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Original Article |
Department of Medicine, Division of Gerontology, University of Maryland School of Medicine, and the Geriatric Research, Education and Clinical Center, Baltimore VA Medical Center, Baltimore, Maryland 21201
Address all correspondence and requests for reprints to: Cynthia Ferrara, Ph.D., Baltimore VA Medical Center, VAMC/GRECC BT/18/GR, 10 North Greene Street, Baltimore, Maryland 21201. E-mail: . cferrara{at}grecc.umaryland.edu
Abstract
Changes in adipose tissue metabolism may contribute to the changes in body fat distribution seen during the menopause transition. We compared in vitro abdominal and gluteal sc adipose tissue metabolism [basal and stimulated lipolysis and activity of adipose tissue lipoprotein lipase (AT-LPL)] in postmenopausal and perimenopausal women (n = 12/group), matched for race, body mass index (29.5 ± 3.8 kg/m2; mean ± SD), and percentage body fat (42 ± 6%). The postmenopausal women were older (54 ± 3 vs. 48 ± 3 yr; P < 0.01) and had higher FSH (55.5 ± 26.4 vs. 16.6 ± 22.5 IU/ml; P < 0.01) and lower estradiol (33.8 ± 14.9 vs. 97.4 ± 61.7 pmol/liter; P < 0.05) concentrations than the perimenopausal women. Despite similar fat cell size and ß-adrenergic receptor and postreceptor (dibutyryl-cAMP)-stimulated lipolysis, basal lipolysis was 77% lower in gluteal adipose cells from postmenopausal compared with perimenopausal women (P < 0.05). Within each group, AT-LPL activity in the gluteal region was significantly higher than in the abdominal region (P < 0.05). In addition, AT-LPL activity was significantly higher in the postmenopausal compared with perimenopausal women in both gluteal (4.9 ± 3.6 vs. 2.0 ± 1.4 nmol free fatty acid/g·min; P < 0.05) and abdominal (3.2 ± 2.6 vs. 1.3 ± 0.9 nmol free fatty acid/g·min; P < 0.05) adipose cells. The results of this study suggest that menopause status is associated with differences in adipose tissue metabolism in both the abdominal and gluteal fat depots. The lower lipolysis and higher AT-LPL activity in postmenopausal women may predispose them to gain body fat after menopause.
PERIMENOPAUSE, DEFINED AS the 2- to 8-yr period preceding menopause and the 1 yr after the final menses (1), is characterized by irregular menstrual cycles with increased periods of amenorrhea (2), declining ovarian function, and variable estradiol and FSH levels (3). Changes in body composition, including an increase in total adiposity and a redistribution of fat to the abdominal region (4, 5) also occur during the transition to menopause. Although it is well established that menopause results in changes in body fat distribution, the biological mechanisms contributing to this change are not known.
Changes in adipose tissue metabolism may significantly contribute to the changes in body fat distribution seen during the menopause transition. Adipose tissue lipoprotein lipase (AT-LPL), responsible for the hydrolysis of circulating triglyceride into free fatty acids (FFAs) for uptake and storage by adipose tissue, plays a major role in accumulation and distribution of fat stores (6). Previous studies show higher AT-LPL activity in femoral adipose cells compared with abdominal or mammary adipose cells in premenopausal women (7) or postmenopausal women on hormone replacement therapy (8, 9), but no regional differences in AT-LPL activity in postmenopausal women not on hormone replacement therapy (7, 10). Although prior studies show there are no differences in AT-LPL activity in premenopausal compared with postmenopausal women (7, 11), it is not known whether AT-LPL activity differs in perimenopausal compared with postmenopausal women.
Menopause status may also affect adipose tissue lipolysis, which, in turn, could contribute to changes in body composition. Rebuffe-Scrive et al. (7) observed a higher lipolytic response and sensitivity in abdominal and mammary compared with femoral adipose cells in premenopausal women, but not in postmenopausal women. We also showed a greater lipolytic response and sensitivity in abdominal compared with gluteal adipose cells in postmenopausal women (10). Whether there are regional differences in the lipolytic sensitivity and responsiveness of the abdominal and gluteal adipose cells in perimenopausal women is not known.
Previous studies examining the effects of menopause status or estradiol levels on adipose tissue metabolism have compared premenopausal and postmenopausal women who were not matched for race, body composition, or the phase of the menstrual cycle, factors that may affect adipose tissue metabolism (7, 11). In addition, studies have not clearly identified those women who may be perimenopausal or addressed how changes in hormone levels may affect adipose tissue metabolism in perimenopausal women. The purpose of this investigation was to compare gluteal and abdominal AT-LPL activity and basal and stimulated lipolysis in perimenopausal compared with postmenopausal women matched for race and body composition.
Subjects and Methods
Subjects
Twelve perimenopausal women were recruited to participate in the study. On the basis of previous research (2), the present study used documentation of menstrual cycle irregularities and symptoms of the menopause transition to determine perimenopausal status. Irregular menstrual cycles were defined as a variable amount of time between menstrual periods (compared with a 28-d cycle), or missed periods during the past 6 months. In addition, five of the perimenopausal women also reported having hot flashes, and four women reported having heavier flow during periods or shorter periods than before. FSH levels were not used to define perimenopause, because levels can fluctuate from month to month during perimenopause, which limits their utility as a predictor (1, 12). Twelve postmenopausal women (no menstruation for at least 1 yr, FSH > 30 IU/ml), who had been previously studied in our laboratory, were matched to the perimenopausal women for race (four African-American and eight Caucasian), weight, body mass index (BMI), and percentage body fat. All women were sedentary (exercising no more than 20 min twice a week), weight stable (<2.0 kg weight change in the past year), and had not smoked for at least 5 yr. None of the women were on birth control pills, hormone replacement therapy, or medications affecting lipid metabolism. Menstruating perimenopausal women were tested in the follicular phase (d 4–8) of the menstrual cycle.
All women provided informed consent to participate in the study according to the guidelines of the University of Maryland Institutional Review Board for Human Research and underwent initial screening evaluations, which included a medical history, physical examination, fasting blood profile, and 12-lead resting electrocardiogram. Women with evidence of diabetes (fasting plasma glucose level > 6.4 mM), hypertension (blood pressure higher than 140/90 mm Hg), hyperlipidemia (triglyceride 
400 mg/dl), heart disease, cancer, liver, renal, or hematological disease were excluded.
Body composition measurements
Waist and hip circumferences were measured in duplicate. The waist-to-hip ratio (WHR) was calculated as the ratio of the minimal waist circumference to the circumference at the maximal gluteal protuberance. All participants received a total body scan using dual energy x-ray absorptiometry (DXA, Model DPX-L, Lunar Corp., Madison, WI) to determine percentage body fat, fat mass, and fat-free mass (FFM, total body bone mineral content plus lean tissue mass). A single-slice computed tomography scan taken midway between L4 and L5 was performed using a PQ6000 scanner (Marconi Medical Systems, Cleveland, OH) to measure visceral adipose tissue area and sc abdominal adipose tissue area.
Hormone assays
Plasma and serum were stored at -70 C for subsequent hormone analysis. All determinations were performed in duplicate. FSH was measured in serum using an immunoradiometric assay (Diagnostic Products Corp., Los Angeles, CA). Estradiol (ultra sensitive), free testosterone, and SHBG were measured in serum using an immunoradiometric assay (Diagnostic Systems Laboratories, Inc., Webster, TX). Insulin was measured in serum using a radioactive assay (Linco Research, Inc. St. Louis, MO). The intra- and interassay coefficients of variation (CV) for the measurement of estradiol were 7.5% and 9.4%, CV of free testosterone were 5.0% and 8.3%, and CV of SHBG were 2.0% and 8.3%, respectively.
Adipose cell metabolism
Nutrient intake was controlled for 2 d before the fat biopsy by providing each subject with a weight-maintaining diet composed of 50–55% carbohydrate, 15–20% protein, and 30% fat with 300–400 mg of cholesterol and a polyunsaturated to saturated ratio of 0.6 to 0.8. After an overnight fast, 2–3 g of sc adipose tissue were obtained under local anesthesia (1% xylocaine) from both the abdominal and gluteal regions by aspiration with a 16-gauge needle. Adipose cells were isolated using a modification of the Rodbell method, as previously described (13, 14).
AT-LPL activity and lipolysis assay
Heparin-releasable lipoprotein lipase (LPL) activity was measured as previously described (10). AT-LPL activity was expressed as nanomoles of FFA produced per gram of tissue, or nanomoles of FFA per 106 cells in 1 min. Glycerol released from adipose cells was used as the index of lipolysis, because it is not reused by the adipose cell (15). Pharmacological agents were added just before the beginning of the incubation to stimulate lipolysis. After 2 h, the reaction was stopped with perchloric acid, and the glycerol concentration was measured in the infranatant using an enzymatic fluorometric technique (16). The lipolytic response of isolated adipose cells was tested with isoproterenol, a ß-adrenergic agonist, and with dibutyryl 3', 5' cyclic monophosphate (db-cAMP, 2 mmol/liter), a nonhydrolyzable analog of cAMP. Variations in adenosine metabolism have a major influence on the measurement of lipolysis (17, 18); therefore, in experiments measuring the stimulatory effect of isoproterenol, adenosine deaminase (ADA) was added to remove adenosine present in the medium. However, to control for the activation of lipolysis after adenosine removal, which might prevent or attenuate the response to isoproterenol, isoproterenol stimulation of lipolysis was evaluated in the presence of 1 U/ml ADA and 100 nmol/liter N6-(1,2-phenylisopropyl)-adenosine, a potent adenosine receptor agonist that is neither a substrate nor an inhibitor of ADA, as shown previously (19). Lipolysis was expressed as micromoles of glycerol per 106 cells/2 h. The maximal lipolytic effect, or responsiveness, was calculated as the difference between basal glycerol released and the glycerol released at the maximally effective concentration of the lipolytic agent. The concentration of isoproterenol giving half-maximal response (EC50), an index of sensitivity, was obtained by computer fitting of individual dose-response curves to isoproterenol using Sigma-Plot software (Jandel Scientific, Chicago, IL).
Statistics
All data analyses were completed using Statview for Windows (SAS Institute, Inc., Cary, NC). Statistically significant differences in body composition and hormone concentrations between perimenopausal and postmenopausal women were determined by unpaired t tests. Statistically significant differences in adipose cell metabolism were determined by a 2 x 2 ANOVA, with group x adipose tissue depot as the independent variables. Statistically significant differences between groups or depots were then determined by post hoc t tests. In addition, the dose response curves were analyzed using ANOVA (group x adipose tissue depot x dose of isoproterenol). Pearson correlation coefficients were calculated between selected variables and LPL activity. All data are presented as mean ± SD, with the level of significance set at P less than 0.05 for all analyses.
Results
Subject characteristics
The postmenopausal women (50–59 yr of age) were older than the perimenopausal women (44–55 yr of age) and were on average 5.8 ± 3.3 yr postmenopause (range, 1–11 yr; Table 1
). By design, there were no differences in body weight, BMI, or percentage body fat between the two groups of women. In addition, fat mass, FFM, WHR, sc and intra-abdominal fat areas did not differ between the two groups (Table 1). As expected, FSH levels were lower (P < 0.001) and estradiol levels were higher (P < 0.05) in the perimenopausal compared with the postmenopausal women (Table 2). There were no differences in SHBG, free testosterone, or insulin levels in the perimenopausal compared with the postmenopausal women (Table 2).
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There were no differences in gluteal or abdominal fat cell size (micrograms of triglyceride per cell) in the perimenopausal compared with the postmenopausal women (Table 3). Basal lipolysis in the gluteal adipose cells was higher (P < 0.05) in the perimenopausal women compared with the postmenopausal women. There were no differences in the sensitivity (EC50) or in the maximal lipolytic response to the ß-adrenergic agonist isoproterenol or in the maximal lipolytic capacity, measured by the db-cAMP-stimulated response between the perimenopausal and postmenopausal women (when basal lipolysis was subtracted; Table 3). However, when basal lipolysis was not subtracted, isoproterenol-stimulated lipolysis in the gluteal adipose cells was significantly higher in the perimenopausal compared with the postmenopausal women (4.0 ± 2.4 vs. 2.3 ± 1.9 µmol glycerol/106 cells·2 h; P < 0.05). The isoproterenol dose response curves for both fat depots were compared within each subject and between groups. As expected, there was a dose effect, but no interaction between dose and group, dose and depot, or dose, group and depot. There were no regional differences in basal or stimulated lipolysis between the abdominal and gluteal fat depots in either group.
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). In addition, within each group, the LPL activity in the gluteal region was significantly higher than LPL activity in the abdominal region (P < 0.05; Fig. 1). When expressed per 106 cells, LPL activity in the abdominal region was significantly lower in the perimenopausal compared with the postmenopausal women, but did not reach statistical significance in the gluteal region (P = 0.2). In addition, LPL activity per 106 cells remained higher in the gluteal compared with the abdominal region within each group (P < 0.05).
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Discussion
The results of this study suggest that menopause status, i.e. perimenopause compared with early postmenopause, is associated with significant differences in sc adipose tissue metabolism in both the abdominal and gluteal fat depots. The lower basal lipolysis in gluteal adipose cells and the higher LPL activity in both the gluteal and abdominal adipose cells in the postmenopausal compared with the perimenopausal women may predispose them to gain body fat after menopause.
To our knowledge, this is the first investigation to examine the effects of menopause status on AT-LPL activity in perimenopausal and postmenopausal women matched for race, body weight, and percentage body fat, thus controlling for the effects of obesity on LPL activity. Our results suggest that menopause status is associated with significant differences in LPL activity in both the abdominal and gluteal fat depots. Previous studies comparing abdominal AT-LPL activity in premenopausal compared with postmenopausal women observed no difference between the two groups (7, 11). The postmenopausal women in the present study were in early menopause; thus, although they were significantly older than the women in the perimenopausal group, the difference was on average 6 yr compared with 15–20 yr in previous studies (7, 11). In the present investigation, all of the perimenopausal women were studied during the follicular phase. In previous investigations, premenopausal women were studied in luteal phase (11) or at any point during the menstrual cycle (7). Because the hormone levels are different during the menstrual cycle, this may also contribute to the metabolic differences observed in the present study compared with previous studies.
We observed higher AT-LPL activity in the gluteal compared with the abdominal region in both perimenopausal and postmenopausal women. This conflicts with previous research from our laboratory (10), in which AT-LPL activity was similar in the two fat depots in postmenopausal women. The women in the present study were perimenopausal or in early menopause (1–11 yr postmenopause), whereas the women in our previous study were older (51–67 yr) and were between 1 and 31 yr postmenopause. It is possible that regional differences in AT-LPL activity in the women in the present study may be related to the perimenopausal or early menopause status.
The physiological mechanisms contributing to higher AT-LPL activity in the postmenopausal compared with the perimenopausal women are currently unknown. Although changes in hormone levels, in particular, estradiol, may be a likely explanation, the present study observed no relationship between AT-LPL activity and estradiol levels. This conflicts with the results of Iverius and Brunzell (20), who observed a significant negative relationship between AT-LPL activity and estradiol levels in women 22–66 yr of age. The difference between the two studies may be related to the wide range of age and estradiol levels of the women in the Iverius and Brunzell study compared with those in the present study. In addition, there is conflicting evidence as to whether hormone replacement therapy or sc estrogen patches, which may elevate estrogen levels, affect AT-LPL activity (8, 9, 21, 22). In our study, there was no relationship between AT-LPL activity and other sex hormones. Circulating insulin levels affect AT-LPL levels and activity (6). In the present study, we observed no relationship between insulin concentrations and LPL activity in the two groups of women. Thus, the mechanisms contributing to the changes in AT-LPL activity with menopause require further investigation in a longitudinal study.
To our knowledge, the present study is the first investigation to describe the differences in adipose tissue lipolysis between perimenopausal and postmenopausal women. We found that gluteal adipose tissue lipolysis under basal conditions was higher in the perimenopausal compared with the postmenopausal women despite similar responsiveness and sensitivity to a ß-adrenergic agonist and responsiveness to a postreceptor agonist, when the rate of basal lipolysis was subtracted. Interestingly, when basal lipolysis was not subtracted, the isoproterenol-stimulated rate of lipolysis in gluteal adipose cells was higher in the perimenopausal compared with postmenopausal women, suggesting that the menopause transition may also affect isoproterenol-stimulated lipolysis. Previous studies examining the effects of menopause status and estrogen levels on adipose tissue lipolysis have reported conflicting results. Rebuffe-Scrive et al. (7) observed higher isoproterenol-stimulated lipolysis in the abdominal and mammary depots in premenopausal compared with the postmenopausal women, although estrogen replacement (with or without progesterone) in the same postmenopausal women had no effect on lipolysis. In contrast, estradiol treatment decreased norepinephrine-stimulated lipolysis in the abdominal depot in postmenopausal women (8). Jensen et al. (23) also observed that in postmenopausal women, estrogen deficiency increased whole body FFA release compared with estrogen treatment. Although these investigators (23) did not measure adipose tissue lipolysis directly, they suggest that the increase in FFA release in the postmenopausal women not on estrogen therapy is related to an increase in adipose tissue lipolysis. Unlike the studies of Rebuffe-Scrive et al. (7) and Lindberg et al. (8), we studied postmenopausal and perimenopausal women and matched our subjects for race and overall body fat. In addition, unlike the study of Jensen et al. (23), we measured in vitro adipose tissue lipolysis. These differences in study design may contribute to the differences in the results of this study compared with previous investigations.
In conclusion, the results of the present study suggest that menopause status is associated with significant differences in sc adipose tissue metabolism in both the abdominal and gluteal fat depots. The lower basal lipolysis and higher LPL activity in the postmenopausal women may predispose these women to gain body fat after menopause.
Acknowledgments
We thank all of the subjects who volunteered and the nursing, dietary, and laboratory staff at the Geriatric Research, Education and Clinical Center at the Baltimore VA Medical Center for assistance with research studies. We also thank Ellen M. Rogus, Ph.D., for expert assistance and Andrew P. Goldberg, M.D., for expert advice and support.
Footnotes
Present address for B.J.N.: Section on Gerontology and Geriatric Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157.
This work was supported by a T32 AG00219 training grant; National Institutes of Health Grants R29 AG14066-02, K01 AG00747, K01 AG00685, and R01 AG00608; a VA Merit Review Entry Program grant; and the Department of Veterans Affairs Baltimore Geriatric Research, Education and Clinical Center.
Abbreviations: ADA, Adenosine deaminase; AT-LPL, adipose tissue LPL; BMI, body mass index; CV, coefficient(s) of variation; db-cAMP, dibutyryl cAMP; FFA, free fatty acid; FFM, fat-free mass; LPL, lipoprotein lipase; WHR, waist-to-hip ratio.
Received December 18, 2001.
Accepted June 3, 2002.
References
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