using Nutritionist Pro (version 2.2, First DataBank, Inc., San Bruno, CA). The test-retest CVs were 5% for total energy, 7% for protein, 3% for CHO, and 9% for fat intake.

lipid oxidation is expressed in grams per minute, VCO2 is the volume of CO2 production expressed in liters per minute, VO2 is the volume of VO2 consumption expressed in liters per minute, and N is urinary urea-N excretion expressed in grams per minute. To validate the use of urea-N measurements from different sources to calculate energy expenditure, CHO oxidation, lipid oxidation, and the ratio of lipid/CHO oxidation, we tested the relationship between substrate oxidation values calculated using urinary urea-N of samples collected throughout the study day vs. the same values calculated using 24-h urea-N excretion collected the previous day and found very minor differences: energy expenditure, 0.2% (r ⫽ 0.9999; slope ⫽ 1.002); CHO oxidation, 0.5% (r ⫽ 0.9998; slope ⫽ 1.005); lipid oxidation, 1.6% (r ⫽ 0.9992; slope ⫽ 1.016); and ratio of lipid/CHO oxidation, 4.6% (r ⫽ 0.9971; slope ⫽ 1.046). Similarly, the differences between the substrate oxidation values calculated using urinary urea-N of samples collected throughout the study day vs. the same values calculated assuming daily intake of 102 g protein (equivalent to a population average of ⬃1.5 g protein/kg body weight) were minor: energy expenditure, 0.2% (r ⫽ 0.9999; slope ⫽ 1.002); CHO oxidation, 0.6% (r ⫽ 0.9998; slope ⫽ 1.006); lipid oxidation, 0.7% (r ⫽ 0.9998; slope ⫽ 1.007); and ratio of lipid/CHO oxidation, 0.97% (r ⫽ 0.9996; slope ⫽ 0.9903). The contribution to total energy expenditure due to CHO, lipid, and leucine oxidation was calculated during the resting period and the last 30 min of exercise.

Calculations

Diet and body weight

Leucine kinetics were calculated using steady-state equations. The leucine rate of appearance (Ra) was calculated as: Ra ⫽ (i/plasma 13 [ C]␣KIC TTR), where Ra is also leucine turnover or flux at steady state in micromoles per kilogram per hour, i is the rate of infusion of tracer leucine in micromoles per kilogram per hour, and plasma [13C]␣KIC TTR is the tracer to tracee ratio of plasma [13C]␣KIC at steady state. Leucine oxidation was calculated as: oxidation ⫽ (VCO2 ⫻ breath 13CO2 enrichment)/(bicarbonate retention factor ⫻ plasma [13C]␣KIC TTR), where leucine oxidation is expressed in micromoles per kilogram per hour, VCO2 is expressed in micromoles per kilogram per hour, and the bicarbonate retention factor is 0.81 at rest and 1.00 during exercise, as previously reported (4, 30, 31). The fraction of leucine oxidized was calculated as: fraction oxidized ⫽ (leucine oxidation/leucine Ra) ⫻ 100, where the fraction of leucine oxidized is expressed as a percentage. The nonoxidative leucine disposal (NOLD) was calculated as: NOLD ⫽ Ra ⫺ leucine oxidation, where NOLD is an estimate of whole body protein synthesis in micromoles per kilogram per hour. Whole body protein synthesis (NOLD) and breakdown were calculated in milligrams of protein per kilogram per hour, with the assumption that tissue protein contains 590 ␮mol leucine/g. Protein balance was calculated as: protein balance ⫽ protein synthesis ⫺ protein breakdown. CHO and lipid oxidation were calculated as previously reported by Frayn (32), where urinary urea nitrogen (urea-N) was used as a measure of N excretion, as follows: CHO oxidation ⫽ (4.55 ⫻ VCO2) ⫺ (3.21 ⫻ VO2) ⫺ (2.87 ⫻ N). Lipid oxidation ⫽ (1.67 ⫻ VO2) ⫺ (1.67 ⫻ VCO2) ⫺ (1.92 ⫻ N), where CHO oxidation, as glucose oxidation, is expressed in grams per minute,

Subjects consumed 9798 ⫾ 1647 kJ (2344 ⫾ 394 kcal; mean ⫾ sd), 289 ⫾ 72 g CHO, 91 ⫾ 29 g protein, and 85 ⫾ 18 g fat daily. CHO, protein, and fat constituted 49 ⫾ 8%, 16 ⫾ 4%, and 33 ⫾ 7%, respectively, of the total daily energy intake. There were no significant differences in total daily energy and macronutrient intakes between the placebo and estrogen treatments for the subset of three subjects who completed diet records during both trials (Table 1). Body

Statistical analyses A two-way repeated measures ANOVA was used to determine significant differences in leucine Ra, leucine oxidation, fraction of leucine oxidized, NOLD, whole body protein breakdown, RER, energy expenditure, and the rate of fat and CHO oxidation; the factors were treatment (placebo vs. estrogen supplementation) and time (Statistica version 5.0, StatSoft, Inc., Tulsa, OK). When significance occurred, Tukey’s honestly significant difference test was used post hoc to determine the source of difference. Student’s paired t test was used to determine significant differences in serum hormones in all subjects and in total daily energy and macronutrient intakes for a subset of three subjects between the placebo and estrogen treatments. One-tailed tests were used for RER, leucine oxidation, and substrate oxidation, because we hypothesized a priori that estrogen supplementation will decrease RER and CHO and leucine oxidation, but will increase lipid oxidation and ratio of lipid to CHO oxidation. Two-tailed tests were used for all other statistical analyses. Differences were considered significant at P ⱕ 0.05. Results are presented as the mean ⫾ sem unless otherwise indicated.

Results

TABLE 1. Total daily energy and macronutrient intakes for a subset of three subjects on both the placebo and estrogen treatments Treatment

Placebo

Estrogen

Energy kJ/d kcal/d Carbohydrate (g/d) Protein (g/d) Fat (g/d)

8870 ⫾ 1229 2122 ⫾ 294 239 ⫾ 30 98 ⫾ 21 87 ⫾ 12

8653 ⫾ 1580 2070 ⫾ 378 234 ⫾ 46 92 ⫾ 22 87 ⫾ 11

Data are presented as the mean ⫾ SEM (n ⫽ 3). There was no significant difference between the two treatments (by paired t test, P ⬎ 0.05).



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weights were similar on both treatments. Hormone concentrations

Estrogen supplementation significantly decreased the serum total testosterone concentration (P ⫽ 0.002) and increased the serum estradiol concentration (P ⫽ 0.0008; Table 2). There was a trend toward a decrease in the serum progesterone concentration with estrogen supplementation (P ⫽ 0.09). Estrogen supplementation also significantly decreased the testosterone/estradiol molar ratio and the (testosterone plus progesterone)/estradiol molar ratio (both P ⬍ 0.0001). RER, energy expenditure, and CHO and lipid oxidation

Estrogen supplementation decreased RER (P ⫽ 0.037) at rest and during exercise (Fig. 1). Exercise increased energy expenditure and CHO and lipid oxidation by more than 5-fold, more than 6-fold, and more than 4-fold (all P ⬍ 0.0001), respectively, whereas it decreased the ratio of lipid to CHO oxidation by 50 –70% (P ⫽ 0.003) compared with those at rest (Table 3). Energy expenditure was similar between the two treatments. Estrogen supplementation significantly decreased CHO oxidation by 5–15% (P ⫽ 0.018) and increased lipid oxidation by 22– 42% (P ⫽ 0.012) and the ratio of lipid to CHO oxidation by 31–75% (P ⫽ 0.029) at rest and during exercise. Whole body leucine kinetics

The plasma [13C]␣KIC/[12C]␣KIC and breath 13CO2/ CO2 enrichments are shown in Figs. 2 and 3. Exercise significantly increased leucine oxidation by 2.2-fold and the fraction of leucine oxidized by 2.5-fold (both P ⬍ 0.0001), whereas it decreased whole body leucine Ra by 8% (P ⫽ 0.003), NOLD by 37% (P ⬍ 0.0001), and protein breakdown by 8% (P ⫽ 0.003; Table 4). Estrogen supplementation significantly decreased leucine oxidation by 16% (P ⫽ 0.005) and the fraction of leucine oxidized by 11% (P ⫽ 0.01), but had no significant effect on leucine flux, protein synthesis, or protein breakdown. The estimated protein balance was significantly below zero at rest and during exercise on both treatments; however, it became 2.8-fold more negative during exercise (P ⬍ 0.0001) compared with at rest, but 20% less negative with estrogen supplementation (P ⫽ 0.01) compared with placebo. 12

Contribution of substrate oxidation to energy expenditure

Exercise increased the contribution of CHO oxidation to energy expenditure by 20% and that of protein utilization by 2-fold (P ⬍ 0.0001), whereas it decreased the contribution of TABLE 2. Serum hormone concentrations for 12 men after 8 d of placebo or estrogen supplementation Treatment a

Total testosterone (nmol/liter) Estradiol (pmol/liter)a Progesterone (nmol/liter)b

Placebo

Estrogen

21 ⫾ 1 125 ⫾ 12 3.0 ⫾ 0.3

17 ⫾ 1 876 ⫾ 168 2.5 ⫾ 0.2

Data are the mean ⫾ SEM (n ⫽ 12). a P ⱕ 0.002, estrogen vs. placebo, by paired t test. b P ⫽ 0.09, estrogen vs. placebo, by paired t test.

FIG. 1. RER for 11 men at rest and during 90 min of cycling at an intensity of 65% VO2max during placebo administration (PL; 䡺) or after 8 d of estrogen supplementation (ES; f). Data are the mean ⫾ SEM.

fat oxidation by 16 –23% (Table 5). Estrogen supplementation decreased the contribution of CHO oxidation to energy expenditure by 11% and that of protein utilization by 18% (P ⫽ 0.011), whereas it increased the contribution of fat oxidation by 23–35% compared with placebo. Discussion

We investigated the effect of estradiol supplementation on substrate oxidation and whole body leucine kinetics during moderate intensity endurance exercise in recreationally active, healthy men. Estrogen supplementation resulted in a shift in whole body RER and CHO, lipid, and leucine oxidation toward that found in women, specifically a decrease in RER and CHO and leucine oxidation and an increase in lipid oxidation. In a previous study in which men were provided with a medicated transdermal patch, we found no changes in substrate oxidation during exercise (21). In that study, estradiol was provided at a dose of 0.1– 0.3 mg/d for 11 d before testing, which caused an approximately 2-fold increase in serum estradiol concentrations. It is most likely that the administered dose was not sufficient to counteract the actions of testosterone and effect changes in whole body substrate oxidation in men. Consequently, we provided oral 17␤estradiol tablets in doses 10- to 30-fold higher (an average dose of 3 mg/d) to recreationally active men, increasing serum estradiol 11-fold to levels greater than those observed in women in the luteal phase (22). We observed a significant reduction in glucose Ra, Rd, and metabolic clearance rate, with no effect on glycerol flux. Similarly, estradiol supplementation in amenorrheic women significantly reduced glucose Ra and Rd, but not glycerol Ra and Rd, during exercise (20). This is possibly due to a type II error, because we performed a power analysis using the values reported in the previously mentioned articles and found that the power was less than 50% to detect significance for the reported RER and CHO and lipid oxidation values (21) and glycerol Ra and Rd (22). In the current study of 12 subjects, serum estradiol concentrations increased in men after supplementation with 2 mg/d to levels greater than those observed in women in the luteal phase. Hence, the sample size, length of the treatment

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TABLE 3. Energy expenditure and substrate oxidation in 11 men at rest and during 90 min of cycling at an intensity of 65% VO2max during placebo administration or after 8 d of estrogen supplementation Time

0 mina

30 min

60 min

75 min

90 min

8⫾0 8⫾0

43 ⫾ 3 44 ⫾ 3

45 ⫾ 3 45 ⫾ 3

45 ⫾ 3 46 ⫾ 3

44 ⫾ 3 43 ⫾ 4

2⫾0 2⫾0

10 ⫾ 1 10 ⫾ 1

11 ⫾ 1 11 ⫾ 1

11 ⫾ 1 11 ⫾ 1

10 ⫾ 1 10 ⫾ 1

0.32 ⫾ 0.08 0.27 ⫾ 0.05 ⫺15

2.2 ⫾ 0.2 2.1 ⫾ 0.2 ⫺5

2.2 ⫾ 0.2 2.0 ⫾ 0.2 ⫺8

2.2 ⫾ 0.2 2.0 ⫾ 0.2 ⫺11

2.1 ⫾ 0.2 1.9 ⫾ 0.3 ⫺10

0.055 ⫾ 0.013 0.070 ⫾ 0.015 ⫹29

0.20 ⫾ 0.05 0.25 ⫾ 0.05 ⫹23

0.24 ⫾ 0.05 0.31 ⫾ 0.04 ⫹26

0.24 ⫾ 0.05 0.34 ⫾ 0.04 ⫹42

0.25 ⫾ 0.05 0.30 ⫾ 0.04 ⫹22

0.30 ⫾ 0.09 0.47 ⫾ 0.15 ⫹57

0.10 ⫾ 0.03 0.13 ⫾ 0.03 ⫹31

0.13 ⫾ 0.03 0.17 ⫾ 0.04 ⫹36

0.13 ⫾ 0.03 0.22 ⫾ 0.05 ⫹75

0.15 ⫾ 0.04 0.24 ⫾ 0.08 ⫹67

b

Energy (kJ/min) Placebo Estrogen Energy (kcal/min)b Placebo Estrogen CHO oxidation (g/min)b,c Placebo Estrogend % Change Lipid oxidation (g/min)b Placebo Estrogend % Increase Lipid oxidation/CHO oxidationb Placebo Estrogend % Increase

Data are presented as the mean ⫾ SEM (n ⫽ 11). a Resting values. b Main effect for time, P ⱕ 0.003. c Assumed to be grams of glucose oxidized per minute. d Main effect for estrogen, P ⱕ 0.029.

period, and dosage followed in the current study are adequate to elicit changes in substrate oxidation in men. In contrast to our previously reported research, RER significantly decreased during exercise after estradiol supplementation (22). It is possible that in the previous study we did not reach power with a sample size of eight subjects to detect small changes in whole body substrate oxidation. In the current study, changes in the RER indicate a change toward a decrease in CHO oxidation and an increase in lipid oxidation. The estradiol-induced changes observed in glucose Ra and Rd in previous studies are in accordance with the finding of the current study of a decrease in whole body CHO oxidation during exercise after estradiol supplementation (20, 22). Estradiol supplementation in rats decreases hepatic glucose production through a combination of a reduction in gluconeogenesis and an increase in net glycogen storage (33, 34). The activity of phospoenolpyruvate carboxykinase, the ratelimiting enzyme in the gluconeogenic pathway, is signifi-

cantly reduced after estradiol supplementation (33). Similarly, estradiol supplementation in men decreases the glucose rate of utilization during exercise, and this is not due to a decrease in circulating glucose levels, because plasma glucose concentrations are elevated and the metabolic clearance rate is reduced during exercise compared with placebo (22). Whole body lipid utilization in estradiol-supplemented men and amenorrheic women, as measured using stable isotope glycerol, does not change (20, 22). However, we observed a significant increase in whole body lipid oxidation after estradiol supplementation. Studies in rats show that estradiol has a major influence on lipid metabolism by increasing the bioavailability of fatty acids for oxidation by the exercising muscle (14, 17, 33, 35). Estradiol supplementation in male rats increased plasma free fatty acid and muscle triacylglycerol contents, reduced adipocyte LPL activity, and increased muscle LPL activity at rest and after exercise (14, 17). In addition, isolated rat fat cells treated with estradiol

FIG. 2. Plasma [13C]␣KIC/[12C]␣KIC enrichment for 12 men at rest and during 90 min of cycling at an intensity of 65% VO2max during placebo administration (PL; 䡺) or after 8 d of estrogen supplementation (ES; f). Data are the mean ⫾ SEM.

FIG. 3. Breath 13CO2/12CO2 enrichment for 12 men at rest and during 90 min of cycling at an intensity of 65% VO2max during placebo administration (PL; 䡺) or after 8 d of estrogen supplementation (ES; f). Data are the mean ⫾ SEM.



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TABLE 4. Whole body leucine metabolism in 12 men at rest and during 90 min of cycling at an intensity of 65% VO2max during placebo administration or after 8 d of estrogen supplementation Placebo

Flux (␮mol/kg䡠h)a Fraction of leucine oxidized (%)a,b Leucine oxidation (␮mol/kg䡠h)a,b Protein synthesis (mg protein/kg䡠h)a Protein breakdown (mg protein/kg䡠h)a Protein balance (mg protein/kg䡠h)a,b

Estrogen

Rest

Exercise

Rest

Exercise

144 ⫾ 5 19 ⫾ 1 28 ⫾ 2 196 ⫾ 7 231 ⫾ 8 ⫺34 ⫾ 4

137 ⫾ 6 45 ⫾ 3 62 ⫾ 5 126 ⫾ 8 219 ⫾ 10 ⫺93 ⫾ 8

145 ⫾ 9 16 ⫾ 1 23 ⫾ 2 206 ⫾ 14 233 ⫾ 15 ⫺26 ⫾ 3

129 ⫾ 6 41 ⫾ 3 53 ⫾ 4 129 ⫾ 10 205 ⫾ 10 ⫺76 ⫾ 7

Data are the mean ⫾ SEM (n ⫽ 12). Significance was determined by two-way repeated measures ANOVA. Protein synthesis was calculated from the NOLD. Protein synthesis and breakdown in milligrams of protein were calculated assuming leucine contributes 590 ␮mol/g protein. Protein balance is calculated as: protein synthesis ⫺ protein breakdown. a Main effect for exercise, P ⱕ 0.003. b Main effect for estrogen, P ⱕ 0.01.

showed a reduction in fatty acid synthesis and increased lipolysis (35). However, estradiol increased the activity of the liver lipogenic enzymes acetyl coenzyme A carboxylase and fatty acid synthetase, an action mediated at least in part by an increase in the insulin to glucagon ratio (33). In agreement with previously reported research, we found an increase in leucine oxidation during exercise (3, 4, 7, 24, 36). We also found a significant decrease in NOLD with exercise, an observation reported in recreationally active men and women exercising in the postabsorptive state (3, 7, 24, 36), but not in fed athletes (4). Training attenuates the increase in leucine oxidation observed during endurance exercise, but gender differences in leucine oxidation and flux are maintained throughout the training period (3). Similarly, 7 wk of endurance training in untrained men and women maintained the gender differences in substrate oxidation (2). Whether gender differences in protein metabolism disappear after intensified training in athletes is not known. Studies in mammalian models indicate that ␤-adrenergic receptors are major mediators of whole body substrate utilization. ␤-Adrenergic receptor agonist administration increases the net uptake of amino acids into the hindlimbs of steers and subsequent muscle protein accretion (37), deTABLE 5. The contribution to total energy expenditure due to CHO, lipid, and leucine oxidation at rest and during the last 30 min of exercise in 12 men during placebo administration or after 8 d of estrogen supplementation Treatment

Carbohydrate (%) Placebo Estrogen % Change Lipid (%) Placebo Estrogen % Change Protein (%)a Placebo Estrogenb % Change

Rest

Exercise

68 ⫾ 13 61 ⫾ 12 ⫺11

82 ⫾ 5 72 ⫾ 5 ⫺11

27 ⫾ 6 34 ⫾ 7 23

21 ⫾ 4 28 ⫾ 4 35

2.4 ⫾ 0.2 2.0 ⫾ 0.2 ⫺17

5.1 ⫾ 0.3 4.2 ⫾ 0.3 ⫺18

Data are the percentage of total energy expenditure and are presented as the mean ⫾ SEM (n ⫽ 11). Protein contribution to total energy expenditure was based on leucine oxidation assuming leucine contributes 590 ␮mol/g protein. Significance was determined by twoway repeated measures ANOVA. a Main effect for exercise, P ⬍ 0.0001. b Main effect for estrogen, P ⫽ 0.011.

creases semitendinosus muscle protein degradation, increases muscle protein content and reduces fat and collagen content in lambs (38), increases the accretion of proteins containing 3-methylhistidine (actin and myosin) through a decrease in the fractional breakdown rate and a reciprocal increase in the fractional synthetic rate in rats (39), increases muscle weight in rats (40), and decreases protein degradation in isolated, perfused rat muscle (41). Conversely, ␣- and ␤-adrenergic receptor blockade after injury in dogs inhibited skeletal muscle protein catabolism without affecting whole body nitrogen loss (42). In humans, gender differences in ␤-adrenergic regulation of substrate oxidation during exercise have been reported. Administration of ␤-adrenergic receptor blockers increases whole body leucine oxidation and protein catabolism and reduces free fatty acid mobilization at rest and during exercise and recovery (24, 43), with no change in catecholamine levels. This effect is mediated by both ␤1- and ␤2-receptors and is limited to exercising men, not women (24, 36). Men taking ␤-adrenergic receptor blockers also increase their whole body lysine Ra, indicating an increase in skeletal and nonskeletal (mainly hepatic) amino acid catabolism during exercise (36). Moreover, ␤-adrenergic receptor antagonists increase CHO oxidation and decrease lipolysis and lipid oxidation (23). Conversely, ␤-adrenergic receptor stimulation increases lipolysis and plasma free fatty acid concentration, but decreases fatty acid oxidation, with a concomitant increase in glycogen oxidation (23, 25). In the current study, the responses of substrate oxidation (decreased CHO and leucine oxidation and increased lipid oxidation) suggest that estradiol acts as a ␤-adrenergic receptor agonist. However, estradiol influences the signaling of both the ␣- and ␤-adrenergic receptors, albeit in a reciprocal manner (26, 27). Whereas estradiol decreases ␤-adrenergic receptor function in the estrogen-supplemented female rat brain (26), it increases ␣-adrenergic receptor signaling (27). With no change in total binding density, ␤-adrenergic receptor signaling is attenuated by uncoupling the receptor from the G protein, an action assumed to be due to receptor phosphorylation (26). Simultaneously, ␣1-adrenergic receptor signaling is enhanced due to increased expression of the IGF-I receptor, ensuring increased sensitivity to norepinephrine with no necessary elevation in its concentration (27). This may explain the absence of differences in norepinephrine concentrations between genders (2, 5, 8, 44) and after sup-

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plementation with estradiol in women (20, 45) and men (22) at rest and during moderate intensity endurance exercise. However, epinephrine concentrations in women have been previously reported to be similar to (8, 44) or lower at 90 min of moderate intensity endurance exercise than (2, 5) those in men. Moreover, epinephrine levels are similar after estradiol supplementation in women (20, 45) and men (22). Whole body protein economy at rest and during exercise improved after estradiol supplementation. Estimated protein balance became less negative, resulting in favorable protein retention equivalent to 8 mg protein/kg䡠h at rest and 17 mg protein/kg䡠h during exercise. Extrapolating the values calculated at rest to the nonexercise 22.5 h of the day (14.2 g protein) and assuming that, on the average, recreationally active people exercise for 90 min/d (2 g protein), whole body protein economy is enhanced by an amount equal to 16.2 g protein, equivalent to 16% of the daily North American dietary protein intake. This is corroborated by the reduction in the contribution of protein utilization to energy expenditure at rest (17%) and during exercise (18%).

10.

Conclusion

15.

We conclude that estrogen supplementation in men results in changes in substrate oxidation at rest and during 90 min of moderate intensity endurance exercise. During exercise, estrogen supplementation reduces RER, CHO and leucine oxidation, and estimated protein balance, whereas it increases lipid oxidation. This change in substrate oxidation is similar to the profile observed in women and is possibly mediated by the effects of estradiol on the sympathoadrenergic system.

16.

Acknowledgments We thank Christine Rodriguez, Brian Timmons, Laura Phillips, and Christopher Westbrook for assisting with the subjects, and Jennifer Day for analyzing the diet records. We thank Shire BioChem Inc., St. Laurent, Quebec, Canada, for providing the 17-␤-estradiol and Abbott Laboratories, Ross Division, St. Laurent, Quebec, Canada, for providing the Polycose. Received August 31, 2004. Accepted March 2, 2005. Address all correspondence and requests for reprints to: Dr. Mark A. Tarnopolsky, Room 4U4, Departments of Pediatrics and Medicine, McMaster University Medical Center, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. E-mail: [email protected]. This work was supported by the National Sciences and Engineering Research Council of Canada and the Hamilton Health Sciences Foundation. M.J.H. was recipient of the National Institute of Nutrition 2002– 2004 Postdoctoral Fellowship.

6. 7. 8. 9.

11. 12. 13.

14.

17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

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