Estradiol and Tamoxifen Reverse Ovariectomy-Induced Physical Inactivity in Mice


Medicine & Science in Sports & Exercise:
doi: 10.1249/01.mss.0000241649.15006.b8
BASIC SCIENCES: Original Investigations

Decreased physical activity and increased body mass are associated with estrogen deficiency.

Purpose: To determine whether estrogen or the estrogen analog, tamoxifen, could reverse those detrimental effects after surgical ovariectomy in mice.

Methods: Ten-week-old C57BL/6 mice were sham operated (sham, N = 6) or ovariectomized (OVX, N = 9). After 4 wk of voluntary wheel running, placebo (OVX-P) or 17β-estradiol (OVX-E2) pellets were implanted and the mice ran an additional 4 wk. A second study followed in which mice received placebo, 17β-estradiol, or tamoxifen (OVX-Tam) simultaneously with ovariectomies. Distances run per 24 h and body masses were analyzed by two-way ANOVA with repeated measures.

Results: During the initial 4 wk, OVX mice ran approximately 80% less and had approximately 20% greater body masses compared with sham mice (P < 0.001). Estradiol replacement quickly reversed the inactivity as OVX-E2 mice increased their running from 1.9 ± 0.3 km·24 h−1 to 6.9 ± 0.7 km within a week of replacement, which was equivalent to shams (8.1 ± 0.7 km), whereas OVX-P mice ran only 0.5 ± 0.2 km (P < 0.01). OVX-E2 mice tended to maintain body mass after estradiol replacement, whereas the OVX-P mice continued to increase mass. OVX mice that received tamoxifen had high running activity, approximately 9 km·24 h−1, and maintained body mass.

Conclusion: The removal of ovarian hormones caused mice to become inactive and gain body mass. Hormone therapy in the form of 17β-estradiol or tamoxifen rapidly stimulated voluntary wheel running and reversed body mass gains, indicating that estrogen receptor binding was involved in regulating physical activity.

Author Information

1Program in Physical Therapy; and 2School of Kinesiology, University of Minnesota, Minneapolis, MN

Address for correspondence: Dawn A. Lowe, MMC 388, 420 Delaware St. SE, Minneapolis, MN 55455; E-mail:

Submitted for publication March 2006.

Accepted for publication August 2006.

Article Outline

Physical activity is beneficial to overall health, including positive adaptations of the cardiovascular, neuromuscular, skeletal, and immune systems. Similarly, wheel or treadmill running by rodents results in favorable cardiac and skeletal muscle (1), skeletal (44), and immune adaptations (14). The majority of rodent studies on physical activity have used male rodents, but it was shown recently that C57BL/6J and FVB/NJ female mice outperform their male counterparts in terms of voluntary wheel running and forced treadmill running (18). However, this robust running activity of female rodents is dependent on physiological levels of ovarian hormones (13,27) and intact estrogen receptors (31). For example, 5 wk after the removal of ovaries, rats voluntarily ran 5 km·d−1, but another group of rats that received estradiol replacement at the time of ovariectomy ran 12 km (13). These data suggest that estradiol may be a key ovarian hormone in regulating physical activity in rodents.

How estrogens regulate physical activity in female rodents is not known. Estrogens function primarily by binding to estrogen receptors located in nuclei and cell membranes. Tissues that contain estrogen receptors and that might influence activity include skeletal and cardiac muscle and distinct areas of the central nervous system. As such, regulation may be through direct effects on muscle that, in turn, affect the animal's ability to perform physical activity. Alternatively, the central nervous system could be affected, which could theoretically influence the animal's motivation or willingness to run. An indirect way to get an idea of whether the estrogenic effect is attributable to muscular versus nervous system influences is to better delineate the time course of changes in wheel running activities, because changes in the muscular systems would likely take longer than would changes in the nervous system, as is seen with neuromuscular strength adaptations (28). Cross-sectional study designs have been used in previous studies of voluntary wheel running by ovariectomized rodents, and the time course of changes in running activity was not described (13,27). Thus, a unique aspect of our first study was the use of a longitudinal design that allowed us to precisely delineate the time it took for wheel running to decrease after the removal of ovarian hormones. A second unique aspect of our study was that, in addition to the longitudinal design, we also used a crossover design. That is, 4 wk after the removal of ovarian hormones, in the same group of mice, we determined the time course of increased wheel running in response to estradiol replacement.

Estradiol is also an antioxidant (25), and it is possible that it is this property that somehow influences physical activity. We indirectly investigated this possibility in a second study by using tamoxifen. Tamoxifen is a synthetic compound that works by binding to estrogen receptors, causing a conformational change in the receptor that is distinct from the estradiol-estrogen receptor conformation (24). We are not aware of any data indicating that tamoxifen has any antioxidant properties. Thus, if tamoxifen acts as an estrogen agonist in terms of maintaining physical activity in ovariectomized mice, it would suggest that estradiol influences activity by binding to estrogen receptors, not via its antioxidant property. Tamoxifen is best known for its use in conjunction with chemotherapy for the treatment of breast cancer, where it initially acts as an antagonist in breast tissue. In other tissues, however, it may act as an estrogen antagonist or agonist (23). Until our study, there was no evidence to indicate whether tamoxifen would act as an estrogen agonist or antagonist in relation to physical activity in rodents.

Studies on women have shown that estrogen therapy after menopause has no effect on physical activity (2) or a positive effect on physical activity (34). Obviously, the situation in women is much more complex, particularly because aging reduces physical activity. In our study, we have eliminated the influence of aging by using young mice, such that we directly investigated the effects of estrogenic agents, not aging. We are not aware of any data indicating whether tamoxifen influences the amount of physical activity performed by women. Whether the findings of our studies can be extrapolated to women is yet to be determined. Nonetheless, a better understanding of the mechanisms by which estrogenic agents regulate physical activity in mice will be a good step toward this goal.

Here, we report the effects of ovarian hormone removal, followed by replacement with an estrogenic compound, on voluntary wheel running by adult female C57BL/6 mice. Because body mass is influenced by physical activity and may also be influenced directly by estrogenic compounds, we also measured body mass weekly. We hypothesized that ovarian hormone-deficient mice would have decreased physical activity and increased body mass and that 17β-estradiol and tamoxifen replacement would reverse those effects. We also predicted that increases in heart and hindlimb muscle mass would occur in the hormone-treated mice, indicating beneficial exercise-training responses by mice that were physically active.

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Postpubertal female C57BL/6J mice purchased at 10 wk of age were used in this study. By the end of the studies, mice were less than 6 months old, which is far from the age at which the ovaries fail naturally in mice, that is, at 11-16 months of age (9). Initially, mice were housed four per cage on a 12-h light-dark cycle with the lights on from 6 a.m. until 6 p.m. Mice had free access to water and a phytoestrogen-free commercial rodent chow (2019 Teklad Global 19% Protein Rodent Diet, Harland Teklad, Madison, WI) before any intervention and throughout each study. Approximately 1 wk after the mice had arrived, they were housed individually, and body masses were measured weekly. All protocols and animal care procedures were approved by the institutional animal care and use committee and were in accordance with the policy statement of the American College of Sports Medicine on research with experimental animals.

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Surgical procedures.

Mice were anesthetized via inhalation of 1.75% isoflurane mixed with oxygen at a flow rate of 200 mL·min−1. A 37°C recirculating-water heating-pad was used to maintain body temperature. Under aseptic conditions, bilateral ovariectomy was performed through two small dorsal incisions between the iliac crest and the lower ribs. The abdominal muscle-wall incisions were closed with a single stitch using 6-0 silk suture, and skin incisions were closed with 7-mm wound clips. Approximately 5 min after the isoflurane anesthetic was withdrawn, each mouse was administered 0.15 μg of buprenorphine subcutaneously. Sham operations consisted of the same procedure as the ovariectomy procedure, except that the ovaries were not excised.

Sixty-day time-release pellets (Innovative Research of America, Sarasota, FL) were implanted subcutaneously via an approximately 3-mm incision on the dorsal aspect of the neck of anesthetized mice. Each estradiol pellet contained a total of 0.18 mg of 17β-estradiol so that mice received approximately 3 μg·d−1. This dosage was determined on the basis of previous studies of ovariectomized mice and time-released estradiol pellets (3,8,36) and our preliminary data. Our goal was to mimic the mean physiological level of circulating estradiol in young female mice, that is, approximately 20 pg·mL−1 plasma (30). Preliminary experiments were conducted by implanting estradiol pellets in a subset of female mice (N = 3), collecting blood samples at various time points after implantation, and measuring plasma estradiol levels (Estradiol EIA, DSL-10-4300, Diagnostic Systems Laboratories, Webster, TX). The mean ± SE estradiol level was 46 ± 4 pg·mL−1 plasma between 2 and 30 d after implantation, ranging from 24 to 61 pg·mL−1. Each tamoxifen pellet contained a total of 5 mg of tamoxifen that was released during 60 d. Placebo pellets contained the same matrix as the estradiol or tamoxifen pellets.

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Voluntary wheel running.

Exercise wheels were mounted to the tops of standard mouse cages (47 × 26 × 15 cm). The wheels were 11 cm in diameter, with a running surface of 2 inches (Prevue Mouse Wheel, Pets Warehouse, Copiague, NY). Each wheel had a digital magnetic counter attached that was interfaced to a microprocessor (PIC16F877A Development Board, Custom Computer Systems Inc., Brookfield, WI), which was capable of storing the number of revolutions per 24 h for 8 d.

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Study 1 design.

Mice were randomly assigned to one of three groups: control mice (sham), ovariectomized mice that received placebo (OVX-P), or ovariectomized mice that received estradiol (OVX-E2) (Fig. 1). Exercise wheels were placed in each cage at week 3 to allow mice to acclimate for 1 wk. At week 4, ovarian tissue was surgically removed from mice in two of the three groups (OVX-P, N = 5; OVX-E2, N = 4). The third group of mice underwent sham operations (sham; N = 6). After surgery, mice returned to their individual cages without wheels. At week 5, wheels were replaced and mice were given free access to the exercise wheels thereafter. Mice ran for approximately 4 wk, between weeks 5 and 9, before replacements took place (prereplacement period). Voluntary wheel running continued for 4 wk after pellets were implanted (postreplacement period). At week 13, mice were weighed and anesthetized by an intraperitoneal injection of sodium pentobarbital (100 mg·kg−1 body mass) with supplemental doses given as required. Soleus and extensor digitorum longus (EDL) muscles and the heart were excised, rinsed, blotted dry, weighed, immediately frozen in liquid nitrogen, and stored at −80°C. Mice were euthanized with an overdose of sodium pentobarbital (200 mg·kg−1 body mass).

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Study 2 design.

Mice were randomly assigned to one of three groups: ovariectomized mice that received estradiol (OVX-E2), ovariectomized mice that received placebo (OVX-P), and ovariectomized mice that received tamoxifen (OVX-Tam) (Fig. 2). Exercise wheels were placed in cages for approximately 10 d before surgery (presurgery period). Ovariectomy and pellet implants then occurred simultaneously in each mouse (OVX-E2, N = 5; OVX-P, N = 5; OVX-Tam, N = 6). Mice were given 3 d to recover before wheels were returned to the cages. Mice were then allowed to run voluntarily for approximately 6 wk (postsurgery period). At the end of this period, mice were weighed and anesthetized by an intraperitoneal injection of sodium pentobarbital (100 mg·kg−1 body mass) with supplemental doses given as required. Soleus and EDL muscles and the heart were excised, rinsed, blotted dry, weighed, immediately frozen in liquid nitrogen, and stored at −80°C. Blood was collected from these mice to assess circulating estradiol levels. The mean ± SE plasma estradiol levels for OVX-E2, OVX-P, and OVX-Tam mice were 47 ± 2, 7 ± 0.4, and 7 ± 1 pg·mL−1, respectively. Mice were euthanized with an overdose of sodium pentobarbital (200 mg·kg−1 body mass).

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Statistical analyses.

Two-way repeated-measure ANOVA with Tukey's post hoc tests were used to determine differences in wheel running and in body mass between groups over time. The repeated factor was time, and mean weekly wheel running was calculated and used in this analysis. One-way ANOVA with Tukey's post hoc tests were used to determine differences in final body mass and muscle masses between groups. SigmaStat version 3.0.1 (Systat Software Inc; Point Richmond, CA) was used for statistical analyses with an alpha level of 0.05. Values are reported as means ± SE.

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Study 1
Voluntary wheel running.

There was a significant interaction between group and time (P < 0.001), meaning that wheel running changed differently over time depending on estrogen status. Within the sham group, running was fairly stable over time, with the only differences occurring between week 6 and weeks 9, 12, and 13 (P ≤ 0.028; Fig. 3). Within the OVX-P group, there was no difference in weekly running at any time (P ≥ 0.810). As expected, within the OVX-E2 group, there were many significant differences over time, primarily between pre- and postreplacement weeks. Significant differences in running between groups at each time point are depicted in Figure 3. Overall, mice with low circulating levels of estradiol voluntarily ran considerably less than mice with normal levels. During the prereplacement period, ovariectomized mice ran approximately 80% less than sham mice. Estradiol replacement quickly reversed this inactivity, with the OVX-E2 mice increasing their running from 1.9 ± 0.3 km·d−1 during week 9 to 6.9 ± 0.7 km·d−1 during week 10, which was significantly more than OVX-P mice (Fig. 3). During weeks 11, 12, and 13, voluntary wheel running by the OVX-E2 mice was statistically equivalent to that of sham mice (6.7 ± 0.6 vs 8.3 ± 0.2 km, respectively), whereas the OVX-P mice continued to run very little (0.5 ± 0.01 km).

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Body, soleus, and EDL muscle and heart masses.

Mean body mass at the beginning of study 1 was 18.7 ± 0.3 g for all mice and did not differ between the three groups (P = 0.481). Again, there was a significant interaction between group and time (P < 0.001), indicating that body mass changed over time differently depending on estrogen status. All mice gained mass over time, but body mass of ovariectomized mice increased more than for sham mice during the prereplacement period (Fig. 4). During the postreplacement period, body mass of the OVX-P mice continued to increase to 16% above that during the prereplacement period, whereas body masses of sham and OVX-E2 mice changed from pre- to postreplacement by 3% and −5%, respectively, such that during weeks 11, 12, and 13, the OVX-E2 mice weighed less than the OVX-P mice and were not different from the shams (P ≤ 0.022; Fig. 4). At the end of the study, OVX-P mice had approximately 30% greater body mass than sham and OVX-E2 mice (Table 1).

At the end of the study, soleus muscle mass was 14% greater in the OVX-P mice compared with sham and OVX-E2 mice (Table 1). When normalized to body mass, soleus and EDL muscle masses of OVX-P mice tended to be lower than those of sham and OVX-E2 mice, but they were not statistically different. Heart mass and normalized heart mass of OVX-P mice were less that those of sham and OVX-E2 mice (Table 1).

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Study 2
Voluntary wheel running.

For daily running during the presurgery period, there was no interaction between group and time, indicating that all mice increased running by the same amount during wheel acclimation (Fig. 5; P = 0.089). Running did increase during the first 5 d as the mice acclimated to the wheels (P < 0.001). Overall, there was no difference in running distance between the three groups during the entire presurgery period (Fig. 5; P = 0.864). During the last 3 d of the presurgery period, mice ran an average of 8.4 ± 0.5 km.

The interaction between time and group (i.e., replacement treatment) was close to being statistically significant (P = 0.052) during the postsurgery period (including the final week of the presurgery period, depicted as week 5 on Fig. 5), suggesting that the change in running depended on whether ovariectomized mice received estradiol, placebo, or tamoxifen. There was a main effect of time during postsurgery (P < 0.001) with running during weeks 7, 8, 10, and 11 being different from running during week 5, and weeks 6 and 9 being different from week 11 (Fig. 5). The surgical removal of ovarian hormones and replacement with placebo caused a precipitous decline in voluntary wheel running, reaching a nadir at about week 9. The mean voluntary wheel running of OVX-P mice decreased 87% from peak levels during the presurgery acclimatization period. There was also a main effect of replacement treatment (P < 0.001). Ovariectomy with immediate replacement of estradiol or tamoxifen resulted in maintenance of high running activity during the postsurgery period that was not different between the two groups (P = 0.166) but that was significantly greater than that by the OVX-P mice (P ≤ 0.029; Fig. 5).

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Body, soleus, and EDL muscle and heart masses.

Body mass during the presurgery period was 21.5 ± 0.4 g for all mice and did not differ between the three groups (P = 0.733). During the postsurgery period, body mass gains over time depended on group, that is, what type of pellet the mice received (Fig. 6; interaction, P < 0.001). Body mass of OVX-P mice was 8% greater than for OVX-E2 and OVX-Tam mice at the end of the study (Fig. 6 and Table 2).

Soleus and EDL muscle masses, absolute and normalized to body masses, were not different between OVX-E2, OVX-P, or OVX-Tam mice (Table 2). Heart mass and heart mass normalized to body mass of OVX-P mice were less than those of the OVX-E2 and OVX-Tam mice (Table 2).

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The results from these studies show that mice voluntarily participate in physical activity significantly less after the loss of ovarian hormones. However, after these ovarian hormone-deficient mice received an estrogenic agent, physical activity quickly returned to normal levels. We investigated two different ovarian hormone-like agents on the basis of their prevalence in clinical use and their commercial availability: 1) estradiol, commonly used in hormone therapy for symptomatic menopausal women; and 2) tamoxifen, an estrogen analog that is used in breast cancer treatment. The commonality of these two agents is that both bind and affect estrogen receptors. Before this study, there was no indication of whether tamoxifen would affect physical activity. Our results clearly show that both of these agents acutely increase physical activity in ovariectomized female mice and that estrogen receptor binding mediates the effect.

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Study 1

Mice are athletic and run voluntarily when given the opportunity. In our first study, mice with circulating estradiol approximating physiological levels ran approximately 9 km·d−1. Mice that had low levels of estradiol because of surgical ovariectomy decreased their voluntary running to <1 km·d−1, similar to what has been shown previously in ovariectomized rodents (10,13,27,42). The unique aspect of our study is the demonstration that the drastic decline in running attributable to loss of ovarian hormones can be completely and quickly reversed in the same group of mice by restoring estradiol. The loss of ovarian hormones for 60 d in mature female mice results in decrements in skeletal muscle contractile function (26), but these effects likely did not occur in the short time span (< 1 wk) between ovariectomies and the low wheel running we observed in the present study. In other words, it is probable that removing and then replacing estradiol affected the willingness of the mice to run, not the ability of the mice to run based on skeletal or cardiac muscle function (see "Potential mechanisms" later in the Discussion). Overall, results from this study indicate that the lack of estrogen causes mice to rapidly become sedentary but that estrogen replacement can stimulate ovariectomized mice to resume voluntary wheel running within a few days.

A second measure of ovarian hormone loss and supplementation we examined was body mass. Ovariectomized mice had higher body masses during the prereplacement period compared with sham mice. The ovariectomized mice that later received estradiol stopped gaining body mass such that the ovariectomized, placebo-receiving mice had significantly greater body masses than both the sham and estradiol-receiving mice by the end of the study. Body mass of the estradiol-treated mice became statistically different from that of the placebo-treated mice at week 11, 1 wk later than the drastic change in wheel running by these mice, suggesting that physical activity may have influenced body mass. Whether these changes in body mass were entirely caused by physical activity, caused directly by hormonal effects, or caused by a combination of the two cannot be determined from our study. There is good evidence that estrogen directly affects adiposity (6) and that the loss of estrogen decreases the transport of the satiety factor, leptin, into the brain-impacting food intake and, ultimately, body mass (15). We did not measure food intake, so we do not know whether this contributed to the body mass gains, but it likely did. We speculate that the body mass gains we measured in ovariectomized mice resulted from both hormonal effects, including increased food intake, and reduced physical activity.

Additional measurements were taken on heart and hindlimb muscles as indices of endurance training. Mice that had high running activity (i.e., sham and OVX-E2 mice) had heart masses that were significantly greater than those of mice that ran little (OVX-P). An increase in the ratio of heart mass to body mass has previously been shown to occur in mice in response to voluntary endurance exercise and is interpreted as a beneficial cardiac muscle hypertrophic adaptation (1). This adaptation is not evident until after 2 wk of wheel running (1) and is an example of the relatively long period of time required for a muscle adaptation that would impact an animal's physical ability to perform exercise. Absolute soleus muscle mass was greater in OVX-P mice, but muscles undergo false hypertrophy by gaining nonprotein mass in response to ovariectomy (22,26), which likely explains this finding. Soleus and EDL muscles from mice that ran a lot tended to be larger than those muscles from mice that ran little when normalized to body mass, suggesting that a true muscle hypertrophic response to running did occur, as has been reported previously (43). This suggestion is supported by the significant positive correlations between mean daily distances run during the last week (i.e., week 13) and muscle masses normalized to body mass (soleus muscle, r = 0.521, P = 0.046; EDL muscle, r = 0.534, P = 0.041). Taken together, these data suggest that the quick transitions from running to nonrunning caused by the loss of ovarian hormones, and then the reversal caused by estradiol replacement, were not likely caused by immediate muscular adaptations, but that the downstream, longer-term beneficial effects of physical activity on muscle size were evident by the end of the study.

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Study 2

The results of our second study show that treatment with tamoxifen, a selective estrogen receptor modulator (SERM), maintained wheel-running activity in mice that had lost ovarian hormones. Thus, tamoxifen had effects on wheel-running activity that were similar to those of estradiol, indicating that it acts as an agonist in tissues related to physical activity. We are not aware of any other reports of tamoxifen affecting wheel-running activity in mice, or even in more general terms of an SERM affecting physical activity levels in rodents or women. These data also indicate that the mechanism by which estradiol affects physical activity in mice is through estrogen receptor binding, because this is the only known mode of action for tamoxifen. That is, the effects of estradiol do not seem to be through its antioxidant property.

The wheel-running response induced by tamoxifen displayed different characteristics than that induced by estradiol; essentially, the response seemed to be more robust with tamoxifen (Fig. 5). Ovariectomized mice that immediately received tamoxifen seemed to maintain higher running levels than mice that received estradiol, although the two groups were not statistically different. There are several possibilities that could explain the apparent difference. First, it is notable that during the last presurgery day, mice that were to receive tamoxifen ran approximately 11 km, whereas mice that were to receive estradiol ran only approximately 7 km, which could have influenced the early postsurgery running distances. Second, the daily dosage of tamoxifen released by the pellets was nearly 28 times greater than that of estradiol. We chose a relatively high dose based on a suggestion by the manufacturer and because tamoxifen binds estrogen receptors much weaker than does estradiol (4). However, despite the receptor binding difference, tamoxifen might have been more potent than estradiol because the half-life of tamoxifen is 5-7 d versus 18-24 h for estradiol (32). Any of these differences between the tamoxifen and estradiol groups could have contributed to the differential responses of the mice in terms of wheel-running activity. Nonetheless, the results do confirm that tamoxifen is an estrogen agonist in tissues regulating physical activity in mice.

We found that tamoxifen also acted like estrogen by preventing ovariectomized mice from increasing body mass, similar to what has been shown in ovariectomized rats (11,35). As in study 1, whether this maintenance of body mass was a pharmacological result, was attributable to the greater wheel running of the tamoxifen-treated mice, or was attributable to a combination of those two factors cannot be ascertained. Likewise, a marker of an endurance-training adaptation (i.e., increased heart mass) was observed in the tamoxifen-treated mice that had high running activity, but the differential effects of treatment versus physical activity cannot be determined from this study.

A potential limitation to our studies is that the estradiol pellets we selected resulted in circulating levels of estradiol about twice as high as normal levels in mice (i.e., approximately 40 pg·mL−1 plasma instead of approximately 20 pg·mL−1). We initially based the estradiol dosage on previous studies of ovariectomized mice treated with estradiol via time-release pellets (36). We contend that our results are physiological, as opposed to pharmacological, because supraphysiological 17β-estradiol treatment for mice used to elicit pharmacological manifestations are in the range of 250-600 pg·mL−1 plasma (20), and the circulating estradiol levels in our mice were far from that. In addition, estradiol levels can reach 40-50 pg·mL−1 during proestrus (38).

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Potential mechanisms of altered running activity by estradiol and tamoxifen.

Others have reported that rodent activity levels decrease with removal of ovarian hormones and have suggested possible mechanisms. Kadi and coworkers (13) showed that myosin heavy chain composition of hindlimb muscle fibers was altered when rats reduced wheel-running activity after the loss of ovarian hormones for 5 wk and that myosin heavy chain isoform composition was maintained when rats were given estradiol at the time of ovariectomy. Thus, it was suggested that the effects of estradiol on myosin heavy chain isoform expression might underlie changes in physical activity levels in mice. However, changes in myosin heavy chain isoform composition cannot explain the quick transition between running and nonrunning (and vice versa) in our studies because those isoform changes take at least 2 wk to occur in response to wheel running in mice (1). Therefore, a more direct mechanism such as an estrogenic effect on the central nervous system influencing the animal's willingness to run is more likely the cause of the rapid changes in physical activity.

Several brain areas regulate physical activity in rodents, including forebrain regions such as the medial preoptic area, hypothalamic paraventricular nucleus, lateral hypothalamic area and nucleus accumbens, and the midbrain ventral tegmental area (7,37,41). Neuromodulators such as dopamine, orexin A, and nerve growth factors are connected to physical activity in rodents, and they affect activity acutely, that is, within minutes to hours when injected into specific areas of the brain (17,39,41). Dopamine in the midbrain is associated with reward-centered activities, and behavioral studies show that wheel running in rodents is rewarding (19). In addition, dopamine has connections with estrogenic compounds (5,21). Another neuromodulator, pancreatic polypeptide, also has been shown to affect physical activity (29). Thorburn and Proietto (40) suggest a complicated pathway beginning with estradiol binding to estrogen receptors in the medial preoptic area of the hypothalamus, causing increased nitric oxide, leading to increased pancreatic polypeptide levels, and ending with increased physical activity. Conversely, the lack of estradiol would theoretically result in low levels of pancreatic polypeptide and cause a decrease in wheel running. Either of these mechanisms, as well as others, could apply to studies 1 and 2 because both estradiol and tamoxifen bind to estrogen receptors. From our studies, we cannot deduce the specific cause of the changes in physical activity associated with estrogenic compounds, but we suggest that it is an estrogenic effect on a central nervous system structure and corresponding neuromodulator, not a change in some peripheral structure such as skeletal muscle.

Even less is known about how tamoxifen specifically may affect the central nervous system, so postulating its mechanism of action in regards to regulating physical activity in rodents is even more tentative. Patisaul et al. (32) reported that tamoxifen has a mix of estrogenic agonistic and antagonistic actions in the female rat brain depending on (a) the presence or absence of estrogen, (b) the specific area of the brain in question, and (c) the estrogen receptor isoform (α or β) expressed by cells in that area of the brain. We speculate that the high wheel running of our tamoxifen-treated mice was attributable to agonistic effects of tamoxifen in areas of the brain associated with physical activity. For example, the hypothalamus is a potential area of importance because it is involved in estrogen-related regulation of physical activity in rodents (37), and tamoxifen has an agonistic effect in this area when estrogen is absent (32).

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Estrogen and its effects on physical activity in women.

There is a wide array of conclusions related to the topic of estrogen and physical activity in women. For example, it has been reported that both short-term (2) and long-term (16) estrogen therapy does not affect physical activity, whereas other studies found that estrogen therapy improved physical activity relative to postmenopausal women not on therapy (33,34). Another approach for determining whether estrogen status affects women is to look at normal fluctuating estrogen levels during the menstrual cycle in relation to physical activity. Johnson and coworkers (12) did this and found no correlation between circulating estrogen levels during the menstrual cycle and the amount of energy expended during exercise, concluding that there is no effect of estrogen on physical activity. It is not certain whether (or how) the drastic estradiol-induced changes in physical activity in mice could be extrapolated to women. The data do indicate, however, that the mouse model would be better for detecting the underlying mechanism, because the effects are so robust and consistent in mice.

Overall, our results show relationships between estrogen receptor binding agents, physical activity, and body mass in mice. Again, it is not certain whether there are relationships between these variables in women, particularly postmenopausal women. Estrogen therapy is a common and effective treatment for menopausal symptoms and preventing bone-density loss. If, at the same time, hormone therapy were to increase physical activity in these women, it is likely that menopausal symptoms, bone mineral density, and cardiovascular function, lipid metabolism, and mental health would benefit both from the direct pharmacological effects of the drug and from the increased physical activity. More research is needed to confirm this theory.

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We have shown that both 17β-estradiol and tamoxifen restore wheel-running activity and maintain body mass in young mice that have lost ovarian hormones via ovariectomy. Both of these agents function by binding to estrogen receptors, and this estrogen receptor binding property seems to mediate the effects on physical activity. We speculate that this occurs in some central nervous system structure, causing the large changes in physical activity. Estrogen effects are most commonly studied in skeletal and female reproductive tissues, but our results are important for the basic understanding of estrogenic effects on additional tissues. Our overall results also indicate a potential benefit of hormone therapy (i.e., increased physical activity). In addition, when investigating the effects of estrogenic compounds on molecular and cellular functions in ovariectomized rodents, it should be taken into account that significant changes in physical activity are likely occurring. These changes in physical activity could secondarily contribute to alterations of molecular and cellular functions, especially in estrogen receptor-containing tissues that are modulated by physical activity, such as heart and skeletal muscle.

This research was supported by grants from NIH (AG20990 and AG025861) to DAL and ALM was supported by a training grant from NIH (T32 AR07612).

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