HEALTHY AGING AND THE ENDOCRINE ENVIRONMENT: THE ASSOCIATION BETWEEN THE ENDOCRINE ENVIRONMENT AND BODY COMPOSITION IN POSTMENOPAUSAL WOMEN

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1 HEALTHY AGING AND THE ENDOCRINE ENVIRONMENT: THE ASSOCIATION BETWEEN THE ENDOCRINE ENVIRONMENT AND BODY COMPOSITION IN POSTMENOPAUSAL WOMEN by AMY K. MISKIMON BARBARA A. GOWER, COMMITTEE CHAIR M. AMANDA BROWN BETTY E. DARNELL ROBERT A. OSTER A THESIS Submitted to the graduate faculty of the University of Alabama at Birmingham, in partial fulfillment of the requirements for the degree of Master of Science BIRMINGHAM, ALABAMA 2009

2 HEALTHY AGING AND THE ENDOCRINE ENVIRONMENT: THE ASSOCIATION BETWEEN THE ENDOCRINE ENVIRONMENT AND BODY COMPOSITION IN POSTMENOPAUSAL WOMEN AMY K. MISKIMON CLINICAL NUTRITION ABSTRACT It is well known that with menopause, comes endocrine disturbances and changes in body composition. These menopause-related changes in hormonal parameters and body composition are thought to increase disease risk and perhaps predispose this population to functional impairment. The objective of this study was to investigate the relationships between hormone concentrations and measures of body composition both cross-sectionally and longitudinally. We hypothesized that estrogen concentrations would be inversely associated with intra-abdominal adipose tissue (IAAT) and positively associated with thigh fat both cross-sectionally and with change over time. Additionally, we hypothesized that androgen concentrations would be positively associated with measures of skeletal muscle both cross-sectional and with change over time. All hormone variables were assessed at baseline in 84 postmenopausal women and at a 2- year follow-up in 55 postmenopausal women, 50 of which used hormone replacement therapy (HRT) and the remaining 34 did not use HRT. Skeletal muscle and fat masses were assessed with dual-energy X-ray absorptiometry (DXA), and thigh muscle, thigh fat, and IAAT areas with computed tomography (CT). Partial correlation analysis showed that estradiol and estrone were positively associated with thigh fat area adjusted for total fat mass in non- ii

3 HRT users. HRT users lost ASM over the two year period and also had lower free testosterone than the non-hrt users. Free testosterone was positively associated with ASM and leg lean mass adjusted for total fat mass in HRT users at baseline. Stepwise multiple linear regression analyses did not show any significant hormone predictor variables for change in skeletal muscle and fat distribution over two years. In conclusion, estradiol and estrone may play a role in preferential fat distribution to the thigh region in postmenopausal women. Additionally, in HRT users, free testosterone may be limiting for lean mass. Therefore, we can conclude that because HRT use decreases free testosterone, it may, in fact, be detrimental to maintenance of lean mass in postmenopausal women. ii

4 ACKNOWLEDGMENTS Thank you to everyone that contributed to this thesis. I am overwhelmed by the support, guidance, and patience of everyone involved and I cannot thank you all enough. Therefore, I would like to acknowledge all of the individuals that helped to bring me to this point of completing this thesis project. I would like to give a huge thank you to my committee chair and mentor, Dr. Barbara Gower. Without her guidance and patience, this project would not have been possible. Her vast knowledge of all things endocrine and passion for research in this field are truly inspiring. I have learned more in our Wednesday meetings than in any class I have ever taken. Something I have learned about her, in these two years, is that her passion for research is driven by a desire to change lives for the better, and I feel honored to be able to study under such a remarkable person. I would also like to give a very special thank you to Betty Darnell, my supervisor and mentor at the PCIR. Her constant encouragement and support gave me the confidence I needed to press on and stay on track. Through tears and exciting times, Betty was always there to listen and comfort, and for that, I cannot thank her enough. Thank you to Dr. Amanda Brown, who brought me to Birmingham for the dietetic internship, which marked the beginning of my excitement for nutrition research. Her support, leadership, and belief in me helped to iii

5 bring me where I am today. Thank you to Dr. Robert Oster, our PCIR statistician. His statistical guidance and knowledge helped to make this project a success. Another special thank you goes to the wonderful friends I work with at the PCIR. Suzanne, Nikki, and Jessica have been there for me, through every up and down associated with this experience. They have taught me a lot, been there to answer my many questions, stayed late nights at the office to offer help, and above all they make every day at work fun with their wonderful sense of humors. These girls mean the world to me, and it is my only hope that one day I can return the favor. Finally I would like to acknowledge and thank my family. First of all my wonderful loving and supportive husband, who encouraged me on a daily basis and always was there to listen to me practice a presentation. His words of wisdom comforted me and resonated with me throughout this process. Also thank you to my parents who were 100 percent supportive in every decision I have made. Their love, advice and support molded who I am today and I could not have had two better examples of people that follow their dreams and do it with passion. I love you all very much. iv

6 TABLE OF CONTENTS Page ABSTRACT... ii ACKNOWLEDGMENTS... iv LIST OF TABLES... vii LIST OF FIGURES... viii LIST OF ABBREVIATIONS... ix INTRODUCTION...1 Hypothesis Specific Aims...3 Hypothesis Specifics Aims...4 REVIEW OF LITERATURE...5 Menopause and Sex Hormones...5 Overview...5 Estrogen...5 Estrogen Receptors...7 Androgens...7 Hormone metabolism...9 Hormone Replacement Therapy...10 Menopause and Body Composition...10 Weight and total fat...10 Fat distribution...11 Estrogen and fat metabolism...13 HRT and fat distribution...14 v

7 Aging and Muscle...15 Overview...15 Sarcopenia...16 HRT and change in muscle...18 METHODOLOGY...22 Experimental Subjects...22 Materials and Methods...23 Protocol...23 Body composition and fat distribution...24 Hormone assay...24 Physical activity...25 Statistical Methods...26 Sample Size Considerations...26 Statistical Analyses...27 RESULTS...29 Descriptive Statistics...29 Partial Correlation Analyses...32 Body Composition Correlations...37 Estrogens and Change in Adiposity...39 Androgens and Change in Muscle...39 DISCUSSION...40 REFERENCES...50 APPENDIX: IRB APPROVAL FORMS...62 Current IRB Approval Form...63 vi

8 LIST OF TABLES Table 1 Descriptive statistics by HRT status Body composition outcomes at baseline and 2 year follow-up Hormone outcomes at baseline and 2 year follow-up Partial correlations for hormones with indices of lean mass, fat mass, and fat distribution at baseline for HRT users Partial correlations for hormones with indices of lean mass, fat mass, and fat distribution at baseline for non-hrt users Body composition correlation matrix for HRT users Body composition correlation matrix for non-hrt users...38 vii

9 LIST OF FIGURES Figure 1 Estradiol vs adjusted thigh fat Free testosterone vs adjusted ASM...36 viii

10 LIST OF ABBREVIATIONS ASM andro BIA BMI CT DHEA DHEA-S DXA E1 E1SO4 E2 ER-α ER-β FFM Free T FSH appendicular skeletal muscle adrosteneione bioelectrical impedance analysis body mass index computed tomography dehydroepiandrosterone dehydroepiandrosterone sulfate dual energy X-ray absorptiometry estrone estrone sulfate estradiol estrogen receptor alpha estrogen receptor beta fat free mass free testosterone follicle stimulating hormone ix

11 GCRC HDL HRT IAAT IGF-1 IL-3 LBM PA RIA SAAT SD SHBG T Total T UAB WHR General Clinical Research Center high density lipoprotien hormone replacement therapy Intra-abdominal adipose tissue insulin-like growth factor-1 interleukin-3 lean body mass physical activity radioimmunoassay subcutaneous abdominal adipose tissue standard deviation sex hormone binding globulin testosterone total testosterone University of Alabama at Birmingham waist-hip ratio x

12 1 INTRODUCTION Menopause is a period in a woman s reproductive life defined by loss of ovarian activity, thus leading to a series of endocrine disturbances. Along with changes in the endocrine environment, there is a shift in fat distribution from the gluteofemoral region to the upper body, including the intra-abdominal cavity(1). This increase in central fat has been attributed to the decline in estrogen that occurs with menopause, and increases the risk of heart disease, type II diabetes, and metabolic syndrome (2). There are distinct gender differences in body composition, specifically in skeletal muscle and fat distribution patterns (3). While males generally demonstrate the android type of fat distribution characterized by fat distributed to visceral spaces; females generally demonstrate the gynoid pattern of fat distribution characterized by fat distributed to subcutaneous depots in the gluteofemoral region. Testosterone is well known for having an anabolic affect on muscle deposition; thus, gender differences observed in lean body mass can be attributed to male sex steroid concentrations. Differences observed between genders in fat distribution suggest that estrogen concentrations either directly or indirectly affect fat accrual and distribution (4). While a significant amount of research has been dedicated to the changes in body composition and endocrine hormones with menopause, few researchers have investigated the direct relationships between endocrine hormones and lean body mass retention and visceral fat gain in postmenopausal women over time. These hormones may have a

13 2 distinct implication on postmenopausal women retaining lean muscle mass, thus, preventing falls and hip fracture, maintaining metabolism, and decreasing disease risk factors. The purpose of the current study is to determine relationships between the hormone profile and skeletal muscle and fat distribution in postmenopausal women. The hormones under investigation are estrogens including estradiol, estrone, estrone sulfate and androgens including free testosterone, total testosterone, androstenedione, and DHEA-S. We also examined several other hormones (IGF-1, cortisol) known to affect body composition and body fat distribution for their contribution to 2-yr changes in outcomes of interest. Some researchers have demonstrated that the use of hormone replacement therapy in postmenopausal women may have a significant effect on the distribution of body fat by deterring fat accumulation in the intra-abdominal cavity, although findings are inconsistent (5-8). Thus, it has been hypothesized that hormone replacement therapy may reduce the risk of certain disease states associated with this accrual of central body fat (9, 10). The source of estrogen differs between postmenopausal women using HRT and those not, such that the HRT users receive supraphysiological levels of estrogen from a pharmacologic source, and the non-users produce low levels of circulating estrogens with the main source being conversion of androstenedione to estrone in the periphery (11). Therefore, we examined the aforementioned relationships within HRT users and nonusers separately.

14 3 Hypothesis 1 Among postmenopausal women, estradiol, estrone, and E1SO4 concentrations at baseline will be inversely associated with IAAT at baseline and positively associated with total fat and thigh fat. - Estradiol, estrone, and E1SO4 concentrations at baseline will predict change in IAAT, total fat, and thigh fat over a two year period. Specific Aims 1. Measure estradiol, estrone, and estrone sulfate concentrations of all subjects. 2. Determine total body fat and leg fat via dual-energy X-ray absorptometry (DXA), and intra-abdominal adipose tissue (IAAT) and thigh fat via computed tomography (CT). 3. Determine if there are significant associations between estrogen concentrations (estradiol, estrone, and estrone sulfate) and measures of adiposity (total fat, leg fat, thigh fat, and IAAT). 4. Determine if there are significant associations between estrogen concentrations (estradiol, estrone, and estrone sulfate) at baseline and change in measures of adiposity (total fat, leg fat, thigh fat, and IAAT) over two years.

15 4 Hypothesis II Among postmenopausal women, free testosterone, total testosterone, androstenedione, and DHEA-S concentrations at baseline will be positively associated with thigh muscle area, thigh muscle mass and total appendicular skeletal muscle mass. - Free testosterone, total testosterone, androstenedione, and DHEA-S concentrations at baseline will predict change in thigh muscle area, thigh muscle mass and total appendicular skeletal muscle mass over a two year period. Specific Aims 5. Measure total testosterone, free testosterone, androstenedione, and DHEA-S concentrations of all subjects. 6. Determine appendicular skeletal muscle mass (ASM) (kg) and leg lean (kg) via dualenergy X-ray absorptiometry (DXA), and thigh muscle (cm 2 ) via computed tomography (CT) of all subjects. 7. Determine if there are significant associations between androgen concentrations (total testosterone, free testosterone, androstenedione, and DHEA-S) and measures of lean muscle (ASM (kg), leg lean (kg), and thigh muscle (cm 2 )). 8. Determine if there are significant associations between androgen concentrations (total testosterone, free testosterone, androstenedione, and DHEA-S) at baseline and change in measures of lean muscle (ASM (kg), leg lean (kg), and thigh muscle (cm 2 )) over tw

16 5 REVIEW OF LITERATURE Menopause and Sex Hormones Overview Menopause has been defined as permanent cessation of menstruation after the loss of ovarian activity and is usually identified by clinicians retrospectively after 12 months of amenorrhea (12). The loss of ovarian activity occurs over time and is the result of a series of events leading to ovarian failure. (13-15). The loss of ovarian function is characterized by an increase in FSH reflecting the lack of feedback from estrogens of ovarian origin (16) At the point of menopause, the cyclical increases and decreases in estradiol cease and progesterone concentrations remain low once cessation of cycles occurs (16). Estrogen In women of reproductive age, the ovary produces anywhere from 60 to 600 mg per day of the strongest and most biologically active estrogen, estradiol 17β (E 2 ) (17). Estradiol is found in circulation bound to SHBG (38%), loosely bound to albumin (60%), and the remaining estradiol is free to diffuse across cell membranes (18). The dominant follicle and the corpus luteum produce more than 95% of circulating estradiol in premenopausal women, and the remaining estradiol comes from the conversion of estrone to estradiol in the periphery (11). Estradiol concentrations decline sharply in the first twelve months after cessation of menstruation and continue to decline slowly thereafter (13-15). It is common for small cyclic spikes in estradiol to be observed although

17 6 amenorrhea continues. These elevations could be accounted for by activity of remaining follicles or stromal hyperplasia (19). The main source of estrone, a weaker estrogen, is the metabolism of estradiol and the aromatization of androstenedione in adipose tissue (11). Due to the decreased production of estradiol after menopause, the major source of production of estrone becomes aromatization of androstenedione in the periphery, thus with increased weight and adiposity, there is an increase in the production of estrone (20-22). The concentration of estrone declines after menopause much like estradiol (11). Estrone is similar to estradiol in its decline in concentrations although estrone concentrations are higher than that of estradiol. In premenopausal women the estradiol to estrone ratio is greater than 1.0, and in postmenopausal women the ratio shifts to less than 1.0, resulting in estrone becoming the dominant circulating estrogen after menopause (11). Estrone sulfate concentrations decline in a similar pattern to that of estradiol and estrone (23). Estrone sulfate is a metabolite of estradiol and estrone and is present in much higher concentrations than estradiol and estrone (24). Estrone sulfate is 90% bound to albumin and remains biologically inactive until the sulfate group is removed which occurs within tissues, such as adipose tissue. In women of reproductive age, the ovary is the dominant source of estrogen production. With the loss of ovarian follicles and thus ovarian failure, the ovary no longer is the major source of the production of estrogen. In postmenopausal women

18 7 aromatization of androgens in the peripheral tissues becomes the dominant source of circulating estrogens. Estrogen Receptors Estrogen receptors are members of the nuclear receptor superfamily and are hormone activated regulators of transcription. Estrogen receptors are necessary for estrogenic activity with in the cell. Unbound, an estrogen receptor is associated with a multiprotein complex and these proteins maintain the receptor in a conformation that allows it to bind to the estrogen ligand (25). This binding results in receptor dimerization and binding to the palindromic estrogen receptor response element of the promoter region of estrogen response genes. The receptor then regulates transcription by interacting with the transcription initiation complex (26). Two known estrogen receptors are ER-β and ER-α which demonstrate similar binding affinities for the estrogen ligand; however, ER-β has a higher binding affinity for phytoestrogens. ER-β is expressed in similar tissues to ER-α and also is expressed in tissues lacking ER-α, human granulose cells and spermatids (25). Androgens In women of reproductive age, the primary source of androgen production is in the adrenal and in the ovaries. However, there is conflicting evidence in the literature on postmenopausal production of androgens. While some authors suggest there is negligible androgen synthesis by the ovary after menopause, most suggest that the ovary continues producing testosterone and the adrenal cortex is the main source of other androgens (27).

19 8 In women of reproductive age, 25% of circulating testosterone is produced by the ovary, 25% produced in the adrenal cortex, and the remaining 50% is derived from the conversion of androstenedione in the periphery. Ovarian production of testosterone is unaffected by the menopause transition; however, testosterone concentration decline with age (28). There is a decline in circulating SHBG during the menopause transition, increasing the concentration of free testosterone. Testosterone is found in serum mostly bound to SHBG or bound to albumin; therefore, free testosterone makes up approximately 1-2% of circulating testosterone (29). Oral HRT is known for increasing liver secretion of SHBG, thus decreasing circulating free testosterone (30). Androstenedione concentrations are lower in postmenopausal women compared to younger women of reproductive age in whom androstenedione is the major androgen produced by the ovary. It is thought that this decrease in androstenedione follows menopause (31) while others have reported no decline associated with menopause (14). Most studies have found no change in androstenedione concentrations related to menopause after adjusting for age (13, 32, 33). DHEA-S is an androgen precursor secreted by the adrenal gland (15). The adrenal also secretes DHEA, androstenedione, and testosterone. DHEA-S is an important precursor of androgen synthesis in the periphery (34). The adrenal is the only site of DHEA-S secretion and remains unaffected by menopause status; however, there is a decrease in DHEA-S and DHEA with age independent of menopause (13). Menopause does not seem to have an impact on adrenal secretion of steroid hormones. However,

20 9 clinical deficiencies in DHEA-S have been observed with the use of estrogen replacement therapy (13). Stromal cells, not follicles, are the major source of steroids in the postmenopausal ovary (35). Stromal cells secrete estradiol and androstenedione for up to 30 years after menopause in vitro, but the ovaries of menopausal women secrete testosterone with little to no estradiol or androstenedione (16). Although stromal cells contain the aromatase complex, FSH receptors are almost undetectable on these cells in postmenopausal women, perhaps explaining the absence of estrogen secretion from the ovary in postmenopausal women (16). Hormone Metabolism Although there are abrupt changes in production and secretion of endocrine hormones with menopause, the metabolism and clearance rates of these hormones do not change (36). The metabolism of androgens shifts slightly through the pathway described as aromatization of androgens in the periphery. The main sites of this aromatization of androstenedione to estrone are in the adipose tissue, muscle and possibly the skin (37). This shift in the metabolism pathway of androgens is significantly different in postmenopausal than that of younger premenopausal women, while not greatly impacting overall androgen metabolism. However, this aromatization of androstenedione to estrone becomes the major source of circulating estrogens in postmenopausal women (36).

21 10 Hormone Replacement Therapy Hormone replacement therapy is prescribed for its relief of menopause symptoms including hot flashes and genitourinary symptoms and also prevention of osteoporosis. Despite these benefits, conflicting evidence exists on whether hormone replacement therapy increases or decreases the risk of certain cancers, heart disease, and diabetes. Studies have demonstrated HRT to be beneficial to lipid profile and also shifting fat deposition away from the visceral depots to peripheral depots, thus reducing risk of cardiovascular disease. There have also been associations found between HRT use and decreased insulin sensitivity. Menopause and Body Composition Weight and total body fat There is conflicting evidence on whether or not menopause is associated with weight gain and increase in body fatness. Several studies show that menopause is related to weight gain and increased body fat when adjusting for age (8, 33, 38, 39). In contrast, Wing et al (40) found no significant difference in weight gain between pre- and postmenopausal women in a longitudinal study of middle aged women. In an epidemiological study, Study of Women s Health Across the Nation, approximately 13,000 women including many different ethnicities from different regions of the United States were surveyed. They found BMI to be significantly greater in perimenopausal women and women who had surgical menopause compared to premenopausal women.

22 11 However, they found no difference between naturally postmenopausal women and premenopausal women (41). Fat distribution Intra-abdominal adipose tissue has been associated with increased disease risk for chronic metabolic disease. In postmenopausal women, decreased estrogen concentrations have been linked to increased fat accumulation in the abdominal depots (6); thus, postmenopausal women may be at an increased risk for cardiovascular disease, hypertension, and diabetes (42, 43). Researchers have used anthropometric measures as surrogate measures of intra-abdominal fat (40), radiograph imaging techniques such as the dual-energy X-ray absorptiometry measuring total abdominal fat, and CT scans as a direct measure of intra-abdominal fat by imaging specific fat depots. Findings have been inconsistent in cross-sectional studies using WHR as a measure of intra-abdominal fat deposition (33, 40, 44). Studies using DXA to measure abdominal adiposity found significant increases associated with menopause after adjusting for age (45, 46) and total fat (8), although the DXA imaging cannot differentiate between subcutaneous and visceral adipose depots in the abdomen. Svendsen et al (45) found a significant and independent effect of menopause status on total fat percentage and abdominal fat percentage after controlling for age. Similarly, Gambaccionni et al (47) found perimenopausal women and postmenopausal women had higher total body fat and abdominal adiposity; while the same effect of menopause was still observed in a subgroup of age and BMI matched

23 12 women. Ley et al (8) also reported an increase in abdominal adiposity via DXA scan; however, it was difficult to distinguish between the effects of age vs menopause due to lack of age-matched controls. Studies using computed tomography, a direct measure of abdominal depot fat stores, demonstrate consistent findings that intra-abdominal adipose tissue increases with menopause independent of aging (5, 48, 49). No relationship between menopause and increased central adiposity has been found in few studies. Wang et al (50) found a stronger relationship with age and percentage trunk fat than menopause status and percent trunk fat, implying no independent effect of menopause on increased abdominal adiposity. Schreiner et al (51) used magnetic resonance imaging and found the same proportion of increasing body weight and increasing intra-abdominal adipose tissue before and after menopause. Several studies have examined the relationship between menopause and fat distribution longitudinally. Bjorkelund et al (52) followed a group of 208 women for 6 years. A subgroup of 60 women became postmenopausal prior to follow up and the rest remained premenopausal. In the women who became postmenopausal, significant increases in WHR were observed. In another longitudinal study by Franklin et al (53), MRI was used to determine change in relative volume of visceral and subcutaneous fat depots in the abdominal cavity with menopause. This 8 year study started with 23 subjects at baseline, all premenopausal, and of those subjects 8 were postmenopausal and available for follow up visit. Despite the small sample size, increases in body fat percentage were observed though not significant. Significant increases in total abdominal

24 13 fat, intra-abdominal, and subcutaneous fat were observed. There were not any changes in the percentage of subcutaneous and intra-abdominal fat relative to total abdominal fat with menopause. Estrogen and fat metabolism The mechanisms by which low levels of estrogen impact metabolism and fat distribution are not completely understood. Several pathways have been proposed in which low levels of estrogen may alter lipolytic activity of the adipose tissue. In a study of women receiving HRT, it was demonstrated that there was a reduction of epinephrinestimulated lipolysis in the subcutaneous adipose tissue (54). In a similar study, it was found that treatment with estradiol increases the expression of alpha-2-adrenergic receptors in human subcutaneous adipose tissue through an increase in estrogen receptors (55). This increase in the alpha-2-adrenergic receptors was accompanied by inhibition of epinephrine-induced lipolysis. Estradiol had no influence on the receptor content of the visceral adipose tissue, causing fat to accumulate in the subcutaneous depots instead of the visceral depots (55). One possible explanation for a shift in fat deposition from the periphery to the intra abdominal cavity is a decrease in fat oxidation possibly due to a shift in preferential carbohydrate oxidation in postmenopausal women (56). It has also been suggested that oral estrogen therapy further reduces fat oxidation. (57). However, these effects are seen initially with oral estrogen therapy but may be balanced out over time through beneficial effects on energy intake and physical activity (58).

25 14 HRT and fat distribution Many studies have examined the effects of hormone replacement therapy on body fat distribution in the postmenopausal female both cross-sectionally and longitudinally. Most of these studies compared hormone users to non-users which may be subject to bias assuming that women choosing to use hormone replacement therapy are more health conscious thus impacting the outcomes (59). Many studies examine the impact of HRT on central adiposity via WHR as a surrogate marker for abdominal adiposity and fat distribution. These studies have shown conflicting results with some finding no difference between hormone users and non-users (44, 60). Among intervention studies examining hormone users versus non-users (59), the control groups gained abdominal adiposity while very little or no change was detected in the treatment groups. One intervention study by Aloia et al (61), found no effect of hormone use on trunk/extremity fat ratio measured via DXA in hormone users on combined estrogen and medroxyprogesterone. In a cross-sectional study by Kritz-Silverstein and Barrett-Connor (62), there were no differences in WHR between women who continuously used HRT, intermittently used HRT, and those who had never used HRT. Several studies using direct measure of IAAT via CT scan, suggest HRT might limit IAAT fat deposition. In a study by Gower et al (5), HRT users had less IAAT at baseline than the non users although it was not clear whether this was an effect of the hormone replacement therapy or differences in groups prior to the study. Both groups gained IAF over two years with

26 15 no difference in the rate of gain, and relative proportions of IAAT and total body fat remained stable. Aging and Muscle Overview It is well known that muscle mass and function decline with age in older men and women (63). The decline in muscle mass has been associated linearly with age while individuals over 65 have an accelerated decrease in muscle mass. It has been suggested that this relationship does not remain linear throughout adulthood such that muscle loss may be accelerated with the menopause transition and sarcopenia may be related to loss of ovarian activity. However, investigators have not been able to confirm the hypothesis that estrogen concentrations are related to muscle mass in cross-sectional study design (63). This finding may be due to the elderly population used in the analysis (65-97 years of age), and these relationships may be detected in an early postmenopausal population. Although the association between menopause and loss in fat-free mass has been extensively researched, inconsistencies exist in research methodology with some studies using DXA scans which are unable to differentiate between components of lean mass and CT scans which can give more accurate estimates of regional muscle mass. Toth et al (64) examined total body FFM (fat free mass) and skeletal muscle mass via DXA and results showed no difference between pre- and early postmenopausal women. However,

27 16 most cross-sectional data suggest that postmenopausal women have less fat free mass than pre-menopausal women (45, 47, 65). Several studies have observed a loss in muscle strength in women during the menopause transition (20, 66). However, other studies have found that there is no difference in muscle strength between pre- and post-menopausal women (67). It has also been speculated that a loss in muscle mass may be independent of a loss in muscle strength (68). Rolland et al (68) found that in young postmenopausal women, loss of muscle strength was more directly related to absence of physical activity, high BMI and HDL cholesterol, and a low concentration of hormones (estrone, ipth) and inflammatory markers such as IL-3 receptors. Reduced loss of appendicular muscle mass was more directly associated with anthropometric measures such as lower height, low BMI and high appendicular muscle mass. Greater loss of appendicular muscle mass was also positively associated with estrone and thyroxine, but inversely associated with concentrations of DHEA. Therefore, muscle mass and muscle quality may be independent from each other and perhaps retaining muscle mass and quality are due to different mechanisms (68). Sarcopenia Sarcopenia is the gradual loss of skeletal muscle mass and strength that occurs with advancing age (69). Sarcopenia has also been identified as FFM that is 2SDs below the mean of a young, healthy population (70). This loss in skeletal muscle mass is also associated with loss of muscle strength and quality delineating loss in function as

28 17 age increases. Baumgartner et al (69) found in a study on sarcopenia of the elderly population in New Mexico that 40% of individuals 80 years of age or older can be thought of as sarcopenic and therefore are at risk of morbidity and other disabilities associated with the disease. Although no cause and effect relationship was established between sarcopenia and an increased risk of morbidity or functional impairment, there is an association between these factors (69). This is a very prominent problem among older adults although the magnitude of which is still under investigation. Although two modifiable behavior factors have been identified as level of physical activity including resistance exercise and smoking status (70), much is to be studied on implications from protein intake and muscle turn over of protein and changes in hormone parameters. In a study by Castillo et al (70), prevalence of sarcopenia in both men and women was the lowest for the age group (1.9% in males and 2.5% in females) while prevalence for the condition increased after the age of and was found in 16% of males and 13% of women that were 85 years of age or older. It was also found that those individuals with sarcopenia had lower BMI and fat mass and also significantly higher age than subjects without sarcopenia. Women with sarcopenia exercised significantly less and smoked significantly more than women without sarcopenia and other factors such as estrogen use and alcohol intake were not significant. Exercise and smoking status were not significant in men between the sarcopenic and non-sarcopenic groups. Therefore, sarcopenia can be identified as a very gender specific condition occurring in elderly adults (70).

29 18 The mechanism of sarcopenia is not fully understood. Researchers have speculated a link in IGF-1 to decrease in lean mass, differences in older and young muscle associated with apoptosis, or even declines in anabolic hormones and less myonuclei due to an apoptotic-like mechanism (71). Physical activity and diet are apparent factors contributing to muscle gain and loss, but endocrine factors also play a role in determining body composition. Studies have demonstrated the impact of Vitamin D and Calcium on muscle strength and quality. When in a deficient state, a relationship between an increase in falls in an elderly sarcopenic population has been demonstrated (71). Growth hormone has also been shown to produce a number of anabolic effects, including increasing fat free mass and reducing fat mass in children. Growth hormone replacement in adults with growth hormone deficiency has proven to be beneficial in improving muscle mass and quality. Androgens have also been linked to improved muscle quality and strength, in younger and older subject populations (71). HRT and Change in Muscle Numerous studies have attempted to show that HRT may slow or even stop the loss of muscle in postmenopausal women by comparing body composition in hormone users and non-users (30, 72-74), These studies assessed lean mass and body cell mass via CT scan, DXA, and BIA. Results have been inconsistent in determining whether HRT preserves or accelerates the loss of muscle in postmenopausal women. In most studies, no difference in muscle mass was found between hormone users and non-users, although these finding could be the result of the use of DXA measures which do not differentiate

30 19 between lean muscle mass and other lean components. Inconsistent findings may also be due to differences in study design, including type of HRT given, years of use and method of analysis of body composition. It has been suggested that associations between HRT and body composition outcomes are highly dependent on the type of hormones included in the HRT. One analysis on body cell mass found that it was higher in women using HRT, including estrogen-based and testosterone derived formulas, compared to non-users (73). However, women in this study using CEE and progesterone derived gestagens were no different from the non-user controls. Kenny et al (75) examined the prevalence of sarcopenia in a group of 189 postmenopausal women taking HRT for at least two years. It was found that prevalence of sarcopenia in women using HRT for at least two years (23.8%) was similar to that of women not using HRT (22.6%). It was also shown that HRT use was not a predictor of ASM but that BMI, strength, and testosterone level were significant predictors. In a study by Hanggi et al (7), three different treatment regimens of HRT were observed, comparing differences in lean and fat distribution among groups. Body composition was measured with a DXA analysis. In the controls and oral estrogen group, lean mass significantly decreased while no significant decrease was seen in the trandermal estrogen and tibolone group. This decrease in lean mass was due to a decrease in trunk lean and not lean mass of the head, arms and legs. The oral estrogen group lost weight over the duration of the study due to a loss in lean mass, while the controls weights remained stable due to a gain in fat mass that accompanied the loss in

31 20 lean mass. They also found that the percentage change of total body fat was inversely associated with changes in serum estradiol for all treatment groups (7). In a longitudinal study by Greeves et al (66), they concluded that accelerated decline in muscle mass following menopause may be prevented with the use of HRT. This study was limited by the small sample sizes in each group. Additionally, they found that dynamic and isometric strength decreased in the control group while in the HRT users strength did not change (66). Ronkainen et al (76) examined effects of long term HRT use on muscle composition and function in postmenopausal monozygotic twins. One twin was exposed to HRT and the other had never been exposed. Among the women on HRT, five took estrogen only preparations, six took combined treatment including estrogenic and progestogenic compounds, and four used tibolone. It was found that HRT use was associated with increased mobility and muscle power in the women aged yr. Also, the twin taking the HRT had lower relative proportion of fat in the thigh and greater muscle size than their genetically identical sister. They found that the tibolone users had more lean body mass and larger thigh muscle. The twins taking the estrogen containing formulas demonstrated significantly smaller thigh fat area, subcutaneous fat area, and lower percentage body fat compared to their sisters. It has been reported that the hepatic protein, SHBG, increases with age, thus reducing the concentration of bioavailable testosterone (77). It has also been shown that oral HRT affects hepatic protein production characterized by significant increases SHBG,

32 21 thus reducing circulating free testosterone (30). This presents a possible explanation to the differences in free testosterone concentrations between HRT users and non-users.

33 22 METHODS Experimental Subjects Subjects were 84 postmenopausal women aged yr. Only women who experienced a natural menopause, with the time of menopause known to occur at least 6 months prior to contact, were recruited. Both women using HRT and women not using HRT were recruited. Among hormone users (n=50), subjects predominantly used conjugated equine estrogens, mg/day, and medroxyprogesterone acetate, 2.5 mg/day. Four women used unopposed oral estrogen (conjugated equine estrogens), and one used a transdermal estradiol patch in combination with an oral progestin. In cases where usage was cyclic, testing was conducted during the combined (estrogen + progestin) portion of the cycle. No HRT use (n=34) was defined as no current use, and no use within the past 6 months. Over the 2 year observation period, several women in the HRT group changed their hormone use: five women changed dosage of the same preparation; two women switched to a different formulation of the same hormone (e.g., from Provera to Prometrium); three women altered their hormone use (e.g., from estrogen only to estrogen plus progestin); and two women discontinued HRT. Two women in the control group started using HRT during the course of the study (one used Premarin vaginal cream; the other used Estrace/Prometrium). Among the women recruited, 12% were African American (n=10) and 88% European American (n=74). Five of the African American women were HRT users and the remaining five were non-hrt users. Due to the low number of African American women recruited in each group for this study, unique effects of race could not

34 23 be determined. None of the women smoked. Data were collected over a 27 h period during an in-patient visit to the Department of Nutrition Sciences and the General Clinical Research Center at the University of Alabama at Birmingham (UAB). The protocol was approved by the Institutional Review Board for Human Use at UAB, and all subjects signed an informed consent prior to testing (protocol # X ) Material and Methods Protocol. Testing took place over several years (03/97 06/01). At baseline, and after 2 years, subjects came to UAB for an overnight visit with a comprehensive metabolic evaluation (body composition, fat distribution, insulin sensitivity testing). One year after the baseline visit, subjects reported to UAB for a brief, outpatient evaluation (fasting blood draw and body composition analysis only). At each comprehensive visit, subjects arrived at the Department of Nutrition Sciences at approximately 9:00 am in the fasted condition (12-hour fast). Body composition was determined by dual-energy X-ray absorptiometry (DXA). At approximately 12:00 pm, subjects were escorted to UAB s General Clinical Research Center (GCRC). Subjects remained at the GCRC for approximately 24 hours, departing at noon the following day. At approximately 1900 h, subjects were escorted to Radiology for computed tomography scanning. While at the GCRC, all food was provided. The evening snack was consumed prior to 7:00 pm.

35 24 Subjects then remained fasted until fasting blood draw and intravenous glucose tolerance test the following morning (~7:00 am). Body composition and fat distribution Total and regional body composition [fat mass, ASM, and lean body mass (LBM)] were measured by DXA using a Lunar DPX-L densitometer (LUNAR Radiation Corp., Madison, WI). Subjects were scanned in light clothing while lying flat on their backs with arms at their sides. DXA scans were performed and analyzed with adult software version 1.5g. Intra-abdominal fat (IAAT), thigh muscle, SAAT, and thigh fat, were analyzed by computed tomography scanning with a HiLight/Advantage Scanner (General Electric, Milwaukee) as previously described (78, 79). A scout scan was first performed to locate the L4-L5 inter-vertebral space. Subsequently, a 5mm scan of this abdominal site was taken. Thigh fat and thigh muscle were assessed in a cross-section at mid-thigh. Scans were later analyzed for cross-sectional area (cm 2 ) of adipose tissue using the density contour program with Hounsfield units for adipose tissue set at -190 to -30. We have shown the test-retest reliability for IAAT to be 1.7 percent (80). Scans were analyzed by an individual blinded to the hormone use status of the study participants. Hormone assay Serum E2 was measured using a double-antibody RIA (Diagnostic Products, Los Angeles, CA). Sera were first extracted in diethyl ether. Assay sensitivity was determined to be pmol/l, intraassay coefficient of variation to be 5.3%, and

36 25 interassay coefficient of variation to be 6.0%. Serum total T was measured with a coatedtube RIA (Diagnostic Products); assay sensitivity was nmol/l, intraassay coefficient of variation was 7.7%, and interassay coefficient of variation was 8.2%. For measurement of SHBG, sera were first diluted 1:101, then assayed in duplicate 25-mL aliquots using an immunoradiometric assay (Diagnostic Systems Laboratories, Inc., Webster, TX); the lower limit was 5 nmol/l, the intraassay coefficient of variation was 8.2%, and the interassay coefficient of variation was 8.2%. Free T (pmol/l) was calculated from serum concentrations of total T and SHBG using the method of Sodergard et al (81). This method is based on the concentration of albumin, the binding capacity of SHBG, and the association constants of T for SHBG and albumin, as determined in a sample of normal men and women. Physical activity Physical activity was assessed at baseline using a modification of the technique published by Sallis et al (82). Details have been published (83). In brief, the women were interviewed regarding their participation (hours per week) in physical activity over the previous week. Study personnel subsequently categorized activities as light, moderate, hard,, or very hard, and assigned energy expenditure values to each activity. Measured resting metabolic rate was then used to calculate total and activityrelated energy expenditure for the week. For the purpose of this study, kcal per week of light and moderate physical activity were tested in statistical models for potential contribution to variance in body composition and risk factors.

37 26 Statistical Methods Sample Size Considerations The sample size for this study was 84 postmenopausal females; of these, 50 were using HRT and 34 were not. This sample size was partly based on feasibility considerations, such as the number of subjects that we were able to examine with the resources available to the study. Power calculations were performed using nquery Advisor, version 7. Each calculation assumed a type I error rate of 0.05 and a two-sided statistical test. We looked at associations between hormone levels and various measures of body composition. Assuming the approximate normality of the transformed correlation coefficient as implemented in nquery Advisor, we had at least 80% power to detect significant correlations of 0.31, 0.39, and 0.45, and at least 90% power to detect significant correlations of 0.35, 0.47, and 0.53, in the overall group of 84 women, in the group of 50 HRT using women, and in the group of 34 non-hrt using women, respectively. Power calculations for multiple linear regression models were based on a test of the squared multiple correlation coefficient (R2). The overall group of 84 women, the group of 50 HRT using women, and the group of 34 non-hrt using women provided 80% power to detect an R2 of 0.141, 0.226, and 0.272, respectively, and 90% power to detect an R2 of 0.174, 0.315, and 0.371, respectively, in a 5-parameter model as being statistically significant. Effect sizes of 0.309, 0.404, and for the overall group of 84 women, the group of 50 HRT using women, and the group of 34 non-hrt using women, respectively, were required to provide 80% power, and effect sizes of 0.358,

38 , and for the overall group of 84 women, the group of 50 HRT using women, and the group of 34 non-hrt using women, respectively, were required to provide 90% power, for a test of an individual parameter within a regression model. Statistical Analysis Descriptive statistics were computed for all study variables of interest. Distributions of study variables were examined. Those that were known to deviate from a normal distribution, such as hormone measurements, were log transformed (using the log base 10 transformation) prior to statistical analysis. All statistical tests were twosided and were performed using a type I error rate of Statistical analyses were performed using SAS (version 9.1; SAS Institute, Inc., Cary, NC). Overall comparisons between baseline and follow-up were performed using the paired t-test analysis. Two-way ANOVA repeated measures analyses were used to examine differences from baseline to two year follow-up and group differences between HRT users and non-hrt users in measures of body composition and hormone concentrations. Partial correlation coefficients were calculated to determine associations between variables of interest (hormone measurements including estradiol, estrone sulfate, total testosterone, free testosterone, androstenedione, DHEAS, cortisol, and IGF-1and measures of body composition including total fat adjusted for total lean mass, and IAAT, thigh fat, leg fat, thigh muscle, leg lean, and ASM adjusted for total fat mass). Physical activity was analyzed as a potential confounder in a multiple linear regression model with significant predictors of lean mass at baseline. Stepwise multiple linear regression

39 28 analyses were used for the larger set of potential predictor variables accounting for change over time to help us determine initial models to further consider in the multiple regression analyses. Regression coefficients and correlation coefficients were tested for statistical significance.

40 29 RESULTS Descriptive Statistics Descriptive statistics for the postmenopausal women using HRT and not using HRT in the study are in Tables 1-3. Relevant demographic variables are shown in Table 1, and, with the except of physical activity being higher in the HRT users, there were no differences in the groups at baseline. Baseline and follow up measures of body composition are described in Table 2. From baseline to the two year follow up, significant decreases in ASM and lean leg mass (kg) in the HRT users were noted. In both HRT users and non-users, IAAT was the only measure of fat to increase significantly over the two years. HRT users had significantly less IAAT at baseline than the non-users and this remained true over two years to follow up. HRT users also had significantly less total fat than non-users at baseline and over the course of the study. Hormone concentration variables of interest are reported in Table 3. E1SO4, total testosterone, SHBG, and cortisol increased significantly from baseline to follow up in the HRT users. IGF-1 and androstenedione decreased significantly in both groups. Free testosterone, DHEA-S, FSH, and Insulin concentrations were significantly higher in non-users than in the HRT users. Estradiol, estrone, E1SO4, SHBG, and cortisol were all significantly higher in the HRT users.