Effects of estradiol and progesterone on body composition, protein synthesis, and lipoprotein lipase in rats

Michael J. Toth, Eric T. Poehlman, Dwight E. Matthews, André Tchernof, and Michael J. MacCoss

Division of Clinical Pharmacology and Metabolic Research, Department of Medicine, University of Vermont, Burlington, Vermont 05405


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, 17beta -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 17beta -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.

S animals were permitted ad libitum access to food and water for the next 16 days. Food intake was monitored by weighing the amount of chow (powdered to facilitate food intake measurement) consumed over a 24-h period. The average daily food intake for the S group (n = 8 rats) was fed to the three OVX groups for 16 days after surgery. The average daily food intake during the 16-day study period was similar among S (19 ± 3 g), OVX (18 ± 1 g), OVX+E (18 ± 1), and OVX+P (18 ± 0.5 g) groups. On the afternoon of day 16, body composition was measured with animals under methoxyflurane anesthesia.

On day 17, skeletal muscle protein measurements were performed, and adipose tissue was collected. Rats were anesthetized using acepromazine (2.5 mg/kg) and ketamine (75 mg/kg). Approximately 10 min after injection of anesthesia, [ring-13C6]phenylalanine (5 mg/100 g body wt; 99% 13C; Cambridge Isotope Laboratories, Andover, MA) combined with unlabeled phenylalanine (32 mg/100 g body wt; Sigma Chemical, St. Louis, MO) was injected via a tail vein. Exactly 10 min later, rats were decapitated, blood was collected, and the right hindlimbs were stripped of skin, removed, and placed in ice-cold saline. The gastrocnemius muscle was dissected, blotted dry, and frozen in liquid nitrogen. Parametrial adipose tissue was dissected, blotted dry, and frozen in liquid nitrogen. All samples were kept at -80°C until analysis.

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.

The fractional synthesis rate (ks) of mixed skeletal muscle protein was calculated as
k<SUB>s</SUB><IT>=</IT><FR><NU>E<SUB>M</SUB><IT>·100·1,440</IT></NU><DE><IT>t·</IT>E<SUB>p</SUB></DE></FR>
where EM is the enrichment of phenylalanine in mixed muscle protein, 100 is a constant to convert ks to a percentage, 1,440 is a constant to convert minutes to days, t is the incorporation time in minutes, and EP is the enrichment of plasma phenylalanine; ks is expressed in units of percentage per day.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Baseline body size and composition in growing female rats

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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Fig. 1.   Changes in body mass, fat mass, and fat-free mass expressed as a % of baseline values. Sham-operated animals, solid bars; ovariectomized (OVX) animals, open bars. a, P < 0.05 vs. OVX + estradiol (OVX+E) and OVX + progesterone (OVX+P) groups; b, P < 0.05 vs. OVX+E group; c, P < 0.01 vs. all OVX groups; d, P < 0.01 vs. SHAM, OVX+E, and OVX+P groups.

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 · h-1 · 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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Fig. 2.   Differences in fractional synthesis rate (FSR) of gastronemius muscle protein. Sham-operated animals, solid bars; OVX animals, open bars. a, P < 0.01 vs. SHAM, OVX+E, and OVX+P groups; b, P < 0.05 vs. OVX+P group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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

We thank Susan Malley, Smitha Kizhake, and Walter DeNino for their technical expertise, and Drs. Galbraith, Cipolla, and Rocca for their helpful comments.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Belfrage, P, and Vaughan M. Simple liquid-liquid partition system for isolation of labeled oleic acid from mixtures with glycerides. J Lipid Res 10: 341-344, 1969[Abstract/Free Full Text].

2.   Bracco, EF, Yang MU, Segal K, Hashim SA, and Van Itallie TB. A new method for estimation of body composition in the live rat. Proc Soc Exp Biol Med 174: 143-146, 1983[Abstract].

3.   Callés-Escandon, J, and Malley S. Estrogen replacement decreases lean body mass accretion, preserves fat mass gain and decreases fat oxidation. Obes Res 7: 128S, 1999.

4.   Chehab, FF, Lim ME, and Lu R. Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet 12: 318-320, 1996[ISI][Medline].

5.   Cianflone, K. The acylation stimulating protein pathway: clinical implications. Clin Biochem 30: 301-312, 1997[ISI][Medline].

6.   Clark, RG, and Tarttelin MF. Some effects of ovariectomy and estrogen replacement on body composition in the rat. Physiol Behav 28: 963-969, 1982[ISI][Medline].

7.   Cryer, A, and Jones HM. The development of white adipose tissue. Effect of litter size on the lipoprotein lipase activity of four adipose tissue depots, serum immunoreactive insulin and tissue cellularity during the first year of life in male and female rats. Biochem J 186: 805-815, 1980[ISI][Medline].

8.   Dagnault, A, and Richard D. Involvement of the medial preoptic area in the anorectic action of estrogens. Am J Physiol Regulatory Integrative Comp Physiol 272: R311-R317, 1997[Abstract/Free Full Text].

9.   Dahlberg, E. Characterization of the cytosolic estrogen receptor in rat skeletal muscle. Biochim Biophys Acta 717: 65-75, 1982[ISI][Medline].

10.   Dionne, FT, Lesage RL, Dubé JY, and Tremblay RR. Estrogen binding proteins in rat skeletal and perineal muscles: in vitro and in vivo studies. J Steroid Biochem 11: 1073-1080, 1979[ISI][Medline].

11.   Eckel, RH. Lipoprotein lipase: a multifunctional enzyme relevant to common metabolic diseases. N Engl J Med 320: 1060-1068, 1989[Abstract].

12.   Edén, S. Age- and sex-related differences in episodic growth hormone secretion in the rat. Endocrinology 105: 555-560, 1979[ISI][Medline].

13.   Garlick, PJ, Maltin CA, Baillie AGS, Delday MI, and Grubb DA. Fiber-type composition of nine rat muscles. II. Relationship to protein turnover. Am J Physiol Endocrinol Metab 257: E828-E832, 1989[Abstract/Free Full Text].

14.   Gray, JM, and Greenwood MRC Time course of effects of ovarian hormones on food intake and metabolism. Am J Physiol Endocrinol Metab 243: E407-E412, 1982[Abstract/Free Full Text].

15.   Guyard, B, Fricker J, Brigant L, Betoulle D, and Apfelbaum M. Effects of ovarian steroids on energy balance in rats fed a highly palatable diet. Metabolism 40: 529-533, 1991[ISI][Medline].

16.   Hamosh, M, and Hamosh P. The effect of estrogen on the lipoprotein lipase activity of rat adipose tissue. J Clin Invest 55: 1132-1135, 1975[ISI][Medline].

17.   Hietanen, E, and Greenwood MRC A comparison of lipoprotein lipase activity and adipocyte differentiation in growing male rats. J Lipid Res 18: 480-490, 1977[Abstract].

18.   Kim, HJ, and Kalkhoff RK. Sex steroid influence on triglyceride metabolism. J Clin Invest 56: 888-896, 1975[ISI][Medline].

19.   Linder, C, Chernick SS, Fleck TR, and Scow RO. Lipoprotein lipase and uptake of chylomicron triglyceride by skeletal muscle of rats. Am J Physiol 231: 860-864, 1976[ISI][Medline].

20.   Lobo, MJ, Remesar X, and Alemany M. Effect of chronic intravenous injection of steroid hormones on body weight and composition of female rats. Biochem Mol Biol Int 29: 349-358, 1993[ISI][Medline].

21.   Matthews, DE, Ben-Galim E, and Bier DM. Determination of stable isotopic enrichment in individual plasma amino acids by chemical ionization mass spectrometry. Anal Chem 51: 80-84, 1979[ISI][Medline].

22.   Matthews, DE, Pesola G, and Campbell RG. Effect of epinephrine on amino acid and energy metabolism in humans. Am J Physiol Endocrinol Metab 258: E948-E956, 1990[Abstract/Free Full Text].

23.   McNurlan, MA, and Garlick PJ. Influence of nutrient intake on protein turnover. Diabetes Metab Rev 5: 165-189, 1989[ISI][Medline].

24.   Millward, DJ, Bates PC, Grimble GK, Brown JG, Nathan M, and Rennie MJ. Quantitative importance of nonskeletal-muscle sources of N'-methyl-histidine in urine. Biochem J 190: 225-228, 1980[ISI][Medline].

25.   Millward, DJ, Garlick PJ, Nnanyelugo DO, and Waterlow JC. The relative importance of muscle protein synthesis and breakdown in the regulation of muscle mass. Biochem J 156: 186-188, 1976.

26.   Olivecrona, T, and Bengtsson-Olivecrona G. Lipoprotein lipase and hepatic lipase. Curr Opin Lipidol 1: 222-230, 1990.

27.   Ramirez, I. Estradiol-induced changes in lipoprotein lipase, eating, and body weight in rats. Am J Physiol Endocrinol Metab 240: E533-E538, 1981[Abstract/Free Full Text].

28.   Richard, D. Effects of ovarian hormones on energy balance and brown adipose tissue thermogenesis. Am J Physiol Regulatory Integrative Comp Physiol 250: R245-R249, 1986[Abstract/Free Full Text].

29.   Santidrian, S, and Thompson JR. Effect of estradiol benzoate on the rate of myofibrillar protein degradation in growing ovariectomized female rats. Arch Pharmacol Toxicol 7: 215-221, 1981.

30.   Tan, MH, Sata T, and Havel RJ. The significance of lipoprotein lipase in rat skeletal muscles. J Lipid Res 18: 363-370, 1977[Abstract].

31.   Taskinen, MR, Nikkila M, Huttunen JK, and Hilden H. A micromethod for assay of lipoprotein lipase activity in needle biopsy samples of human adipose tissue and skeletal muscle. Clin Chim Acta 104: 107-117, 1980[ISI][Medline].

32.   Vikman-Adolfsson, K, Oscarsson J, Nilsson-Ehle P, and Edén S. Growth hormone but not gonadal steroids influence lipoprotein lipase and hepatic lipase activity in hypophysectomized rats. J Endocrinol 140: 203-209, 1994[Abstract].

33.   Wade, GN, and Gray JM. Gonadal effects on food intake and adiposity: a metabolic hypothesis. Physiol Behav 22: 583-593, 1979[ISI][Medline].

34.   Wade, GN, and Schneider JE. Metabolic fuels and reproduction in female mammals. Neurosci Biobehav R 16: 235-272, 1992[ISI][Medline].

35.   Wilson, DE, Flowers CM, Carlile SI, and Udall KS. Estrogen treatment and gonadal function in the regulation of lipoprotein lipase. Atherosclerosis 24: 491-499, 1976[ISI][Medline].

36.   Yura, S, Ogawa Y, Sagawa N, Masuzaki H, Itoh H, Ebihara K, Aizawa-Abe M, Fujii S, and Nakao K. Accelerated puberty and late-onset hypothalamic hypogonadism in female transgenic skinny mice overexpressing leptin. J Clin Invest 105: 749-755, 2000[Abstract/Free Full Text].

37.   Zhang, Y, Proenca R, Maffei M, Barone M, Leopold L, and Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 372: 425-432, 1994[ISI][Medline].


Am J Physiol Endocrinol Metab 280(3):E496-E501
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