Division of Clinical Pharmacology and Metabolic Research, Department of Medicine, University of Vermont, Burlington, Vermont 05405
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ABSTRACT |
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Prior studies
suggest that estradiol and progesterone regulate body composition in
growing female rats. Because these studies did not consider the
confounding effect of changes in food intake, it remains unclear
whether ovarian hormones regulate body composition independently of
their effects on food intake. We utilized a pair-feeding paradigm to
examine the effects of these hormones on body composition. In addition,
skeletal muscle protein fractional synthesis rate and adipose tissue
lipoprotein lipase activity were measured to examine pathways of
substrate deposition into fat and fat-free tissue. Female
Sprague-Dawley rats [pubertal: 7-8 wk old; 190 ± 0.5 (SE)
g] were separated into four groups: 1) sham-operated (S;
n = 8), 2) ovariectomized plus placebo (OVX;
n = 8), 3) ovariectomized plus estradiol
(OVX+E; n = 8), and 4) ovariectomized plus
progesterone (OVX+P; n = 8). All ovariectomized groups
were pair-fed to the S group. Body composition was measured using total
body electrical conductivity. The relative increase in fat-free mass
was greater (P < 0.01) in the OVX group (31 ± 2%) than in the S (17 ± 2%), OVX+E (18 ± 2%), and OVX+P
(22 ± 2%) groups. The fractional synthetic rates of
gastrocnemius muscle protein paralleled changes in fat-free mass: OVX
had a higher (P < 0.05) synthesis rate (21 ± 3%/day) than S (12 ± 2%/day), OVX+E (11 ± 2%/day), and
OVX+P (8 ± 1%/day) groups. Body fat increased in the S group
(31 ± 7%; P < 0.01), whereas the OVX groups
lost fat (OVX: 10 ± 7%; OVX+E:
15 ± 7%; OVX+P:
13 ± 7%). No differences in lipoprotein lipase were found. Our
results suggest that estradiol and progesterone may regulate the growth
of fat and fat-free tissues in female rats. Moreover, ovarian hormones
may influence skeletal muscle growth through their effects on skeletal
muscle protein synthesis.
skeletal muscle; adipose tissue; flooding dose
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INTRODUCTION |
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OVARIAN HORMONES REGULATE body composition in female rats (28, 33). The removal of ovarian hormones by ovariectomy causes an increase in both body lipid and protein content (28, 33). Replacement of estradiol after ovariectomy prevents the increase in body lipid and protein content (28), whereas replacement of progesterone does not alter these changes (28, 33). The effects of ovarian hormones on body composition are thought to be derived primarily from changes in energy intake. In support of this notion, Richard (28) demonstrated that ovariectomy promotes positive energy imbalance by stimulating food intake and that estradiol replacement prevents these changes. Further studies have shown that ovarian hormones regulate food intake through direct effects on neuronal pathways (8). These results suggest that estradiol and progesterone regulate body composition primarily through their effects on feeding behavior.
Ovarian hormones have been shown to affect the metabolism of organ and tissue components of body composition independently of their effects on energy intake. For example, previous studies have shown that ovariectomy stimulates and estradiol replacement inhibits adipose tissue lipoprotein lipase (LPL) (14, 16, 18, 27, 35), the rate-limiting enzyme controlling the hydrolysis of circulating triglycerides and their uptake into adipocytes (11). Although changes in LPL activity are likely due to changes in energy intake that accompany ovariectomy (17, 19, 30), ovarian hormones have been shown to affect LPL independently of food intake (14, 27). Over time, these changes in LPL activity would be expected to promote changes in body fat levels by altering the uptake and storage of fat in adipocytes. Thus it is plausible to hypothesize that ovarian hormones affect body composition independently of changes in food intake.
To examine the effects of ovarian hormones on the regulation of body composition, we measured body composition in growing female rats before and after ovariectomy with or without hormone replacement. All ovariectomized animals were pair-fed to sham-operated rats to control for the effects of ovarian hormones on food intake. This experimental paradigm permits the examination of the effects of estradiol and progesterone on the growth of fat and fat-free tissue independent of the confounding effects of changes in food intake. In addition, we examined metabolic pathways controlling the entry of energy substrates into body fat and protein (i.e., skeletal muscle) compartments by measuring adipose tissue LPL activity and skeletal muscle protein synthesis, respectively.
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MATERIALS AND METHODS |
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Animals. Female Sprague-Dawley rats weighing 175-200 g (7-8 wk old) were purchased from Taconic (Germantown, NY) and were housed singly in wire-bottom cages. Rats were maintained on a 12:12-h light-dark cycle in a temperature-controlled (21.1 ± 0.2°C) room. Tap water and chow (20% protein, 6% fat, 74% carbohydrate; Harlan-Teklad) were available ad libitum before initiation of the study. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Vermont and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, Washington, DC).
Protocol.
Rats were divided into four groups: sham-operated (S), ovariectomized
plus placebo (OVX), ovariectomized plus estradiol (OVX+E), and
ovariectomized plus progesterone (OVX+P). Four days after their
delivery to the laboratory, baseline body composition was measured by
total body electrical conductivity with animals under methoxyflurane
anesthesia. Ovariectomy was conducted on the following morning with
animals under acepromazine (2.5 mg/kg) and ketamine (75 mg/kg)
anesthesia. Placebo, 17-estradiol (0.1 mg), or progesterone (4-pregnene-3,20-dione; 50 mg) pellets (all pellets were 21-day release; Innovative Research of America, Sarasota, FL) were placed subcutaneously in the subscapular region. These pellets produce plasma
17
-estradiol levels between 10 and 30 pg/ml and progesterone levels
between 10 and 20 ng/ml (personal communication, Innovative Research of
America). Buprenorphine was administered subcutaneously (0.025 mg/kg)
directly after surgery and every 12 h thereafter for 36 h.
Body composition. Fat mass and fat-free mass were measured by total body electrical conductivity by use of a small animal body composition analyzer (model SA-2, EM-SCAN, Springfield, IL). During the measurement, animals were under light anesthesia (methoxyflurane). The average of four measurements was used to calculate fat-free mass (g) from the following equation: 22.69 + (0.1185 × E0.513) L1.42, where E is the electrical conductivity measure, and L is the nasoanal length (cm). Body fat was calculated by subtracting fat-free mass from body mass. Previous studies have shown that body composition estimated using electrical conductivity correlates closely with body composition derived from densitometry, total body water, and chemical analysis (2).
LPL activity. Heparin-releasable adipose tissue LPL activity was determined with the method of Taskinen et al. (31). Adipose tissue (30 mg) was placed in Krebs Ringer-phosphate buffer containing heparin. Eluates were incubated with a substrate mixture of 14C-labeled and unlabeled triolein in a lecithin-Tris-albumin buffer emulsified by ultrasound. Pooled human plasma was used as a source of apo-CII to stimulate LPL activity. The reaction was carried out at 37°C for 45 min. The resulting free fatty acids were isolated by the Belfrage extraction (1). Recovery of radioactivity is >95% in our laboratory. The intra-assay and interassay coefficients of variation were 4.5 and 12.1%, respectively.
Skeletal muscle protein synthesis.
A sample of gastrocnemius muscle tissue (~50 mg) was homogenized in
solubilization buffer (100 mM sodium pyrophosphate, 1% SDS, and 4 mM
EGTA, pH 7.4). The resulting homogenate was centrifuged. The
supernatant was decanted, treated with ice-cold 10% TCA, allowed to
precipitate overnight (14 h), and then centrifuged. The precipitate was washed with petroleum ether, the ether was evaporated under N2, 6 M HCl was added, and the tube was capped and heated
for 24 h at 110°C. The acid and water from the protein
hydrolysate were removed by drying under N2. The sample was
reacidified with 1 M acetic acid. Amino acids were isolated by ion
exchange chromatography and derivatized to their N-acetyl,
n-methyl esters (NAM) using a modification of a procedure
previously described (21). NAM-derivatized amino acids
were reconstituted in ethyl acetate. NAM-phenylalanine isotopic
enrichment (mole percent excess) was measured by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Plasma amino acids were derivatized to their
N-heptafluorobutyryl, n-propyl, or HFBP,
derivative and then measured by GC-MS, as previously described
(22). We used plasma phenylalanine enrichment as a proxy
measure of the enrichment of the precursor pool for protein synthesis.
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Statistical analysis. Analysis of variance was used to examine differences among groups. If a significant group effect was found, a Duncan's multiple range test was used to identify the location of differences among groups. Because differences in baseline body size and composition measures were found, variables were expressed as a relative change from their baseline preintervention value by subtracting the preintervention value from the postintervention value, dividing by the preintervention value, and multiplying by 100. All data are expressed as means ± SE, unless otherwise specified.
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RESULTS |
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Nasoanal length, body mass, fat mass, and fat-free mass data at
baseline (i.e., before surgery) are shown in Table
1. Nasoanal length was lower
(P < 0.05) in the OVX+E group compared with all other
groups. No differences in body mass or fat mass were found. Fat-free
mass was greater (P < 0.05) in OVX+P compared with OVX and OVX+E groups.
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Because differences in baseline body size and composition measures were
found, variables were expressed as a relative change from their
baseline presurgery value (Fig. 1).
Relative body mass gain was higher (P < 0.05) in the S
(20 ± 2%) and OVX (23 ± 1%) groups compared with the
OVX+P (14 ± 1%) group and was higher (P < 0.05)
in the OVX+P compared with the OVX+E (9 ± 1%) group. The
relative increase in body mass for S animals was similar to growth
rates reported by the supplier for rats of similar strain, sex, and
size over a 16-day period (personal communication; Taconic). Fat mass
increased (P < 0.01) in the S (30 ± 5%) group
but decreased in all OVX groups (OVX: 13 ± 6%; OVX+E:
13 ± 5%; OVX+P:
10 ± 5%). The increase in fat-free
mass was greater (P < 0.01) in the OVX (34 ± 2%) group compared with S (19 ± 4%), OVX+E (16 ± 2%),
and OVX+P (20 ± 2%) groups. No differences in the relative change in nasoanal length were found among groups (S: 5.4 ± 1.1%; OVX: 5.9 ± 0.4%; OVX+E: 4.3 ± 0.5%; OVX+P:
4.1 ± 0.7%; data not shown in Fig. 1). Similar group effects
were found when body mass and composition were expressed on an absolute
(g) basis. The increase in body mass was greater (P < 0.01) in S (38 ± 4 g) and OVX (43 ± 2 g) compared
with OVX+P (27 ± 2 g) and was greater (P < 0.05) in OVX+P compared with OVX+E (16 ± 2 g). The increase
in fat mass was greater (P < 0.01) in S (11 ± 2 g) compared with OVX (
6 ± 2 g), OVX+E (
6 ± 3 g), and OVX+P (
4 ± 4 g). The increase in fat-free
mass was greater (P < 0.01) in OVX (48 ± 2 g) than in all other groups (S: 27 ± 6 g; OVX+E: 22 ± 2 g; OVX+P: 31 ± 4 g).
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No differences in LPL activity were found among S (3.00 ± 0.8),
OVX (3.15 ± 0.6), OVX+E (2.68 ± 0.53), and OVX+P (2.55 ± 0.4 µmol · h1 · g
tissue
1) groups.
The fractional synthesis rates of gastrocnemius muscle protein are
shown in Fig. 2. Differences in protein
fractional synthetic rate paralleled differences in fat-free mass among
groups. Fractional synthesis rates were higher (P < 0.01) in the OVX (21 ± 3%/day) group compared with S (12 ± 2%/day), OVX+E (11 ± 2%/day), and OVX+P (8 ± 1%/day)
groups. In addition, fractional synthesis rates were higher
(P < 0.05) in the S compared with the OVX+P group. Protein synthesis rates in S rats compare well with data from rats at a
similar developmental stage (13).
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DISCUSSION |
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The primary goal of this study was to examine the effects of estradiol and progesterone on body composition in growing female rats. Our pair-feeding paradigm allowed us to examine the role of estradiol and progesterone in the regulation of fat and fat-free components of body mass without the confounding effect of changes in energy intake. We found that 1) OVX prevents fat accumulation, and neither estradiol nor progesterone replacement can restore the normal pattern of fat gain; 2) OVX stimulates the accumulation of fat-free mass, whereas both estradiol and progesterone replacement inhibit the growth of fat-free tissue; and 3) removal of ovarian hormones by OVX is associated with greater, and replacement of either estradiol or progesterone is associated with lower, rates of skeletal muscle protein synthesis. Collectively, our results suggest that ovarian hormones play an important role in regulating the deposition of energy substrates into adipose and nonadipose tissues in growing female rats and that these effects are independent of changes in food intake. In addition, this is the first study to demonstrate a role for estradiol and progesterone in the modulation of skeletal muscle protein metabolism.
Fat mass and LPL activity. Body fat increased in the S group but decreased in all OVX groups (Fig. 1). Our results differ from previous studies showing that ovariectomy and ovariectomy with progesterone replacement increased body fat and that estradiol replacement prevented these changes (28, 33). Changes in body fat noted in previous studies, however, are largely due to the effects of ovarian hormones on energy intake (28). Our results suggest a different role for estradiol and progesterone in the regulation of adiposity. Specifically, removal of ovarian hormones by OVX prevents normal fat accumulation, and neither estradiol nor progesterone replacement alone can recover normal patterns of fat accumulation. Our findings differ, however, from those of Guyard et al. (15), who found similar relative increases in fat and fat-free mass in OVX and OVX+E rats by use of a pair-feeding paradigm. It should be noted, however, that Guyard et al. used a higher dose of estradiol than that used in the present study. Moreover, the highly palatable diet employed by Guyard et al. caused a significant increase (27%) in energy intake. Differences in the hormone replacement dose and dietary conditions may partially explain differing results among studies.
Adipose tissue contains the largest store of oxidizable energy substrates in the body. Moreover, it is the primary source for the hormone leptin (37). The availability of both oxidizable energy substrates and circulating leptin concentrations has been postulated to control reproductive function (4, 34, 36). Thus estradiol and progesterone may partially regulate reproductive function indirectly through their effects on body fat. The mechanism through which ovarian hormones regulate body fat stores remains unclear. LPL is the rate-limiting enzyme controlling the hydrolysis and uptake of circulating triglyceride fatty acids into adipocytes (11) and has been shown to partially regulate the accumulation of body fat in growing rats (17). Interestingly, however, we found no differences in parametrial adipose tissue LPL activity among groups. Our findings differ from previous studies showing that ovariectomy increases and estradiol replacement decreases adipose tissue LPL activity (16, 18, 35). However, because rats were allowed to feed ad libitum in these prior studies, alterations in LPL activity were likely due to changes in energy intake, given that feeding is a potent stimulus for adipose tissue LPL (19, 30). Ramirez (27) showed that the aforementioned effects of ovarian hormones on LPL precede and, therefore, occur independently of changes in food intake. The changes in LPL noted by Ramirez, however, were acute, occurring one day after estradiol administration. To our knowledge, the present study is the first to investigate the prolonged (>2 wk) effect of ovariectomy with or without hormone replacement on adipose tissue LPL activity while controlling for changes in energy intake by use of a pair-feeding design. Interestingly, adipose tissue LPL activity did not reflect group differences in the relative change in fat mass. This may be explained by the fact that LPL activity was measured in only one adipose tissue depot. However, prior work suggests that developmental changes in LPL activity are remarkably similar among various fat depots (7). Moreover, estradiol has been shown to have similar effects on LPL activity in several depots (27). Thus, if parametrial LPL activity is representative of other adipose tissue depots, our results suggest that ovarian hormones may regulate fat mass through other mechanisms. Recently, several lines of evidence suggest that triglyceride synthesis is a stronger predictor of lipid accumulation in adipocytes than LPL activity (5, 26). Thus ovarian hormones may regulate adipose tissue accumulation by affecting triglyceride synthesis (5) or other processes that control adipose tissue lipid balance (e.g., lipolysis).Fat-free mass and skeletal muscle protein synthesis. The relative increase in fat-free mass was greater in the OVX group compared with all other groups (Fig. 1). Our results agree with previous studies showing that ovariectomy potentiates the growth of fat-free tissue, whereas replacement of estradiol prevents the increase in fat-free mass (28). In contrast to our findings, previous studies have shown that replacement of progesterone does not prevent the ovariectomy-induced increase in fat-free mass (28). However, as with changes in adiposity, it is unclear whether alterations in fat-free mass observed in prior studies are due to the effects of the hormones, per se, or alterations in energy intake (23). Our findings suggest that, independently of changes in food intake, ovariectomy potentiates and estradiol or progesterone replacement inhibits the growth of fat-free mass.
Skeletal muscle is the largest and most malleable component of fat-free mass. Under normal physiological conditions, changes in skeletal muscle protein levels are primarily determined by changes in skeletal muscle protein synthesis (25). The fractional synthesis rate of gastrocnemius muscle protein was greater in the OVX group compared with all other groups (Fig. 2), suggesting that the removal of ovarian hormones stimulated, and/or replacement of either estradiol or progesterone inhibited, muscle protein synthesis. The marked differences in fractional synthesis rates among groups is probably a function of the developmental stage of the rat studies. During puberty, skeletal muscle fractional synthesis rate is high and decreases dramatically with maturation (13). Hormonal factors that regulate skeletal muscle development, especially those that contribute to sex differences in body composition, such as estradiol and progesterone, would be expected to have pronounced effects on skeletal muscle protein synthesis at this time. Because differences in skeletal muscle protein synthesis paralleled differences in the relative change in fat-free mass, our findings suggest that estradiol and progesterone regulate the growth of fat-free mass by altering skeletal muscle protein synthesis. Ovarian hormones may be acting directly on skeletal muscle (9, 10) or on other physiological or hormonal systems (12, 32) to regulate skeletal muscle protein metabolism. To our knowledge, this is the first study to show that ovarian hormones may partially regulate skeletal muscle protein metabolism. Although early work by Santidrian and Thompson (29) showed that estradiol decreased myofibrillar protein breakdown, as indicated by a reduction in 3-methylhistidine excretion, it is unclear whether these changes were due to estradiol, per se, or changes in energy intake. Moreover, 3-methylhistidine excretion may not be an accurate indicator of skeletal muscle protein breakdown (24). Indeed, a reduction in myofibrillar protein breakdown, which would be expected to potentiate skeletal muscle growth, is in direct contrast to the attenuated growth of fat-free mass observed in estradiol-replaced animals in the present study. Several caveats to our findings should be noted. First, although we controlled energy intake using a pair-feeding design, we cannot discount the possibility that energy expenditure changed in response to the interventions. However, studying female Sprague-Dawley rats of similar age and weight as those used in the present experiment, Richard (28) showed that neither ovariectomy nor hormone replacement affected energy expenditure. Thus any change in energy expenditure was likely minimal. Second, because LPL activity was measured at one time point (i.e., 16 days after surgery), differences in the change in adiposity may have resulted from alterations in LPL that occurred before our measurement. Serial measurements of LPL activity are needed to clarify its role in the regulation of ovarian hormone-induced changes in adiposity. Third, replacement of estradiol and progesterone by use of continuous-release pellets does not mimic the cyclical secretion of estradiol and progesterone that occurs in vivo. Prior studies show that changes in body composition are similar in sham-operated rats and those replaced with both estradiol and progesterone (28), suggesting that constant release of hormones does not alter changes in body composition. No data are available, however, regarding the effect of constant hormone release on adipose tissue LPL activity or skeletal muscle protein synthesis. Fourth, steroid hormone-induced changes in body water could influence body composition measurements if the hydration of fat-free mass were altered. We do not believe this to be the case. Prior studies show no effect of steriod hormones on body water content or the hydration of lean tissue (6, 20). Finally, the effect of estradiol and progesterone on body composition may be age specific. We studied pubertal animals (i.e., 7-8 wk old). The effects of ovarian hormones may be specific to this period of rapid growth with its attendant hormonal milieu. Preliminary studies suggest, however, that similar effects of ovarian hormones on fat and fat-free mass occur in mature (>320 g) female rats (3). Thus our findings may reflect a general action of estradiol and progesterone on the regulation of body composition in female rats. In conclusion, our results show that estradiol and progesterone control the partitioning of energy into fat and fat-free tissue in growing female rats. Specifically, removal of ovarian hormones by ovariectomy prevents normal fat accumulation, and replacement of either estradiol or progesterone is insufficient to promote fat gain. Moreover, ovariectomy potentiates the growth of fat-free tissue, whereas replacement of either estradiol or progesterone alone negatively modulates fat-free mass accretion. No effect of either estradiol or progesterone on adipose tissue LPL activity was found. In contrast, ovariectomy is associated with greater, and replacement of either estradiol or progesterone with lower, rates of skeletal muscle protein synthesis. The possibility exists that these effects of estradiol and progesterone on body composition in growing female rats may partially account for sex-related differences in body composition. ![]() |
ACKNOWLEDGEMENTS |
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We thank Susan Malley, Smitha Kizhake, and Walter DeNino for their technical expertise, and Drs. Galbraith, Cipolla, and Rocca for their helpful comments.
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FOOTNOTES |
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This work was supported by grants from the National Institutes of Health (AM-02125 and AG-13978 supplement to M. J. Toth) and the University of Vermont General Clinical Research Center (RR-00109).
Address for reprint requests and other correspondence: M. J. Toth, Given Bldg. C-247, Univ. of Vermont, Burlington, VT 05405.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 26 July 2000; accepted in final form 27 November 2000.
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